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Sample records for plasma rna viral

  1. HTLV-1 viral RNA is detected rarely in plasma of HTLV-1 infected subjects.

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    Demontis, Maria Antonietta; Sadiq, Maaz Tahir; Golz, Simon; Taylor, Graham P

    2015-12-01

    Plasma of patients infected with HTLV-1 is considered non-infectious but detection of HTLV-1 genomic RNA in plasma has been recently reported. The aim of this project was to detect and quantify HTLV-1 RNA in plasma and assess its potential value in diagnosis and prognosis. RNA from 1 ml of plasma from 65 subjects infected with HTLV-1 (27 asymptomatic carriers [AC]), 17 patients with HTLV-1-associated myelopathy (HAM/TSP), 14 with adult T-cell leukemia/lymphoma (ATLL), two co-infected with HIV, and five with other HTLV-1-associated disease, was extracted and reverse transcribed. HTLV-1 specific nested PCR was performed using primers to amplify the conserved Tax region. All samples were run in quadruplicate, nested PCR products were detected by gel electrophoresis. HTLV-1 RNA was detected in plasma from 18 (28%) patients, always at a very low copy number (3-13 copies viral cDNA per milliliter of plasma). Mean values of HTLV-1 proviral load did not differ between patients in whom HTLV-1 RNA was detected and patients in whom it was not possible to detect HTLV-1 RNA in plasma. HTLV-1 genomic RNA can be detected in the plasma of a minority of patients but not at a level or frequency to be useful clinically or diagnostically. Lack of transmission of HTLV-1 by plasma is due to the rare presence of HTLV-1 virions, regardless of any other factor.

  2. Analysis of plasma viral RNA levels during acute dengue virus infection using quantitative competitor reverse transcription-polymerase chain reaction.

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    Sudiro, T M; Zivny, J; Ishiko, H; Green, S; Vaughn, D W; Kalayanarooj, S; Nisalak, A; Norman, J E; Ennis, F A; Rothman, A L

    2001-01-01

    There is increasing recognition of the potential importance of viral burden in the pathogenesis of dengue hemorrhagic fever (DHF). There is little data available, however, describing the kinetics of viral replication in humans with natural dengue virus (DV) infection. Standard procedures for measuring titers of infectious virus in clinical specimens are either laborious or insensitive. We developed a method for measurement of DV RNA in plasma samples based on reverse transcription-polymerase chain reaction (RT-PCR) using a mutant RNA target as a competitor. This technique was reproducible and accurate for samples containing any of the four DV serotypes, and could be applied to samples containing as few as 250 copies of RNA per reaction. We examined plasma viral RNA levels in 80 children with acute DV infection; sequential plasma samples were tested in 34 of these children. Plasma viral RNA levels ranged as high as 10(9) RNA copies/ml, and correlated with titers of infectious virus measured in mosquitoes (r= 0.69). Plasma viral RNA levels fell rapidly during the last several days of the febrile period. We did not find a significant difference in maximal plasma viral RNA levels between children with DHF and children with dengue fever, but peak viral RNA levels were identified in only 16 subjects. We conclude that this quantitative RT-PCR method will be valuable for further studies of natural DV infections.

  3. Detection of Viral RNA in Tissues following Plasma Clearance from an Ebola Virus Infected Patient

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    Bordi, Licia; Castilletti, Concetta; Colavita, Francesca; Quartu, Serena; Nicastri, Emanuele; Lauria, Francesco Nicola; Petrosillo, Nicola; Lanini, Simone; Kobinger, Gary; Zumla, Alimuddin; Di Caro, Antonino; Ippolito, Giuseppe; Capobianchi, Maria Rosaria; Lalle, Eleonora

    2017-01-01

    An unprecedented Ebola virus (EBOV) epidemic occurred in 2013–2016 in West Africa. Over this time the epidemic exponentially grew and moved to Europe and North America, with several imported cases and many Health Care Workers (HCW) infected. Better understanding of EBOV infection patterns in different body compartments is mandatory to develop new countermeasures, as well as to fully comprehend the pathways of human-to-human transmission. We have longitudinally explored the persistence of EBOV-specific negative sense genomic RNA (neg-RNA) and the presence of positive sense RNA (pos-RNA), including both replication intermediate (antigenomic-RNA) and messenger RNA (mRNA) molecules, in the upper and lower respiratory tract, as compared to plasma, in a HCW infected with EBOV in Sierra Leone, who was hospitalized in the high isolation facility of the National Institute for Infectious Diseases “Lazzaro Spallanzani” (INMI), Rome, Italy. We observed persistence of pos-RNA and neg-RNAs in longitudinally collected specimens of the lower respiratory tract, even after viral clearance from plasma, suggesting possible local replication. The purpose of the present study is to enhance the knowledge on the biological features of EBOV that can contribute to the human-to-human transmissibility and to develop effective intervention strategies. However, further investigation is needed in order to better understand the clinical meaning of viral replication and shedding in the respiratory tract. PMID:28056096

  4. Increased levels of HIV RNA detected in samples with viral loads close to the detection limit collected in Plasma Preparation Tubes (PPT).

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    Griffith, Brigitte P; Mayo, Donald R

    2006-02-01

    The accurate and reliable quantification of HIV RNA is an essential part of the management of HIV infected individuals, and elucidation of factors that may affect HIV RNA measurements, such as the use of Vacutainer Plasma Preparation Tubes (PPT), is crucial. The objective of this study was to determine if plasma samples with viral loads close to the lower limit of the dynamic range of the assay collected in PPT tubes had increased levels of HIV RNA as compared to samples collected in standard EDTA tubes. HIV RNA levels were compared in 112 paired plasma samples collected in PPT and standard EDTA tubes. All samples had been frozen prior to testing. Discrepancies between PPT and EDTA tubes did not occur for samples with high viral loads. However, in samples with viral loads close to the lower limit of the dynamic range, levels of HIV RNA detected were higher in a large proportion of PPT as compared to the corresponding EDTA plasma samples. Forty percent of plasma pairs had no detectable HIV RNA in the EDTA aliquot, but had low levels of HIV RNA in the corresponding PPT aliquot. This prospective study underlines the need for cautious interpretation of small transient viral load changes in samples with values close to the detection limit.

  5. Hepatitis G Viral RNA Co-infection in Plasma and Peripheral Blood Mononuclear Cells in Patients with Hepatitis C

    Institute of Scientific and Technical Information of China (English)

    LI Shuli; ZENG Linglan; LUO Duande; LIU Wei; GUO Jingsong; YANG Xiaoming

    2001-01-01

    The incidence of the co-infection of hepatitis G virus (HGV) and hepatitis C virus(HCV) and its clinical implication was investigated and the difference in the positive rate of HGV RNA and HCV RNA between plasma and peripheral blood mononuclear cells (PBMCs) observed. By using reverse transcriptase polymerase chain reaction (RT-PCR) assay, HCV-RNA and HGV-RNA in plasma and PBMCs of 72 patients with hepatitis C was detected. It was showed that HGV RNA was positive in plasma of 11 patients, in PBMCs of 15 patients, and simultaneously in both of plasma and PBMCs of 10 patients with the co-infection rate being 22.2 %. Nine patients were both HGV RNA and HCV RNA positive in plasma, 11 patients were both HGV RNA and HCV RNA positive in PBMC, and 6 patients were both HGV RNA and HCV RNA positive in both plasma and PBMC with the positive rate being 12.4 %, 15.3 % and 8.3 % respectively. The positive rate of both HGV RNA and HCV RNA in PBMCs was higher than in plasma. It was concluded that the HGV co-infection rate in the patients with hepatitis C was 22. 2 %. Simultaneous examination of plasma and PBMC can improve clinically detectable rate.

  6. RNA silencing and plant viral diseases.

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    Wang, Ming-Bo; Masuta, Chikara; Smith, Neil A; Shimura, Hanako

    2012-10-01

    RNA silencing plays a critical role in plant resistance against viruses, with multiple silencing factors participating in antiviral defense. Both RNA and DNA viruses are targeted by the small RNA-directed RNA degradation pathway, with DNA viruses being also targeted by RNA-directed DNA methylation. To evade RNA silencing, plant viruses have evolved a variety of counter-defense mechanisms such as expressing RNA-silencing suppressors or adopting silencing-resistant RNA structures. This constant defense-counter defense arms race is likely to have played a major role in defining viral host specificity and in shaping viral and possibly host genomes. Recent studies have provided evidence that RNA silencing also plays a direct role in viral disease induction in plants, with viral RNA-silencing suppressors and viral siRNAs as potentially the dominant players in viral pathogenicity. However, questions remain as to whether RNA silencing is the principal mediator of viral pathogenicity or if other RNA-silencing-independent mechanisms also account for viral disease induction. RNA silencing has been exploited as a powerful tool for engineering virus resistance in plants as well as in animals. Further understanding of the role of RNA silencing in plant-virus interactions and viral symptom induction is likely to result in novel anti-viral strategies in both plants and animals.

  7. Dicer-2 processes diverse viral RNA species.

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    Leah R Sabin

    Full Text Available RNA silencing pathways play critical roles in gene regulation, virus infection, and transposon control. RNA interference (RNAi is mediated by small interfering RNAs (siRNAs, which are liberated from double-stranded (dsRNA precursors by Dicer and guide the RNA-induced silencing complex (RISC to targets. Although principles governing small RNA sorting into RISC have been uncovered, the spectrum of RNA species that can be targeted by Dicer proteins, particularly the viral RNAs present during an infection, are poorly understood. Dicer-2 potently restricts viral infection in insects by generating virus-derived siRNAs from viral RNA. To better characterize the substrates of Dicer-2, we examined the virus-derived siRNAs produced during the Drosophila antiviral RNAi response to four different viruses using high-throughput sequencing. We found that each virus was uniquely targeted by the RNAi pathway; dicing substrates included dsRNA replication intermediates and intramolecular RNA stem loops. For instance, a putative intergenic RNA hairpin encoded by Rift Valley Fever virus generates abundant small RNAs in both Drosophila and mosquito cells, while repetitive sequences within the genomic termini of Vaccinia virus, which give rise to abundant small RNAs in Drosophila, were found to be transcribed in both insect and mammalian cells. Moreover, we provide evidence that the RNA species targeted by Dicer-2 can be modulated by the presence of a viral suppressor of RNAi. This study uncovered several novel, heavily targeted features within viral genomes, offering insight into viral replication, viral immune evasion strategies, and the mechanism of antiviral RNAi.

  8. A Viral Noncoding RNA Complements a Weakened Viral RNA Silencing Suppressor and Promotes Efficient Systemic Host Infection

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    Flobinus, Alyssa; Hleibieh, Kamal; Klein, Elodie; Ratti, Claudio; Bouzoubaa, Salah; Gilmer, David

    2016-01-01

    Systemic movement of beet necrotic yellow vein virus (BNYVV) in Beta macrocarpa depends on viral RNA3, whereas in Nicotiana benthamiana this RNA is dispensable. RNA3 contains a coremin motif of 20 nucleotides essential for the stabilization of noncoding RNA3 (ncRNA3) and for long-distance movement in Beta species. Coremin mutants that are unable to accumulate ncRNA3 also do not achieve systemic movement in Beta species. A mutant virus carrying a mutation in the p14 viral suppressor of RNA silencing (VSR), unable to move long distances, can be complemented with the ncRNA3 in the lesion phenotype, viral RNA accumulation, and systemic spread. Analyses of the BNYVV VSR mechanism of action led to the identification of the RNA-dependent RNA polymerase 6 (RDR6) pathway as a target of the virus VSR and the assignment of a VSR function to the ncRNA3. PMID:27782046

  9. Dengue viral RNA levels in peripheral blood mononuclear cells are associated with disease severity and preexisting dengue immune status.

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    Anon Srikiatkhachorn

    Full Text Available BACKGROUND: Infection with dengue viruses (DENV causes a wide range of manifestations from asymptomatic infection to a febrile illness called dengue fever (DF, to dengue hemorrhagic fever (DHF. The in vivo targets of DENV and the relation between the viral burden in these cells and disease severity are not known. METHOD: The levels of positive and negative strand viral RNA in peripheral blood monocytes, T/NK cells, and B cells and in plasma of DF and DHF cases were measured by quantitative RT-PCR. RESULTS: Positive strand viral RNA was detected in monocytes, T/NK cells and B cells with the highest amounts found in B cells. Viral RNA levels in CD14+ cells and plasma were significantly higher in DHF compared to DF, and in cases with a secondary infection compared to those undergoing a primary infection. The distribution of viral RNA among cell subpopulations was similar in DF and DHF cases. Small amounts of negative strand RNA were found in a few cases only. The severity of plasma leakage correlated with viral RNA levels in plasma and in CD14+ cells. CONCLUSIONS: B cells were the principal cells containing DENV RNA in peripheral blood, but overall there was little active DENV RNA replication detectable in peripheral blood mononuclear cells (PBMC. Secondary infection and DHF were associated with higher viral burden in PBMC populations, especially CD14+ monocytes, suggesting that viral infection of these cells may be involved in disease pathogenesis.

  10. Phosphorylation of the viral coat protein regulates RNA virus infection

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    Hoover HS

    2016-11-01

    Full Text Available Haley S Hoover, C Cheng Kao Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, USA Abstract: Coat proteins (CPs are the most abundant protein produced during a viral infection. CPs have been shown to regulate the infection processes of RNA viruses, including RNA replication and gene expression. The numerous activities of the CP in infection are likely to require regulation, possibly through posttranslational modifications. Protein posttranslational modifications are involved in signal transduction, expanding and regulating protein function, and responding to changes in the environment. Accumulating evidence suggests that phosphorylation of viral CPs is involved in the regulation of the viral infection process from enabling virion disassembly to regulation of viral protein synthesis and replication. CP phosphorylation also affects viral trafficking and virion assembly. This review focuses on the regulatory roles that phosphorylation of CPs has in the life cycle of viruses with RNA genomes. Keywords: viral capsid protein, posttranslational modification, phosphorylation, protein–RNA interaction

  11. Hepatitis E viral loads in plasma pools for fractionation.

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    Baylis, Sally A; Corman, Victor M; Ong, Edgar; Linnen, Jeffrey M; Nübling, C Micha; Blümel, Johannes

    2016-10-01

    It is now recognized that blood donors may be silently infected with hepatitis E virus (HEV) and that plasma pools used in the manufacture of plasma-derived medicinal products may also contain detectable virus RNA. The occurrence of HEV-infected blood and plasma donors can vary considerably depending on local epidemiology. Manufacturing plasma pools from North America, Europe, the Middle East, and Asia were examined for the presence of HEV using transcription-mediated amplification of HEV RNA; confirmatory testing was performed using real-time reverse transcription polymerase chain reaction and sequencing. A total of 484 pools were tested. Asian pools were most frequently positive for HEV RNA and had higher viral loads, although none exceeding 300 IU/mL, and the sequenced strains (n = 5) clustered with Genotype 4, including one significantly divergent sequence. Only HEV Genotype 3 was identified in North American (n = 5) and European (n = 5) pools. There was no evidence of HEV in any pools tested from the Middle East. HEV was detected in manufacturing plasma pools from three different continents; viral loads were low-consistent with large pool sizes and moderate levels of HEV viremia at the individual donation level-but are nevertheless informative for risk assessment of plasma-derived medicinal products. Where sequencing was possible, analysis confirmed the presence of viruses consistent with locally circulating genotypes in the respective regions. The absence of HEV in Middle Eastern pools is consistent with the low prevalence of HEV in this region, likely due to low pork consumption. © 2016 AABB.

  12. Viral Evasion and Manipulation of Host RNA Quality Control Pathways.

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    Hogg, J Robert

    2016-08-15

    Viruses have evolved diverse strategies to maximize the functional and coding capacities of their genetic material. Individual viral RNAs are often used as substrates for both replication and translation and can contain multiple, sometimes overlapping open reading frames. Further, viral RNAs engage in a wide variety of interactions with both host and viral proteins to modify the activities of important cellular factors and direct their own trafficking, packaging, localization, stability, and translation. However, adaptations increasing the information density of small viral genomes can have unintended consequences. In particular, viral RNAs have developed features that mark them as potential targets of host RNA quality control pathways. This minireview focuses on ways in which viral RNAs run afoul of the cellular mRNA quality control and decay machinery, as well as on strategies developed by viruses to circumvent or exploit cellular mRNA surveillance.

  13. [RNA silencing and viral disease induction in plants].

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    Shimura, Hanako; Masuta, Chikara

    2012-06-01

    RNA silencing plays an important role in plant resistance against viruses. As a counter-defense against RNA silencing, plant viruses have evolved RNA silencing suppressors (RSSs). RNA silencing is likely to play a major role in disease development. For example, RSSs have been found to disturb the gene expression controlled by miRNAs in plant tissue and organ development, resulting in plant malformation. Mosaic symptoms, which are typical in virus-infected plants, are actually a consequence of local arms race between host RNA silencing and viral RSSs. In addition, recent studies revealed that viral siRNAs could induce RNA silencing even against a certain host gene and thus a disease symptom through a complementary (homologous) sequence coincidentally found between virus and host gene. RNA silencing is the principal mediator of viral pathogenicity and disease induction and therefore should be exploited as a powerful tool for engineering virus resistance in plants as well as in animals.

  14. Influenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis

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    te Velthuis, Aartjan J.W.; Fodor, Ervin

    2016-01-01

    The genome of influenza viruses consists of multiple segments of single stranded negative-sense RNA. Each of these segments is bound by the heterotrimeric viral RNA-dependent RNA polymerase and multiple copies of nucleoprotein, forming viral ribonucleoprotein (vRNP) complexes. It is in the context of these vRNPs that the viral RNA polymerase carries out transcription of viral genes and replication of the viral RNA genome. In this Review, we discuss our current knowledge of the structure of the influenza virus RNA polymerase, how it carries out transcription and replication, and how its activities are modulated by viral and host factors. Furthermore, we discuss how advances in our understanding of polymerase function could help identifying new antiviral targets. PMID:27396566

  15. Impact of collection method on assessment of semen HIV RNA viral load.

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    Brendan J W Osborne

    Full Text Available BACKGROUND: The blood HIV RNA viral load is the best-defined predictor of HIV transmission, in part due to ease of measurement and the correlation of blood and genital tract (semen or cervico-vaginal viral load, although recent studies found semen HIV RNA concentration to be a stronger predictor of HIV transmission. There is currently no standardized method for semen collection when measuring HIV RNA concentration. Therefore, we compared two collection techniques in order to study of the impact of antiretroviral therapy on the semen viral load. METHODOLOGY/PRINCIPAL FINDINGS: Semen was collected by masturbation from HIV-infected, therapy-naïve men who have sex with men (MSM either undiluted (Visit 1 or directly into transport medium (Visit 2. Seminal plasma was then isolated, and the HIV RNA concentration obtained with each collection technique was measured and corrected for dilution if necessary. Collection of semen directly into transport medium resulted in a median HIV RNA viral load that was 0.4 log10 higher than undiluted samples. CONCLUSIONS/SIGNIFICANCE: The method of semen collection is an important consideration when quantifying the HIV RNA viral load in this compartment.

  16. Replicon RNA Viral Vectors as Vaccines

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    Lundstrom, Kenneth

    2016-01-01

    Single-stranded RNA viruses of both positive and negative polarity have been used as vectors for vaccine development. In this context, alphaviruses, flaviviruses, measles virus and rhabdoviruses have been engineered for expression of surface protein genes and antigens. Administration of replicon RNA vectors has resulted in strong immune responses and generation of neutralizing antibodies in various animal models. Immunization of mice, chicken, pigs and primates with virus-like particles, naked RNA or layered DNA/RNA plasmids has provided protection against challenges with lethal doses of infectious agents and administered tumor cells. Both prophylactic and therapeutic efficacy has been achieved in cancer immunotherapy. Moreover, recombinant particles and replicon RNAs have been encapsulated by liposomes to improve delivery and targeting. Replicon RNA vectors have also been subjected to clinical trials. Overall, immunization with self-replicating RNA viruses provides high transient expression levels of antigens resulting in generation of neutralizing antibody responses and protection against lethal challenges under safe conditions. PMID:27827980

  17. Favipiravir (T-705), a novel viral RNA polymerase inhibitor

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    Furuta, Yousuke; Gowen, Brian B.; Takahashi, Kazumi; Shiraki, Kimiyasu; Smee, Donald F.; Barnard, Dale L.

    2013-01-01

    Favipiravir (T-705; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) is an antiviral drug that selectively inhibits the RNA-dependent RNA polymerase of influenza virus. It is phosphoribosylated by cellular enzymes to its active form, favipiravir-ribofuranosyl-5′-triphosphate (RTP). Its antiviral effect is attenuated by the addition of purine nucleic acids, indicating the viral RNA polymerase mistakenly recognizes favipiravir-RTP as a purine nucleotide. Favipiravir is active against a broad range of ...

  18. Role of RNase MRP in viral RNA degradation and RNA recombination.

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    Jaag, Hannah M; Lu, Qiasheng; Schmitt, Mark E; Nagy, Peter D

    2011-01-01

    RNA degradation, together with RNA synthesis, controls the steady-state level of viral RNAs in infected cells. The endoribonucleolytic cleavage of viral RNA is important not only for viral RNA degradation but for RNA recombination as well, due to the participation of some RNA degradation products in the RNA recombination process. To identify host endoribonucleases involved in degradation of Tomato bushy stunt virus (TBSV) in a Saccharomyces cerevisiae model host, we tested eight known endoribonucleases. Here we report that downregulation of SNM1, encoding a component of the RNase MRP, and a temperature-sensitive mutation in the NME1 gene, coding for the RNA component of RNase MRP, lead to reduced production of the endoribonucleolytically cleaved TBSV RNA in yeast. We also show that the highly purified yeast RNase MRP cleaves the TBSV RNA in vitro, resulting in TBSV RNA degradation products similar in size to those observed in yeast cells. Knocking down the NME1 homolog in Nicotiana benthamiana also led to decreased production of the cleaved TBSV RNA, suggesting that in plants, RNase MRP is involved in TBSV RNA degradation. Altogether, this work suggests a role for the host endoribonuclease RNase MRP in viral RNA degradation and recombination.

  19. Anti-viral RNA silencing: do we look like plants ?

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    Lecellier Charles-Henri

    2006-01-01

    Full Text Available Abstract The anti-viral function of RNA silencing was first discovered in plants as a natural manifestation of the artificial 'co-suppression', which refers to the extinction of endogenous gene induced by homologous transgene. Because silencing components are conserved among most, if not all, eukaryotes, the question rapidly arose as to determine whether this process fulfils anti-viral functions in animals, such as insects and mammals. It appears that, whereas the anti-viral process seems to be similarly conserved from plants to insects, even in worms, RNA silencing does influence the replication of mammalian viruses but in a particular mode: micro(miRNAs, endogenous small RNAs naturally implicated in translational control, rather than virus-derived small interfering (siRNAs like in other organisms, are involved. In fact, these recent studies even suggest that RNA silencing may be beneficial for viral replication. Accordingly, several large DNA mammalian viruses have been shown to encode their own miRNAs. Here, we summarize the seminal studies that have implicated RNA silencing in viral infection and compare the different eukaryotic responses.

  20. Colocalization of different influenza viral RNA segments in the cytoplasm before viral budding as shown by single-molecule sensitivity FISH analysis.

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    Yi-ying Chou

    Full Text Available The Influenza A virus genome consists of eight negative sense, single-stranded RNA segments. Although it has been established that most virus particles contain a single copy of each of the eight viral RNAs, the packaging selection mechanism remains poorly understood. Influenza viral RNAs are synthesized in the nucleus, exported into the cytoplasm and travel to the plasma membrane where viral budding and genome packaging occurs. Due to the difficulties in analyzing associated vRNPs while preserving information about their positions within the cell, it has remained unclear how and where during cellular trafficking the viral RNAs of different segments encounter each other. Using a multicolor single-molecule sensitivity fluorescence in situ hybridization (smFISH approach, we have quantitatively monitored the colocalization of pairs of influenza viral RNAs in infected cells. We found that upon infection, the viral RNAs from the incoming particles travel together until they reach the nucleus. The viral RNAs were then detected in distinct locations in the nucleus; they are then exported individually and initially remain separated in the cytoplasm. At later time points, the different viral RNA segments gather together in the cytoplasm in a microtubule independent manner. Viral RNAs of different identities colocalize at a high frequency when they are associated with Rab11 positive vesicles, suggesting that Rab11 positive organelles may facilitate the association of different viral RNAs. Using engineered influenza viruses lacking the expression of HA or M2 protein, we showed that these viral proteins are not essential for the colocalization of two different viral RNAs in the cytoplasm. In sum, our smFISH results reveal that the viral RNAs travel together in the cytoplasm before their arrival at the plasma membrane budding sites. This newly characterized step of the genome packaging process demonstrates the precise spatiotemporal regulation of the

  1. RNA Viruses and RNAi: Quasispecies Implications for Viral Escape

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    John B. Presloid

    2015-06-01

    Full Text Available Due to high mutation rates, populations of RNA viruses exist as a collection of closely related mutants known as a quasispecies. A consequence of error-prone replication is the potential for rapid adaptation of RNA viruses when a selective pressure is applied, including host immune systems and antiviral drugs. RNA interference (RNAi acts to inhibit protein synthesis by targeting specific mRNAs for degradation and this process has been developed to target RNA viruses, exhibiting their potential as a therapeutic against infections. However, viruses containing mutations conferring resistance to RNAi were isolated in nearly all cases, underlining the problems of rapid viral evolution. Thus, while promising, the use of RNAi in treating or preventing viral diseases remains fraught with the typical complications that result from high specificity of the target, as seen in other antiviral regimens.

  2. Intracellular Detection of Viral Transcription and Replication Using RNA FISH

    Science.gov (United States)

    2016-05-26

    Chapter 14. Intracellular detection of viral transcription and replication using RNA FISH i. Summary/Abstract Many hemorrhagic fever viruses...examine the mechanisms in which viruses replicate, assemble, and traffic through the cell. An additional benefit of this method is that the robust...Visualization of single RNA transcripts in situ. Science, 1998. 280(5363): p. 585-90. 4. Jambo, K.C., et al., Small alveolar macrophages are infected

  3. Quantification of infectious HIV-1 plasma viral load using a boosted in vitro infection protocol.

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    Rusert, Peter; Fischer, Marek; Joos, Beda; Leemann, Christine; Kuster, Herbert; Flepp, Markus; Bonhoeffer, Sebastian; Günthard, Huldrych F; Trkola, Alexandra

    2004-08-15

    Methods currently used for HIV-1 viral load measurements are very sensitive, but cannot distinguish between infectious and noninfectious particles. Here we describe the development of a novel, sensitive, and highly reproducible method that allows rapid isolation and quantification of infectious particles from patient plasma. By immobilizing HIV-1 particles in human plasma to platelets using polybrene, we observed a 10- to 1000-fold increase in infectivity over infection protocols using free virus particles. Using this method, we evaluated infectivity in plasma from 52 patients at various disease stages. At plasma viral loads of 1000-10000 HIV-1 RNA copies/ml 18%, at 10,000-50,000 copies/ml 73%, at 50,000-100,000 copies/ml 90%, and above 100,000 copies 96% of cultures were positive. We found that infectious titers among patients vary distinctively but are characteristic for a patient over extended time periods. Furthermore, we demonstrate that by evaluating infectious titers in conjunction with total HIV RNA loads, subtle effects of treatment intervention on viremia levels can be detected. The immobilization procedure does not interfere with viral entry and does not restore the infectivity of neutralized virus. Therefore, this assay system can be utilized to investigate the influence of substances that specifically affect virion infectivity such as neutralizing antibodies, soluble CD4, or protease inhibitors. Measuring viral infectivity may thereby function as an additional, useful marker in monitoring disease progression and evaluating efficacy of antivirals in vivo.

  4. In-depth characterization of viral isolates from plasma and cells compared with plasma circulating quasispecies in early HIV-1 infection.

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    Judith Dalmau

    Full Text Available BACKGROUND: The use of in vitro models to unravel the phenotypic characteristics of circulating viral variants is key to understanding HIV-1 pathogenesis but limited by the availability of primary viral isolates from biological samples. However, overall in vivo genetic variability of HIV-1 within a subject may not be reflected in the viable viral population obtained after isolation. Although several studies have tried to determine whether viral populations expanded in vitro are representative of in vivo findings, the answer remains unclear due to the reduced number of clonal sequences analyzed or samples compared. In order to overcome previous experimental limitations, here we applied Deep Pyrosequencing (DPS technology in combination with phenotypic experiments to analyze and compare with unprecedented detail the composition of viral isolates and in vivo quasispecies. METHODOLOGY/PRINCIPAL FINDINGS: We amplified by DPS HIV-1 genomic regions covering gag, protease, integrase and env-V3 to characterize paired isolates from plasma and peripheral blood mononuclear cells and compare them with total plasma viral RNA in four recently HIV-1 infected subjects. Our study demonstrated the presence of unique haplotypes scattered between sample types with conservation of major variants. In addition, no differences in intra- and inter-population encoded protein variability were found between the different types of isolates or when these were compared to plasma viral RNA within subjects. Additionally, in vitro experiments demonstrated phenotypic similarities in terms of replicative capacity and co-receptor usage between viral isolates and plasma viral RNA. CONCLUSION: This study is the first in-depth comparison and characterization of viral isolates from different sources and plasma circulating quasispecies using DPS in recently HIV-1 infected subjects. Our data supports the use of primary isolates regardless of their plasma or cellular origin to define

  5. Favipiravir (T-705), a novel viral RNA polymerase inhibitor.

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    Furuta, Yousuke; Gowen, Brian B; Takahashi, Kazumi; Shiraki, Kimiyasu; Smee, Donald F; Barnard, Dale L

    2013-11-01

    Favipiravir (T-705; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) is an antiviral drug that selectively inhibits the RNA-dependent RNA polymerase of influenza virus. It is phosphoribosylated by cellular enzymes to its active form, favipiravir-ribofuranosyl-5'-triphosphate (RTP). Its antiviral effect is attenuated by the addition of purine nucleic acids, indicating the viral RNA polymerase mistakenly recognizes favipiravir-RTP as a purine nucleotide. Favipiravir is active against a broad range of influenza viruses, including A(H1N1)pdm09, A(H5N1) and the recently emerged A(H7N9) avian virus. It also inhibits influenza strains resistant to current antiviral drugs, and shows a synergistic effect in combination with oseltamivir, thereby expanding influenza treatment options. A Phase III clinical evaluation of favipiravir for influenza therapy has been completed in Japan and two Phase II studies have been completed in the United States. In addition to its anti-influenza activity, favipiravir blocks the replication of many other RNA viruses, including arenaviruses (Junin, Machupo and Pichinde); phleboviruses (Rift Valley fever, sandfly fever and Punta Toro); hantaviruses (Maporal, Dobrava, and Prospect Hill); flaviviruses (yellow fever and West Nile); enteroviruses (polio- and rhinoviruses); an alphavirus, Western equine encephalitis virus; a paramyxovirus, respiratory syncytial virus; and noroviruses. With its unique mechanism of action and broad range of antiviral activity, favipiravir is a promising drug candidate for influenza and many other RNA viral diseases for which there are no approved therapies.

  6. Accurate strand-specific quantification of viral RNA.

    Directory of Open Access Journals (Sweden)

    Nicole E Plaskon

    Full Text Available The presence of full-length complements of viral genomic RNA is a hallmark of RNA virus replication within an infected cell. As such, methods for detecting and measuring specific strands of viral RNA in infected cells and tissues are important in the study of RNA viruses. Strand-specific quantitative real-time PCR (ssqPCR assays are increasingly being used for this purpose, but the accuracy of these assays depends on the assumption that the amount of cDNA measured during the quantitative PCR (qPCR step accurately reflects amounts of a specific viral RNA strand present in the RT reaction. To specifically test this assumption, we developed multiple ssqPCR assays for the positive-strand RNA virus o'nyong-nyong (ONNV that were based upon the most prevalent ssqPCR assay design types in the literature. We then compared various parameters of the ONNV-specific assays. We found that an assay employing standard unmodified virus-specific primers failed to discern the difference between cDNAs generated from virus specific primers and those generated through false priming. Further, we were unable to accurately measure levels of ONNV (- strand RNA with this assay when higher levels of cDNA generated from the (+ strand were present. Taken together, these results suggest that assays of this type do not accurately quantify levels of the anti-genomic strand present during RNA virus infectious cycles. However, an assay permitting the use of a tag-specific primer was able to distinguish cDNAs transcribed from ONNV (- strand RNA from other cDNAs present, thus allowing accurate quantification of the anti-genomic strand. We also report the sensitivities of two different detection strategies and chemistries, SYBR(R Green and DNA hydrolysis probes, used with our tagged ONNV-specific ssqPCR assays. Finally, we describe development, design and validation of ssqPCR assays for chikungunya virus (CHIKV, the recent cause of large outbreaks of disease in the Indian Ocean

  7. A Viral RNA Structural Element Alters Host Recognition of Nonself RNA

    Energy Technology Data Exchange (ETDEWEB)

    Hyde, J. L.; Gardner, C. L.; Kimura, T.; White, J. P.; Liu, G.; Trobaugh, D. W.; Huang, C.; Tonelli, M.; Paessler, S.; Takeda, K.; Klimstra, W. B.; Amarasinghe, G. K.; Diamond, M. S.

    2014-01-30

    Although interferon (IFN) signaling induces genes that limit viral infection, many pathogenic viruses overcome this host response. As an example, 2'-O methylation of the 5' cap of viral RNA subverts mammalian antiviral responses by evading restriction of Ifit1, an IFN-stimulated gene that regulates protein synthesis. However, alphaviruses replicate efficiently in cells expressing Ifit1 even though their genomic RNA has a 5' cap lacking 2'-O methylation. We show that pathogenic alphaviruses use secondary structural motifs within the 5' untranslated region (UTR) of their RNA to alter Ifit1 binding and function. Mutations within the 5'-UTR affecting RNA structural elements enabled restriction by or antagonism of Ifit1 in vitro and in vivo. These results identify an evasion mechanism by which viruses use RNA structural motifs to avoid immune restriction.

  8. Liposomal nanocontainers as models for viral infection: monitoring viral genomic RNA transfer through lipid membranes.

    Science.gov (United States)

    Bilek, Gerhard; Matscheko, Nena M; Pickl-Herk, Angela; Weiss, Victor U; Subirats, Xavier; Kenndler, Ernst; Blaas, Dieter

    2011-08-01

    After uptake into target cells, many nonenveloped viruses undergo conformational changes in the low-pH environment of the endocytic compartment. This results in exposure of amphipathic viral peptides and/or hydrophobic protein domains that are inserted into and either disrupt or perforate the vesicular membranes. The viral nucleic acids thereby gain access to the cytosol and initiate replication. We here demonstrate the in vitro transfer of the single-stranded positive-sense RNA genome of human rhinovirus 2 into liposomes decorated with recombinant very-low-density lipoprotein receptor fragments. Membrane-attached virions were exposed to pH 5.4, mimicking the in vivo pH environment of late endosomes. This triggered the release of the RNA whose arrival in the liposomal lumen was detected via in situ cDNA synthesis by encapsulated reverse transcriptase. Subsequently, cDNA was PCR amplified. At a low ratio between virions and lipids, RNA transfer was positively correlated with virus concentration. However, membranes became leaky at higher virus concentrations, which resulted in decreased cDNA synthesis. In accordance with earlier in vivo data, the RNA passes through the lipid membrane without causing gross damage to vesicles at physiologically relevant virus concentrations.

  9. Who Regulates Whom? An Overview of RNA Granules and Viral Infections

    Directory of Open Access Journals (Sweden)

    Natalia Poblete-Durán

    2016-06-01

    Full Text Available After viral infection, host cells respond by mounting an anti-viral stress response in order to create a hostile atmosphere for viral replication, leading to the shut-off of mRNA translation (protein synthesis and the assembly of RNA granules. Two of these RNA granules have been well characterized in yeast and mammalian cells, stress granules (SGs, which are translationally silent sites of RNA triage and processing bodies (PBs, which are involved in mRNA degradation. This review discusses the role of these RNA granules in the evasion of anti-viral stress responses through virus-induced remodeling of cellular ribonucleoproteins (RNPs.

  10. Who Regulates Whom? An Overview of RNA Granules and Viral Infections

    Science.gov (United States)

    Poblete-Durán, Natalia; Prades-Pérez, Yara; Vera-Otarola, Jorge; Soto-Rifo, Ricardo; Valiente-Echeverría, Fernando

    2016-01-01

    After viral infection, host cells respond by mounting an anti-viral stress response in order to create a hostile atmosphere for viral replication, leading to the shut-off of mRNA translation (protein synthesis) and the assembly of RNA granules. Two of these RNA granules have been well characterized in yeast and mammalian cells, stress granules (SGs), which are translationally silent sites of RNA triage and processing bodies (PBs), which are involved in mRNA degradation. This review discusses the role of these RNA granules in the evasion of anti-viral stress responses through virus-induced remodeling of cellular ribonucleoproteins (RNPs). PMID:27367717

  11. RNA interference strategies as therapy for respiratory viral infections.

    Science.gov (United States)

    DeVincenzo, John P

    2008-10-01

    RNA interference (RNAi) is a recently discovered, naturally occurring intracellular process that regulates gene expression through the silencing of specific mRNAs. Methods of harnessing this natural pathway are being developed that allow the catalytic degradation of targeted mRNAs using specifically designed complementary small-interfering RNAs (siRNA). siRNAs are being chemically modified to acquire drug-like properties. Numerous recent high profile publications have provided proofs of concept that RNAi may be of therapeutic use. Much of the design of these siRNAs can be accomplished bioinformatically, thus potentially expediting drug discovery and opening new avenues of therapy for many uncommon, orphan, or emerging diseases. Although endogenous human disease targets can theoretically be affected by RNAi therapeutics, nonendogenous targets (eg, viral targets) are attractive and RNAi therapeutics have been shown to act as antivirals in vivo and in vitro. Respiratory viral infections are particularly attractive targets for RNAi therapeutics because the infected cells exist at the air-lung interface, thereby positioning these cells to be accessible to topical administration of siRNA, for example by aerosol. RNAi therapeutics have been shown to be active against respiratory syncytial virus, parainfluenza and influenza in vitro and in vivo resulting in profound antiviral effects. The first RNAi therapeutic to be designed as an anti-infective medication has now entered proof of concept clinical trials in man. A discussion of the science behind RNAi is followed by a presentation of the potential practical issues in applying this technology to respiratory viral diseases. RNAi may offer new strategies for the treatment of respiratory syncytial virus and other respiratory viruses.

  12. Interferon Induction by RNA Viruses and Antagonism by Viral Pathogens

    Directory of Open Access Journals (Sweden)

    Yuchen Nan

    2014-12-01

    Full Text Available Interferons are a group of small proteins that play key roles in host antiviral innate immunity. Their induction mainly relies on host pattern recognition receptors (PRR. Host PRR for RNA viruses include Toll-like receptors (TLR and retinoic acid-inducible gene I (RIG-I like receptors (RLR. Activation of both TLR and RLR pathways can eventually lead to the secretion of type I IFNs, which can modulate both innate and adaptive immune responses against viral pathogens. Because of the important roles of interferons, viruses have evolved multiple strategies to evade host TLR and RLR mediated signaling. This review focuses on the mechanisms of interferon induction and antagonism of the antiviral strategy by RNA viruses.

  13. Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome

    Science.gov (United States)

    Lu, Rui; Folimonov, Alexey; Shintaku, Michael; Li, Wan-Xiang; Falk, Bryce W.; Dawson, William O.; Ding, Shou-Wei

    2004-11-01

    Viral infection in both plant and invertebrate hosts requires a virus-encoded function to block the RNA silencing antiviral defense. Here, we report the identification and characterization of three distinct suppressors of RNA silencing encoded by the 20-kb plus-strand RNA genome of citrus tristeza virus (CTV). When introduced by genetic crosses into plants carrying a silencing transgene, both p20 and p23, but not coat protein (CP), restored expression of the transgene. Although none of the CTV proteins prevented DNA methylation of the transgene, export of the silencing signal (capable of mediating intercellular silencing spread) was detected only from the F1 plants expressing p23 and not from the CP- or p20-expressing F1 plants, demonstrating suppression of intercellular silencing by CP and p20 but not by p23. Thus, intracellular and intercellular silencing are each targeted by a CTV protein, whereas the third, p20, inhibits silencing at both levels. Notably, CP suppresses intercellular silencing without interfering with intracellular silencing. The novel property of CP suggests a mechanism distinct to p20 and all of the other viral suppressors known to interfere with intercellular silencing and that this class of viral suppressors may not be consistently identified by Agrobacterium coinfiltration because it also induces RNA silencing against the infiltrated suppressor transgene. Our analyses reveal a sophisticated viral counter-defense strategy that targets the silencing antiviral pathway at multiple steps and may be essential for protecting CTV with such a large RNA genome from antiviral silencing in the perennial tree host. RNA interference | citrus tristeza virus | virus synergy | antiviral immunity

  14. Coordinated function of cellular DEAD-box helicases in suppression of viral RNA recombination and maintenance of viral genome integrity.

    Directory of Open Access Journals (Sweden)

    Chingkai Chuang

    2015-02-01

    Full Text Available The intricate interactions between viruses and hosts include an evolutionary arms race and adaptation that is facilitated by the ability of RNA viruses to evolve rapidly due to high frequency mutations and genetic RNA recombination. In this paper, we show evidence that the co-opted cellular DDX3-like Ded1 DEAD-box helicase suppresses tombusviral RNA recombination in yeast model host, and the orthologous RH20 helicase functions in a similar way in plants. In vitro replication and recombination assays confirm the direct role of the ATPase function of Ded1p in suppression of viral recombination. We also present data supporting a role for Ded1 in facilitating the switch from minus- to plus-strand synthesis. Interestingly, another co-opted cellular helicase, the eIF4AIII-like AtRH2, enhances TBSV recombination in the absence of Ded1/RH20, suggesting that the coordinated actions of these helicases control viral RNA recombination events. Altogether, these helicases are the first co-opted cellular factors in the viral replicase complex that directly affect viral RNA recombination. Ded1 helicase seems to be a key factor maintaining viral genome integrity by promoting the replication of viral RNAs with correct termini, but inhibiting the replication of defective RNAs lacking correct 5' end sequences. Altogether, a co-opted cellular DEAD-box helicase facilitates the maintenance of full-length viral genome and suppresses viral recombination, thus limiting the appearance of defective viral RNAs during replication.

  15. Viral suppressors of RNA silencing hinder exogenous and endogenous small RNA pathways in Drosophila.

    Directory of Open Access Journals (Sweden)

    Bassam Berry

    Full Text Available BACKGROUND: In plants and insects, RNA interference (RNAi is the main responder against viruses and shapes the basis of antiviral immunity. Viruses counter this defense by expressing viral suppressors of RNAi (VSRs. While VSRs in Drosophila melanogaster were shown to inhibit RNAi through different modes of action, whether they act on other silencing pathways remained unexplored. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that expression of various plant and insect VSRs in transgenic flies does not perturb the Drosophila microRNA (miRNA pathway; but in contrast, inhibits antiviral RNAi and the RNA silencing response triggered by inverted repeat transcripts, and injection of dsRNA or siRNA. Strikingly, these VSRs also suppressed transposon silencing by endogenous siRNAs (endo-siRNAs. CONCLUSIONS/SIGNIFICANCE: Our findings identify VSRs as tools to unravel small RNA pathways in insects and suggest a cosuppression of antiviral RNAi and endo-siRNA silencing by viruses during fly infections.

  16. Multiple functions of DDX3 RNA helicase in gene regulation, tumorigenesis, and viral infection

    OpenAIRE

    2014-01-01

    The DEAD-box RNA helicase DDX3 is a multifunctional protein involved in all aspects of RNA metabolism, including transcription, splicing, mRNA nuclear export, translation, RNA decay and ribosome biogenesis. In addition, DDX3 is also implicated in cell cycle regulation, apoptosis, Wnt-β-catenin signaling, tumorigenesis, and viral infection. Notably, recent studies suggest that DDX3 is a component of anti-viral innate immune signaling pathways. Indeed, DDX3 contributes to enhance the induction ...

  17. SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5'UTR RNA.

    Science.gov (United States)

    Han, Yang; Wang, Lvyin; Cui, Jin; Song, Yu; Luo, Zhen; Chen, Junbo; Xiong, Ying; Zhang, Qi; Liu, Fang; Ho, Wenzhe; Liu, Yingle; Wu, Kailang; Wu, Jianguo

    2016-12-15

    Enterovirus 71 (EV71) possesses a single-stranded positive RNA genome that contains a single open reading frame (ORF) flanked by a 5' untranslated region (5'UTR) and a polyadenylated 3'UTR. Here, we demonstrated that EV71 activates the production of silent mating type information regulation 2 homolog 1 (SIRT1), a histone deacetylase (HDAC). EV71 further stimulates SIRT1 sumoylation and deacetylase activity, and enhances SIRT1 translocation from the nucleus to the cytoplasm. More interestingly, activated SIRT1 subsequently binds with the EV71 3D(pol) protein (a viral RNA-dependent RNA polymerase, RdRp) to repress the acetylation and RdRp activity of 3D(pol), resulting in the attenuation of viral genome replication. Moreover, SIRT1 interacts with the cloverleaf structure of the EV71 RNA 5'UTR to inhibit viral RNA transcription, and binds to the internal ribosome entry site (IRES) of the EV71 5'UTR to attenuate viral RNA translation. Thus, EV71 stimulates SIRT1 production and activity, which in turn represses EV71 genome replication by inhibiting viral polymerase, and attenuates EV71 RNA transcription and translation by interfering with viral RNA. These results uncover a new function of SIRT1 and reveal a new mechanism underlying the regulation of EV71 replication. © 2016. Published by The Company of Biologists Ltd.

  18. Quantification of HIV-1 viral RNA in the blood in needles used for venous puncture in HIV-infected individuals

    Directory of Open Access Journals (Sweden)

    Iris Ricardo Rossin

    2011-12-01

    Full Text Available INTRODUCTION: Occupational HIV infection among healthcare workers is an important issue in exposures involving blood and body fluids. There are few data in the literature regarding the potential and the duration of infectivity of HIV type 1 (HIV-1 in contaminated material under adverse conditions. METHODS: We quantified HIV-1 viral RNA in 25×8mm calibre hollow-bore needles, after punctures, in 25 HIV-1-infected patients selected during the sample collection. All of the patients selected were between the ages of 18 and 55. Five samples were collected from 16 patients: one sample for the immediate quantification of HIV-1 RNA in the plasma and blood samples from the interior of 4 needles to be analyzed at 0h, 6h, 24h, and 72h after collection. In nine patients, another test was carried out in the blood from one additional needle, in which HIV-1 RNA was assessed 168h after blood collection. The method used to assess HIV-1 RNA was nucleic acid sequence-based amplification. RESULTS: Up to 7 days after collection, HIV-1 RNA was detected in all of the needles. The viral RNA remained stable up to 168h, and there were no statistically significant differences among the needle samples. CONCLUSIONS: Although the infectivity of the viral material in the needles is unknown, the data indicate the need to re-evaluate the practices in cases of occupational accidents in which the source is not identified.

  19. Longitudinal serum HIV RNA quantification: correlation to viral phenotype at seroconversion and clinical outcome

    DEFF Research Database (Denmark)

    Katzenstein, T L; Pedersen, C; Nielsen, C

    1996-01-01

    OBJECTIVE: To investigate the longitudinal changes in serum HIV RNA, and to clarify whether the viral load early in infection has a predictive value for the clinical outcome; also, to correlate viral phenotype at seroconversion and changes in CD4 cell counts with viral burden. DESIGN: Twenty...... seroconverters with HIV isolates available at seroconversion had HIV RNA quantified by polymerase chain reaction (PCR) at seroconversion and thereafter every 6 months. Mean follow-up time was 65 months. Patients were classified according to viral phenotype at seroconversion, time to AIDS progression, serum viral....... Harbouring syncytium-inducing (SI) virus at seroconversion was associated with faster progression to AIDS than non-SI (NSI; P RNA. CONCLUSION: Serum HIV RNA is high around the time...

  20. Viral RNA silencing suppressors (RSS): novel strategy of viruses to ablate the host RNA interference (RNAi) defense system.

    Science.gov (United States)

    Bivalkar-Mehla, Shalmali; Vakharia, Janaki; Mehla, Rajeev; Abreha, Measho; Kanwar, Jagat Rakesh; Tikoo, Akshay; Chauhan, Ashok

    2011-01-01

    Pathogenic viruses have developed a molecular defense arsenal for their survival by counteracting the host anti-viral system known as RNA interference (RNAi). Cellular RNAi, in addition to regulating gene expression through microRNAs, also serves as a barrier against invasive foreign nucleic acids. RNAi is conserved across the biological species, including plants, animals and invertebrates. Viruses in turn, have evolved mechanisms that can counteract this anti-viral defense of the host. Recent studies of mammalian viruses exhibiting RNA silencing suppressor (RSS) activity have further advanced our understanding of RNAi in terms of host-virus interactions. Viral proteins and non-coding viral RNAs can inhibit the RNAi (miRNA/siRNA) pathway through different mechanisms. Mammalian viruses having dsRNA-binding regions and GW/WG motifs appear to have a high chance of conferring RSS activity. Although, RSSs of plant and invertebrate viruses have been well characterized, mammalian viral RSSs still need in-depth investigations to present the concrete evidences supporting their RNAi ablation characteristics. The information presented in this review together with any perspective research should help to predict and identify the RSS activity-endowed new viral proteins that could be the potential targets for designing novel anti-viral therapeutics. Copyright © 2010 Elsevier B.V. All rights reserved.

  1. CNOT4-Mediated Ubiquitination of Influenza A Virus Nucleoprotein Promotes Viral RNA Replication

    Directory of Open Access Journals (Sweden)

    Yu-Chen Lin

    2017-05-01

    Full Text Available Influenza A virus (IAV RNA segments are individually packaged with viral nucleoprotein (NP and RNA polymerases to form a viral ribonucleoprotein (vRNP complex. We previously reported that NP is a monoubiquitinated protein which can be deubiquitinated by a cellular ubiquitin protease, USP11. In this study, we identified an E3 ubiquitin ligase, CNOT4 (Ccr4-Not transcription complex subunit 4, which can ubiquitinate NP. We found that the levels of viral RNA, protein, viral particles, and RNA polymerase activity in CNOT4 knockdown cells were lower than those in the control cells upon IAV infection. Conversely, overexpression of CNOT4 rescued viral RNP activity. In addition, CNOT4 interacted with the NP in the cell. An in vitro ubiquitination assay also showed that NP could be ubiquitinated by in vitro-translated CNOT4, but ubiquitination did not affect the protein stability of NP. Significantly, CNOT4 increased NP ubiquitination, whereas USP11 decreased it. Mass spectrometry analysis of ubiquitinated NP revealed multiple ubiquitination sites on the various lysine residues of NP. Three of these, K184, K227, and K273, are located on the RNA-binding groove of NP. Mutations of these sites to arginine reduced viral RNA replication. These results indicate that CNOT4 is a ubiquitin ligase of NP, and ubiquitination of NP plays a positive role in viral RNA replication.

  2. Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins

    Science.gov (United States)

    Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann

    2017-01-01

    RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5′ terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. PMID:28338950

  3. Viral miRNA targeting of bicistronic and polycistronic transcripts.

    Science.gov (United States)

    Zhu, Ying; Huang, Yufei; Jung, Jae U; Lu, Chun; Gao, Shou-Jiang

    2014-08-01

    Successful viral infection entails a choreographic regulation of viral gene expression program. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes numerous miRNAs that regulate viral life cycle. However, few viral targets have been identified due to the lack of information on KSHV 3' untranslated regions (3'UTRs). Recent genome-wide mapping of KSHV transcripts and 3'UTRs has revealed abundant bicistronic and polycistronic transcripts. The extended 3'UTRs of the 5' proximal genes of bicistronic and polycistronic transcripts offer additional regulatory targets. Indeed, a genome-wide screening of KSHV 3'UTRs has identified several bicistronic and polycistronic transcripts as the novel targets of viral miRNAs. Together, these works have expanded our knowledge of the unique features of KSHV gene regulation program and provided valuable resources for the research community.

  4. dsRNA sensing during viral infection: lessons from plants, worms, insects, and mammals.

    Science.gov (United States)

    de Faria, Isaque João da Silva; Olmo, Roenick Proveti; Silva, Emanuele Guimarães; Marques, João Trindade

    2013-05-01

    Host defense systems often rely on direct and indirect pattern recognition to sense the presence of invading pathogens. Patterns can be molecules directly produced by the pathogen or indirectly generated by changes in host parameters as a consequence of infection. Viruses are intracellular pathogens that hijack the cellular machinery to synthesize their own molecules making direct recognition of viral molecules a great challenge. Antiviral systems in prokaryotes and eukaryotes commonly exploit aberrant nucleic acid sensing to recognize virus infection as host and viral nucleic acid metabolism can greatly differ. Indeed, the generation of dsRNA is often associated with viral infection. In this review, we discuss current knowledge on the mechanisms of viral dsRNA sensing utilized by 2 important antiviral defense systems, RNA interference (RNAi) and the vertebrate immune system. The major viral sensors of the vertebrate immune systems are RIG-like receptors, while RNAi pathways depend on Dicer proteins. These 2 families of sensors share a similar helicase domain with high specificity for dsRNA, which is necessary, but not sufficient for efficient recognition by these receptors. Additional intrinsic features to the dsRNA molecule are also necessary for activation of antiviral systems. Studies utilizing synthetic ligands, in vitro biochemistry and reporter systems have greatly helped increase our knowledge on intrinsic features of dsRNA recognition. However, characteristics such as subcellular localization are extrinsic to the dsRNA itself, but certainly influence the recognition in vivo. Thus, mechanisms of viral dsRNA recognition must address how cellular sensors are recruited to nucleic acids or vice versa. Accessory proteins are likely important for in vivo recognition of extrinsic features of viral RNA, but have mostly remained undiscovered due to the limitations of previous strategies. Hence, the identification of novel components of antiviral systems must take

  5. Comparative analysis of RNA silencing suppression activities between viral suppressors and an endogenous plant RNA-dependent RNA polymerase.

    Science.gov (United States)

    Yoon, Ju-Yeon; Han, Kyoung-Sik; Park, Han-Yong; Choi, Seung-Kook

    2012-06-01

    RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in eukaryotes, including higher plants. To counteract this, several plant viruses express silencing suppressors that inhibit RNA silencing in host plants. Here, we show that both 2b protein from peanut stunt virus (PSV) and a hairpin construct (designated hp-RDR6) that silences endogenous RNA-dependent RNA polymerase 6 (RDR6) strongly suppress RNA silencing. The Agrobacterium infiltration system was used to demonstrate that both PSV 2b and hp-RDR6 suppressed local RNA silencing as strongly as helper component (HC-Pro) from potato virus Y (PVY) and P19 from tomato bush stunt virus (TBSV). The 2b protein from PSV eliminated the small-interfering RNAs (siRNAs) associated with RNA silencing and prevented systemic silencing, similar to 2b protein from cucumber mosaic virus (CMV). On the other hand, hp-RDR6 suppressed RNA silencing by inhibiting the generation of secondary siRNAs. The small coat protein (SCP) of squash mosaic virus (SqMV) also displayed weak suppression activity of RNA silencing. Agrobacterium-mediated gene transfer was used to investigate whether viral silencing suppressors or hp-RDR6 enhanced accumulations of green fluorescence protein (GFP) and β-glucuronidase (GUS) as markers of expression in leaf tissues of Nicotina benthamiana. Expression of both GFP and GUS was significantly enhanced in the presence of PSV 2b or CMV 2b, compared to no suppression or the weak SqMV SCP suppressor. Co-expression with hp-RDR6 also significantly increased the expression of GFP and GUS to levels similar to those induced by PVY HC-Pro and TBSV P19.

  6. Intrinsically disordered region of influenza A NP regulates viral genome packaging via interactions with viral RNA and host PI(4,5)P2.

    Science.gov (United States)

    Kakisaka, Michinori; Yamada, Kazunori; Yamaji-Hasegawa, Akiko; Kobayashi, Toshihide; Aida, Yoko

    2016-09-01

    To be incorporated into progeny virions, the viral genome must be transported to the inner leaflet of the plasma membrane (PM) and accumulate there. Some viruses utilize lipid components to assemble at the PM. For example, simian virus 40 (SV40) targets the ganglioside GM1 and human immunodeficiency virus type 1 (HIV-1) utilizes phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2]. Recent studies clearly indicate that Rab11-mediated recycling endosomes are required for influenza A virus (IAV) trafficking of vRNPs to the PM but it remains unclear how IAV vRNP localized or accumulate underneath the PM for viral genome incorporation into progeny virions. In this study, we found that the second intrinsically disordered region (IDR2) of NP regulates two binding steps involved in viral genome packaging. First, IDR2 facilitates NP oligomer binding to viral RNA to form vRNP. Secondly, vRNP assemble by interacting with PI(4,5)P2 at the PM via IDR2. These findings suggest that PI(4,5)P2 functions as the determinant of vRNP accumulation at the PM.

  7. Nuclear imprisonment: viral strategies to arrest host mRNA nuclear export.

    Science.gov (United States)

    Kuss, Sharon K; Mata, Miguel A; Zhang, Liang; Fontoura, Beatriz M A

    2013-07-01

    Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

  8. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

    Science.gov (United States)

    Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.

    2013-01-01

    Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

  9. RNA-binding protein CPEB1 remodels host and viral RNA landscapes.

    Science.gov (United States)

    Batra, Ranjan; Stark, Thomas J; Clark, Elizabeth; Belzile, Jean-Philippe; Wheeler, Emily C; Yee, Brian A; Huang, Hui; Gelboin-Burkhart, Chelsea; Huelga, Stephanie C; Aigner, Stefan; Roberts, Brett T; Bos, Tomas J; Sathe, Shashank; Donohue, John Paul; Rigo, Frank; Ares, Manuel; Spector, Deborah H; Yeo, Gene W

    2016-12-01

    Host and virus interactions occurring at the post-transcriptional level are critical for infection but remain poorly understood. Here, we performed comprehensive transcriptome-wide analyses revealing that human cytomegalovirus (HCMV) infection results in widespread alternative splicing (AS), shortening of 3' untranslated regions (3' UTRs) and lengthening of poly(A)-tails in host gene transcripts. We found that the host RNA-binding protein CPEB1 was highly induced after infection, and ectopic expression of CPEB1 in noninfected cells recapitulated infection-related post-transcriptional changes. CPEB1 was also required for poly(A)-tail lengthening of viral RNAs important for productive infection. Strikingly, depletion of CPEB1 reversed infection-related cytopathology and post-transcriptional changes, and decreased productive HCMV titers. Host RNA processing was also altered in herpes simplex virus-2 (HSV-2)-infected cells, thereby indicating that this phenomenon might be a common occurrence during herpesvirus infections. We anticipate that our work may serve as a starting point for therapeutic targeting of host RNA-binding proteins in herpesvirus infections.

  10. [Presence of autocomplementary RNA with viral specificity in cells infected with herpes virus].

    Science.gov (United States)

    Béchet, J M; Montagnier, L; Latarjet, R

    1975-01-13

    RNA from cells infected with Herpes simplex virus contain a higher percentage of double-stranded RNA than non-infected cells. This percentage increases three-fold upon self-annealing. The complementary RNA sequences were shown to be virus-specific by the following criteria: (1) high melting temperature than double-stranded RNA from non infected cells; (2) higher density in caesium sulphate; (3) specific hybridization with viral DNA.

  11. Novel microRNA-like viral small regulatory RNAs arising during human hepatitis A virus infection.

    Science.gov (United States)

    Shi, Jiandong; Sun, Jing; Wang, Bin; Wu, Meini; Zhang, Jing; Duan, Zhiqing; Wang, Haixuan; Hu, Ningzhu; Hu, Yunzhang

    2014-10-01

    MicroRNAs (miRNAs), including host miRNAs and viral miRNAs, play vital roles in regulating host-virus interactions. DNA viruses encode miRNAs that regulate the viral life cycle. However, it is generally believed that cytoplasmic RNA viruses do not encode miRNAs, owing to inaccessible cellular miRNA processing machinery. Here, we provide a comprehensive genome-wide analysis and identification of miRNAs that were derived from hepatitis A virus (HAV; Hu/China/H2/1982), which is a typical cytoplasmic RNA virus. Using deep-sequencing and in silico approaches, we identified 2 novel virally encoded miRNAs, named hav-miR-1-5p and hav-miR-2-5p. Both of the novel virally encoded miRNAs were clearly detected in infected cells. Analysis of Dicer enzyme silencing demonstrated that HAV-derived miRNA biogenesis is Dicer dependent. Furthermore, we confirmed that HAV mature miRNAs were generated from viral miRNA precursors (pre-miRNAs) in host cells. Notably, naturally derived HAV miRNAs were biologically and functionally active and induced post-transcriptional gene silencing (PTGS). Genomic location analysis revealed novel miRNAs located in the coding region of the viral genome. Overall, our results show that HAV naturally generates functional miRNA-like small regulatory RNAs during infection. This is the first report of miRNAs derived from the coding region of genomic RNA of a cytoplasmic RNA virus. These observations demonstrate that a cytoplasmic RNA virus can naturally generate functional miRNAs, as DNA viruses do. These findings also contribute to improved understanding of host-RNA virus interactions mediated by RNA virus-derived miRNAs.

  12. Measurement of HIV-1 viral load for drug resistance surveillance using dried blood spots: literature review and modeling of contribution of DNA and RNA.

    Science.gov (United States)

    Parkin, Neil T

    2014-01-01

    World Health Organization-recommended surveys of acquired HIV-1 drug resistance include assessment of HIV-1 viral load suppression to levels below 1,000 copies/ml and drug resistance-associated mutation patterns in subjects on antiretroviral therapy. Surveys are being conducted in regions of the world that cannot support the collection, storage, and shipping of frozen plasma. Therefore, dried blood spots are often the specimen type of choice for both genotyping and viral load measurement. Furthermore, viral load testing for individual patient management in these regions is being scaled-up in accordance with WHO 2013 Guidelines for Antiretroviral Treatment. Technical issues related to the adaptation of viral load assays to dried blood spots, especially with respect to sensitivity (limit of detection), specificity (cell-free RNA vs. cell-associated DNA or RNA), and assay method, affect the interpretation of a viral load result from dried blood spots. Amongst published studies of commercial assay performance with dried blood spots, the bioMérieux EasyQ® and Abbott RealTime assays tended to show high (> 90%) specificity and sensitivity; the Biocentric Generic or Roche TaqMan® assays tended to show high sensitivity but lower specificity, using a 1,000 copies/ml threshold. The relative contribution of cell-associated DNA or RNA to a viral load measurement is likely to vary between patients, depending on clinical parameters and treatment status. A model was developed that predicts that in patients on antiretroviral therapy with low plasma viral load, cellular DNA is the predominant source of non-plasma virus-derived nucleic acid in dried blood spots. The extent of viral load overestimation from dried blood spots becomes less important when plasma viral load is over about 5,000 copies/ml. To avoid misclassifying subjects with plasma viral load suppression, the World Health Organization-recommended threshold of 1,000 copies/ml can be applied only when an assay that can

  13. Determining mutant spectra of three RNA viral samples using ultra-deep sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H

    2012-06-06

    RNA viruses have extremely high mutation rates that enable the virus to adapt to new host environments and even jump from one species to another. As part of a viral transmission study, three viral samples collected from naturally infected animals were sequenced using Illumina paired-end technology at ultra-deep coverage. In order to determine the mutant spectra within the viral quasispecies, it is critical to understand the sequencing error rates and control for false positive calls of viral variants (point mutantations). I will estimate the sequencing error rate from two control sequences and characterize the mutant spectra in the natural samples with this error rate.

  14. Kaposi's Sarcoma-Associated Herpesvirus Hijacks RNA Polymerase II To Create a Viral Transcriptional Factory.

    Science.gov (United States)

    Chen, Christopher Phillip; Lyu, Yuanzhi; Chuang, Frank; Nakano, Kazushi; Izumiya, Chie; Jin, Di; Campbell, Mel; Izumiya, Yoshihiro

    2017-06-01

    Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional "factories," which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of "viral transcriptional factories" decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an "all-in-one" factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells.IMPORTANCE B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the physical distribution of

  15. Gene expression regulation in retinal pigment epithelial cells induced by viral RNA and viral/bacterial DNA

    Science.gov (United States)

    Brosig, Anton; Kuhrt, Heidrun; Wiedemann, Peter; Kohen, Leon; Bringmann, Andreas

    2015-01-01

    Purpose The pathogenesis of age-related macular degeneration (AMD) is associated with systemic and local inflammation. Various studies suggested that viral or bacterial infection may aggravate retinal inflammation in the aged retina. We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells. Methods Cultured human RPE cells were stimulated with poly(I:C; 500 µg/ml) or CpG-ODN (500 nM). Alterations in gene expression and protein secretion were determined with real-time RT–PCR and ELISA, respectively. Phosphorylation of signal transduction molecules was revealed by western blotting. Results Poly(I:C) induced gene expression of the pattern recognition receptor TLR3, transcription factors (HIF-1α, p65/NF-κB), the angiogenic factor bFGF, inflammatory factors (IL-1β, IL-6, TNFα, MCP-1, MIP-2), and complement factors (C5, C9, CFB). Poly(I:C) also induced phosphorylation of ERK1/2 and p38 MAPK proteins, and the secretion of bFGF and TNFα from the cells. CpG-ODN induced moderate gene expression of transcription factors (p65/NF-κB, NFAT5) and complement factors (C5, C9), while it had no effect on the expression of various TLR, angiogenic factor, and inflammatory factor genes. The activities of various signal transduction pathways and transcription factors were differentially involved in mediating the poly(I:C)-induced transcriptional activation of distinct genes. Conclusions The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina. The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit

  16. Separation and isolation of BTV dsRNA segments and viral proteins.

    Science.gov (United States)

    Li, Joseph K-K; Huang, I-Jen; Hayama, Emiko

    2012-05-01

    Bluetongue virus (BTV) genome contains ten double-stranded RNA segments. The sequence of the plus strand of each of the BTV genomic double-stranded RNAs is the same as that of its mRNA, which encodes for a single viral protein, except the smallest S4 segment which can encode for two nonstructural proteins, primarily for the release assistance of the viral progeny. The separation and isolation of each BTV dsRNA segment and viral protein have provided extensive data related to its viral infection, pathology, suppression of host cellular functions, and eventual apoptosis of the infected host cells. This cytoplasmic virus is also an animal killer that costs the U.S. livestock industry at least $125 million yearly. However, this virus has no known effect on humans. Thus, it is very safe to carry out investigation with the virus, preferably in a BSL-2 laboratory.

  17. The herpes simplex virus host shutoff RNase degrades cellular and viral mRNAs made before infection but not viral mRNA made after infection.

    Science.gov (United States)

    Taddeo, Brunella; Zhang, Weiran; Roizman, Bernard

    2013-04-01

    A herpes simplex virus tegument protein brought into the cell during infection and designated the virion host shutoff protein (VHS) is an endoribonuclease that degrades mRNA. The prevailing view for many years has been that the VHS-RNase does not discriminate between cellular and viral RNAs and that the viruses prevail because the accumulation of viral transcripts outpaces their degradation. Here we report the following. (i) The degradation of viral mRNA made during infection of Vero or HEp-2 cells proceeds at a much-reduced rate compared to that of cellular mRNA. In effect, viral mRNAs are largely stable, whereas cellular mRNAs are rapidly degraded or, in the case of AU-rich mRNA, cleaved and rendered dysfunctional. (ii) In contrast to viral mRNAs made after infection, viral mRNAs expressed by plasmids transfected into cells prior to infection are degraded after infection at a rate comparable to that of cellular mRNAs. Moreover, the mRNA encoded by the transfected plasmid is hyperadenylated in the infected cell. Hyperadenylation but not degradation of mRNAs is blocked by actinomycin D. The results indicate that VHS-mRNA discriminates between viral and cellular mRNA but only in the context of infection and that discrimination is not based on the sequence of the mRNA but most likely on one or more viral factors expressed in the infected cell.

  18. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus

    Directory of Open Access Journals (Sweden)

    Fangquan Wang

    2016-05-01

    Full Text Available Rice black-streaked dwarf virus (RBSDV belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA. By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

  19. Antiviral RNA silencing viral counter defense in plants

    NARCIS (Netherlands)

    Bucher, E.C.

    2006-01-01

    The research described in this thesis centres around the mechanism of RNA silencing in relation to virus-host interaction, an area of increasing importance. It shows how this recently disclosed mechanism can be used to produce virus-resistant plants. Based on the activity of the RNA silencing machin

  20. Antiviral RNA silencing viral counter defense in plants

    NARCIS (Netherlands)

    Bucher, E.C.

    2006-01-01

    The research described in this thesis centres around the mechanism of RNA silencing in relation to virus-host interaction, an area of increasing importance. It shows how this recently disclosed mechanism can be used to produce virus-resistant plants. Based on the activity of the RNA silencing

  1. Antiviral RNA silencing viral counter defense in plants

    NARCIS (Netherlands)

    Bucher, E.C.

    2006-01-01

    The research described in this thesis centres around the mechanism of RNA silencing in relation to virus-host interaction, an area of increasing importance. It shows how this recently disclosed mechanism can be used to produce virus-resistant plants. Based on the activity of the RNA silencing machin

  2. SearchSmallRNA: a graphical interface tool for the assemblage of viral genomes using small RNA libraries data.

    Science.gov (United States)

    de Andrade, Roberto R S; Vaslin, Maite F S

    2014-03-07

    Next-generation parallel sequencing (NGS) allows the identification of viral pathogens by sequencing the small RNAs of infected hosts. Thus, viral genomes may be assembled from host immune response products without prior virus enrichment, amplification or purification. However, mapping of the vast information obtained presents a bioinformatics challenge. In order to by pass the need of line command and basic bioinformatics knowledge, we develop a mapping software with a graphical interface to the assemblage of viral genomes from small RNA dataset obtained by NGS. SearchSmallRNA was developed in JAVA language version 7 using NetBeans IDE 7.1 software. The program also allows the analysis of the viral small interfering RNAs (vsRNAs) profile; providing an overview of the size distribution and other features of the vsRNAs produced in infected cells. The program performs comparisons between each read sequenced present in a library and a chosen reference genome. Reads showing Hamming distances smaller or equal to an allowed mismatched will be selected as positives and used to the assemblage of a long nucleotide genome sequence. In order to validate the software, distinct analysis using NGS dataset obtained from HIV and two plant viruses were used to reconstruct viral whole genomes. SearchSmallRNA program was able to reconstructed viral genomes using NGS of small RNA dataset with high degree of reliability so it will be a valuable tool for viruses sequencing and discovery. It is accessible and free to all research communities and has the advantage to have an easy-to-use graphical interface. SearchSmallRNA was written in Java and is freely available at http://www.microbiologia.ufrj.br/ssrna/.

  3. Mutational Analysis Reveals a Noncontractile but Interactive Role of Actin and Profilin in Viral RNA-Dependent RNA Synthesis▿

    Science.gov (United States)

    Harpen, Mary; Barik, Tiasha; Musiyenko, Alla; Barik, Sailen

    2009-01-01

    As obligatory parasites, viruses co-opt a variety of cellular functions for robust replication. The expression of the nonsegmented negative-strand RNA genome of respiratory syncytial virus (RSV), a significant pediatric pathogen, absolutely requires actin and is stimulated by the actin-regulatory protein profilin. As actin is a major contractile protein, it was important to determine whether the known functional domains of actin and profilin were important for their ability to activate RSV transcription. Analyses of recombinant mutants in a reconstituted RSV transcription system suggested that the divalent-cation-binding domain of actin is critically needed for binding to the RSV genome template and for the activation of viral RNA synthesis. In contrast, the nucleotide-binding domain and the N-terminal acidic domain were needed neither for template binding nor for transcription. Specific surface residues of actin, required for actin-actin contact during filamentation, were also nonessential for viral transcription. Unlike actin, profilin did not directly bind to the viral template but was recruited by actin. Mutation of the interactive residues of actin or profilin, resulting in the loss of actin-profilin binding, also abolished profilin's ability to stimulate viral transcription. Together, these results suggest that actin acts as a classical transcription factor for the virus by divalent-cation-dependent binding to the viral template and that profilin acts as a transcriptional cofactor, in part by associating with actin. This essential viral role of actin is independent of its contractile cellular role. PMID:19710142

  4. Physician experience and rates of plasma HIV-1 RNA suppression among illicit drug users: an observational study

    Directory of Open Access Journals (Sweden)

    Sangsari Sassan

    2012-01-01

    Full Text Available Abstract Background Despite the availability of antiretroviral therapy (ART, suboptimal treatment outcomes have been observed among HIV-seropositive illicit drug users. As there is an urgent need to improve responses to antiretroviral therapy among this population, we undertook this study to evaluate the role of physician experience on rates of plasma HIV-1 RNA suppression following initiation of ART. Methods Using data from a community-recruited cohort of HIV-positive illicit drug users, we used Cox proportional hazards regression to model the time to plasma viral HIV RNA Results Between May 1996 and December 2008, 267 individuals initiated ART among whom 227 (85% achieved a plasma HIV RNA Conclusions In this setting of universal HIV/AIDS care, illicit drug users with more experienced physicians exhibited faster rates of plasma viral load suppression. These findings argue for specialized services to help optimize HIV treatment outcomes among this population.

  5. Viral suppressors of RNA interference impair RNA silencing induced by a Semliki Forest virus replicon in tick cells

    NARCIS (Netherlands)

    Garcia, S.; Billecocq, A.; Crance, J.M.; Prins, M.W.; Garin, D.; Bouloy, M.

    2006-01-01

    It was recently shown that infection of ISE6 tick cells by a recombinant Semliki Forest virus (SFV) expressing a heterologous gene induced small interfering RNAs (siRNAs) and silencing of the gene. To gain information on RNA interference (RNAi) in ticks, three known viral inhibitors that act in diff

  6. Viral suppressors of RNA interference impair RNA silencing induced by a Semliki Forest virus replicon in tick cells

    NARCIS (Netherlands)

    Garcia, S.; Billecocq, A.; Crance, J.M.; Prins, M.W.; Garin, D.; Bouloy, M.

    2006-01-01

    It was recently shown that infection of ISE6 tick cells by a recombinant Semliki Forest virus (SFV) expressing a heterologous gene induced small interfering RNAs (siRNAs) and silencing of the gene. To gain information on RNA interference (RNAi) in ticks, three known viral inhibitors that act in diff

  7. Seminal Plasma HIV-1 RNA Concentration Is Strongly Associated with Altered Levels of Seminal Plasma Interferon-γ, Interleukin-17, and Interleukin-5

    Science.gov (United States)

    Hoffman, Jennifer C.; Anton, Peter A.; Baldwin, Gayle Cocita; Elliott, Julie; Anisman-Posner, Deborah; Tanner, Karen; Grogan, Tristan; Elashoff, David; Sugar, Catherine; Yang, Otto O.

    2014-01-01

    Abstract Seminal plasma HIV-1 RNA level is an important determinant of the risk of HIV-1 sexual transmission. We investigated potential associations between seminal plasma cytokine levels and viral concentration in the seminal plasma of HIV-1-infected men. This was a prospective, observational study of paired blood and semen samples from 18 HIV-1 chronically infected men off antiretroviral therapy. HIV-1 RNA levels and cytokine levels in seminal plasma and blood plasma were measured and analyzed using simple linear regressions to screen for associations between cytokines and seminal plasma HIV-1 levels. Forward stepwise regression was performed to construct the final multivariate model. The median HIV-1 RNA concentrations were 4.42 log10 copies/ml (IQR 2.98, 4.70) and 2.96 log10 copies/ml (IQR 2, 4.18) in blood and seminal plasma, respectively. In stepwise multivariate linear regression analysis, blood HIV-1 RNA level (pplasma HIV-1 RNA level. After controlling for blood HIV-1 RNA level, seminal plasma HIV-1 RNA level was positively associated with interferon (IFN)-γ (p=0.03) and interleukin (IL)-17 (p=0.03) and negatively associated with IL-5 (p=0.0007) in seminal plasma. In addition to blood HIV-1 RNA level, cytokine profiles in the male genital tract are associated with HIV-1 RNA levels in semen. The Th1 and Th17 cytokines IFN-γ and IL-17 are associated with increased seminal plasma HIV-1 RNA, while the Th2 cytokine IL-5 is associated with decreased seminal plasma HIV-1 RNA. These results support the importance of genital tract immunomodulation in HIV-1 transmission. PMID:25209674

  8. Viral Oncogenes, Noncoding RNAs, and RNA Splicing in Human Tumor Viruses

    Directory of Open Access Journals (Sweden)

    Zhi-Ming Zheng

    2010-01-01

    Full Text Available Viral oncogenes are responsible for oncogenesis resulting from persistent virus infection. Although different human tumor viruses express different viral oncogenes and induce different tumors, their oncoproteins often target similar sets of cellular tumor suppressors or signal pathways to immortalize and/or transform infected cells. Expression of the viral E6 and E7 oncogenes in papillomavirus, E1A and E1B oncogenes in adenovirus, large T and small t antigen in polyomavirus, and Tax oncogene in HTLV-1 are regulated by alternative RNA splicing. However, this regulation is only partially understood. DNA tumor viruses also encode noncoding RNAs, including viral microRNAs, that disturb normal cell functions. Among the determined viral microRNA precursors, EBV encodes 25 from two major clusters (BART and BHRF1, KSHV encodes 12 from a latent region, human polyomavirus MCV produce only one microRNA from the late region antisense to early transcripts, but HPVs appears to produce no viral microRNAs.

  9. Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins

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    Marcio Hedil

    2016-07-01

    Full Text Available The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far.

  10. Nidovirus replication structures : hijacking membranes to support viral RNA synthesis

    NARCIS (Netherlands)

    Knoops, Kèvin

    2011-01-01

    Positive-stranded RNA viruses replicate in the cytoplasm of host cells and their replication complexes are associated with modified cell membranes. We investigated the structure of the nidovirus-induced membrane modifications and found that nidoviruses transform the endoplasmic reticulum into a reti

  11. Sensitive, Specific Complementary - Strand Optical Detection of Viral RNA

    Science.gov (United States)

    1997-06-01

    promise Its components have been characterized and proven to show unparalleled sensitivity; in addition, an innovative method for improving...sensing. 14. SUBJECT TERMS 15. NUMBER OF PAGES RNA, enterovirii, biosensor, evanscent waves, refractometry , 31 phase detection, complementary strand...Phase Measurements (10) Tri-Phase Detection Method . (11) Phase Detectors (12) Phase Processor Board (13) Laboratory Experim ents

  12. Advancing viral RNA structure prediction: measuring the thermodynamics of pyrimidine-rich internal loops.

    Science.gov (United States)

    Phan, Andy; Mailey, Katherine; Sakai, Jessica; Gu, Xiaobo; Schroeder, Susan J

    2017-02-17

    Accurate thermodynamic parameters improve RNA structure predictions and thus accelerate understanding of RNA function and the identification of RNA drug binding sites. Many viral RNA structures, such as internal ribosome entry sites, have internal loops and bulges that are potential drug target sites. Current models used to predict internal loops are biased towards small, symmetric purine loops, and thus poorly predict asymmetric, pyrimidine-rich loops with more than 6 nucleotides that occur frequently in viral RNA. This paper presents new thermodynamic data for 40 pyrimidine loops, many of which can form UU or protonated CC base pairs. Protonated cytosine and uracil base pairs stabilize asymmetric internal loops. Accurate prediction rules are presented that account for all thermodynamic measurements of RNA asymmetric internal loops. New loop initiation terms for loops with more than 6 nucleotides are presented that do not follow previous assumptions that increasing asymmetry destabilizes loops. Since the last 2004 update, 126 new loops with asymmetry or sizes greater than 2x2 have been measured (Mathews 2004). These new measurements significantly deepen and diversify the thermodynamic database for RNA. These results will help better predict internal loops that are larger, pyrimidine-rich, and occur within viral structures such as internal ribosome entry sites.

  13. HIV-1 TAR element is processed by Dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR

    Directory of Open Access Journals (Sweden)

    McCaffrey Timothy

    2007-07-01

    Full Text Available Abstract Background RNA interference (RNAi is a regulatory mechanism conserved in higher eukaryotes. The RNAi pathway generates small interfering RNA (siRNA or micro RNA (miRNA from either long double stranded stretches of RNA or RNA hairpins, respectively. The siRNA or miRNA then guides an effector complex to a homologous sequence of mRNA and regulates suppression of gene expression through one of several mechanisms. The suppression of gene expression through these mechanisms serves to regulate endogenous gene expression and protect the cell from foreign nucleic acids. There is growing evidence that many viruses have developed in the context of RNAi and express either a suppressor of RNAi or their own viral miRNA. Results In this study we investigated the possibility that the HIV-1 TAR element, a hairpin structure of ~50 nucleotides found at the 5' end of the HIV viral mRNA, is recognized by the RNAi machinery and processed to yield a viral miRNA. We show that the protein Dicer, the enzyme responsible for cleaving miRNA and siRNA from longer RNA sequences, is expressed in CD4+ T-cells. Interestingly, the level of expression of Dicer in monocytes is sub-optimal, suggesting a possible role for RNAi in maintaining latency in T-cells. Using a biotin labeled TAR element we demonstrate that Dicer binds to this structure. We show that recombinant Dicer is capable of cleaving the TAR element in vitro and that TAR derived miRNA is present in HIV-1 infected cell lines and primary T-cell blasts. Finally, we show that a TAR derived miRNA is capable of regulating viral gene expression and may be involved in repressing gene expression through transcriptional silencing. Conclusion HIV-1 TAR element is processed by the Dicer enzyme to create a viral miRNA. This viral miRNA is detectable in infected cells and appears to contribute to viral latency.

  14. On-chip purification and detection of hepatitis C virus RNA from human plasma.

    Science.gov (United States)

    Vaghi, V; Potrich, C; Pasquardini, L; Lunelli, L; Vanzetti, L; Ebranati, E; Lai, A; Zehender, G; Mombello, D; Cocuzza, M; Pirri, C F; Pederzolli, C

    2016-01-01

    Hepatitis C virus (HCV) is one of the main causes of chronic liver disease worldwide. The diagnosis and monitoring of HCV infection is a crucial need in the clinical management. The conventional diagnostic technologies are challenged when trying to address molecular diagnostics, especially because they require a complex and time-consuming sample preparation phase. Here, a new concept based on surface functionalization was applied to viral RNA purification: first of all polydimethylsiloxane (PDMS) flat surfaces were modified to hold RNA adsorption. After a careful chemical and morphological analysis of the modified surfaces, the functionalization protocols giving the best RNA adsorbing surfaces were applied to PDMS microdevices. The functionalized microdevices were then used for RNA purification from HCV infected human plasma samples. RNA purification and RT were successfully performed in the same microdevice chamber, saving time of analysis, reagents, and labor. The PCR protocol for HCV cDNA amplification was also implemented in the microdevice, demonstrating that the entire process of HCV analysis, from plasma to molecular readout, could be performed on-chip. Not only HCV but also other microdevice-based viral RNA detection could therefore result in a successful Point-of-Care (POC) diagnostics for resource-limited settings. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

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    Beatriz M. A. Fontoura

    2013-07-01

    Full Text Available Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

  16. Undetectable plasma viral load predicts normal survival in HIV-2-infected people in a West African village

    Directory of Open Access Journals (Sweden)

    Ricard Dominique

    2010-05-01

    Full Text Available Abstract Background There have been no previous studies of the long-term survival and temporal changes in plasma viral load among HIV-2 infected subjects. Methods 133 HIV-2 infected and 158 HIV-uninfected subjects from a rural area in North-west Guinea-Bissau, West Africa were enrolled into a prospective cohort study in 1991 and followed-up to mid-2009. Data were collected on four occasions during that period on HIV antibodies, CD4% and HIV-2 plasma viral load. Results Median age (interquartile range [IQR] of HIV-2 infected subjects at time of enrollment was 47 (36, 60 years, similar to that of HIV-uninfected control subjects, 49 (38, 62 (p = 0.4. Median (IQR plasma viral load and CD4 percentage were 347 (50, 4,300 copies/ml and 29 (22, 35 respectively. Overall loss to follow-up to assess vital status was small, at 6.7% and 6.3% for HIV-2 infected and uninfected subjects respectively. An additional 17 (12.8% and 16 (10.1% of HIV-2 infected and uninfected subjects respectively were censored during follow-up due to infection with HIV-1. The mortality rate per 100 person-years (95% CI was 4.5 (3.6, 5.8 among HIV-2 infected subjects compared to 2.1 (1.6, 2.9 among HIV-uninfected (age-sex adjusted rate ratio 1.9 (1.3, 2.8, p Viral load measurements were available for 98%, 78%, 77% and 61% HIV-2 infected subjects who were alive and had not become super-infected with HIV-1, in 1991, 1996, 2003 and 2006 respectively. Median plasma viral load (RNA copies per ml (IQR did not change significantly over time, being 150 (50, 1,554; n = 77 in 1996, 203 (50, 2,837; n = 47 in 2003 and 171 (50, 497; n = 31 in 2006. Thirty seven percent of HIV-2 subjects had undetectable viraemia ( Conclusions A substantial proportion of HIV-2 infected subjects in this cohort have stable plasma viral load, and those with an undetectable viral load (37% at study entry had a normal survival rate. However, the sequential laboratory findings need to be interpreted with caution given

  17. Repertoire of bovine miRNA and miRNA-like small regulatory RNAs expressed upon viral infection.

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    Evgeny A Glazov

    Full Text Available MicroRNA (miRNA and other types of small regulatory RNAs play a crucial role in the regulation of gene expression in eukaryotes. Several distinct classes of small regulatory RNAs have been discovered in recent years. To extend the repertoire of small RNAs characterized in mammals and to examine relationship between host miRNA expression and viral infection we used Illumina's ultrahigh throughput sequencing approach. We sequenced three small RNA libraries prepared from cell line derived from the adult bovine kidney under normal conditions and upon infection of the cell line with Bovine herpesvirus 1. We used a bioinformatics approach to distinguish authentic mature miRNA sequences from other classes of small RNAs and short RNA fragments represented in the sequencing data. Using this approach we detected 219 out of 356 known bovine miRNAs and 115 respective miRNA* sequences. In addition we identified five new bovine orthologs of known mammalian miRNAs and discovered 268 new cow miRNAs many of which are not identifiable in other mammalian genomes and thus might be specific to the ruminant lineage. In addition we found seven new bovine mirtron candidates. We also discovered 10 small nucleolar RNA (snoRNA loci that give rise to small RNA with possible miRNA-like function. Results presented in this study extend our knowledge of the biology and evolution of small regulatory RNAs in mammals and illuminate mechanisms of small RNA biogenesis and function. New miRNA sequences and the original sequencing data have been submitted to miRNA repository (miRBase and NCBI GEO archive respectively. We envisage that these resources will facilitate functional annotation of the bovine genome and promote further functional and comparative genomics studies of small regulatory RNA in mammals.

  18. Synthesis and Biological Evaluation of Brain-Specific Anti-RNA Viral Agents

    Science.gov (United States)

    1990-03-31

    genetic material either DNA or RNA and the type of nucleic acid give rise to the system of nomenclature for these entities. The viral DNA or RNA can then... anhydride (3) to produce the 5’-trigonellinate iodide of the protected sugar (4). Reduction of this quaternary salt in aqueous basic sodium dithionite...yielded the 5’-(1,4- dihydrotrigonellinate) of the ribavirin acetonide (5, AVS 5505). In the above synthesis, trigonelline anhydride was used to avoid

  19. Rational design of a flavivirus vaccine by abolishing viral RNA 2'-O methylation.

    Science.gov (United States)

    Li, Shi-Hua; Dong, Hongping; Li, Xiao-Feng; Xie, Xuping; Zhao, Hui; Deng, Yong-Qiang; Wang, Xiao-Yu; Ye, Qing; Zhu, Shun-Ya; Wang, Hong-Jiang; Zhang, Bo; Leng, Qi-Bin; Zuest, Roland; Qin, E-De; Qin, Cheng-Feng; Shi, Pei-Yong

    2013-05-01

    Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2'-O cap of their RNA; alternatively, they "snatch" host mRNA cap to form the 5' end of viral RNA. The function of 2'-O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2'-O methylation is replicative, but its viral RNA lacks 2'-O methylation and is recognized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vaccine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2'-O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2'-O methylation was stable in cell culture after being passaged for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and retained the engineered mutation in vivo. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2'-O methylation-defective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2'-O methyltransferases.

  20. Rational Design of a Flavivirus Vaccine by Abolishing Viral RNA 2′-O Methylation

    Science.gov (United States)

    Li, Shi-Hua; Dong, Hongping; Li, Xiao-Feng; Xie, Xuping; Zhao, Hui; Deng, Yong-Qiang; Wang, Xiao-Yu; Ye, Qing; Zhu, Shun-Ya; Wang, Hong-Jiang; Zhang, Bo; Leng, Qi-Bin; Zuest, Roland; Qin, E-De

    2013-01-01

    Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2′-O cap of their RNA; alternatively, they “snatch” host mRNA cap to form the 5′ end of viral RNA. The function of 2′-O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2′-O methylation is replicative, but its viral RNA lacks 2′-O methylation and is recognized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vaccine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2′-O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2′-O methylation was stable in cell culture after being passaged for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and retained the engineered mutation in vivo. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2′-O methylation-defective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2′-O methyltransferases. PMID:23487465

  1. A Novel Mechanism Underlying the Innate Immune Response Induction upon Viral-Dependent Replication of Host Cell mRNA: A Mistake of +sRNA Viruses' Replicases

    Science.gov (United States)

    Delgui, Laura R.; Colombo, María I.

    2017-01-01

    Viruses are lifeless particles designed for setting virus-host interactome assuring a new generation of virions for dissemination. This interactome generates a pressure on host organisms evolving mechanisms to neutralize viral infection, which places the pressure back onto virus, a process known as virus-host cell co-evolution. Positive-single stranded RNA (+sRNA) viruses are an important group of viral agents illustrating this interesting phenomenon. During replication, their genomic +sRNA is employed as template for translation of viral proteins; among them the RNA-dependent RNA polymerase (RdRp) is responsible of viral genome replication originating double-strand RNA molecules (dsRNA) as intermediates, which accumulate representing a potent threat for cellular dsRNA receptors to initiate an antiviral response. A common feature shared by these viruses is their ability to rearrange cellular membranes to serve as platforms for genome replication and assembly of new virions, supporting replication efficiency increase by concentrating critical factors and protecting the viral genome from host anti-viral systems. This review summarizes current knowledge regarding cellular dsRNA receptors and describes prototype viruses developing replication niches inside rearranged membranes. However, for several viral agents it's been observed both, a complex rearrangement of cellular membranes and a strong innate immune antiviral response induction. So, we have included recent data explaining the mechanism by, even though viruses have evolved elegant hideouts, host cells are still able to develop dsRNA receptors-dependent antiviral response. PMID:28164038

  2. Innate immune restriction and antagonism of viral RNA lacking 2'-O methylation

    Energy Technology Data Exchange (ETDEWEB)

    Hyde, Jennifer L. [Departments of Medicine, Washington University School of Medicine, St Louis., MO 63110 (United States); Diamond, Michael S., E-mail: diamond@borcim.wustl.edu [Departments of Medicine, Washington University School of Medicine, St Louis., MO 63110 (United States); Molecular Microbiology, Washington University School of Medicine, St Louis., MO 63110 (United States); Pathology & Immunology, Washington University School of Medicine, St Louis., MO 63110 (United States); The Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, St Louis., MO 63110 (United States)

    2015-05-15

    N-7 and 2′-O methylation of host cell mRNA occurs in the nucleus and results in the generation of cap structures (cap 0, m{sup 7}GpppN; cap 1, m{sup 7}GpppNm) that control gene expression by modulating nuclear export, splicing, turnover, and protein synthesis. Remarkably, RNA cap modification also contributes to mammalian cell host defense as viral RNA lacking 2′-O methylation is sensed and inhibited by IFIT1, an interferon (IFN) stimulated gene (ISG). Accordingly, pathogenic viruses that replicate in the cytoplasm have evolved mechanisms to circumvent IFIT1 restriction and facilitate infection of mammalian cells. These include: (a) generating cap 1 structures on their RNA through cap-snatching or virally-encoded 2′-O methyltransferases, (b) using cap-independent means of translation, or (c) using RNA secondary structural motifs to antagonize IFIT1 binding. This review will discuss new insights as to how specific modifications at the 5′-end of viral RNA modulate host pathogen recognition responses to promote infection and disease.

  3. EBV noncoding RNA EBER2 interacts with host RNA-binding proteins to regulate viral gene expression.

    Science.gov (United States)

    Lee, Nara; Yario, Therese A; Gao, Jessica S; Steitz, Joan A

    2016-03-22

    Epstein-Barr virus (EBV) produces a highly abundant noncoding RNA called EBV-encoded RNA 2 (EBER2) that interacts indirectly with the host transcription factor paired box protein 5 (PAX5) to regulate viral latent membrane protein 1/2 (LMP1/2) gene expression as well as EBV lytic replication. To identify intermediary proteins, we isolated EBER2-PAX5-containing complexes and analyzed the protein components by mass spectrometry. The top candidates include three host proteins splicing factor proline and glutamine rich (SFPQ), non-POU domain-containing octamer-binding protein (NONO), and RNA binding motif protein 14 (RBM14), all reported to be components of nuclear bodies called paraspeckles. In vivo RNA-protein crosslinking indicates that SFPQ and RBM14 contact EBER2 directly. Binding studies using recombinant proteins demonstrate that SFPQ and NONO associate with PAX5, potentially bridging its interaction with EBER2. Similar to EBER2 or PAX5 depletion, knockdown of any of the three host RNA-binding proteins results in the up-regulation of viral LMP2A mRNA levels, supporting a physiologically relevant interaction of these newly identified factors with EBER2 and PAX5. Identification of these EBER2-interacting proteins enables the search for cellular noncoding RNAs that regulate host gene expression in a manner similar to EBER2.

  4. Exploration of sequence space as the basis of viral RNA genome segmentation.

    Science.gov (United States)

    Moreno, Elena; Ojosnegros, Samuel; García-Arriaza, Juan; Escarmís, Cristina; Domingo, Esteban; Perales, Celia

    2014-05-06

    The mechanisms of viral RNA genome segmentation are unknown. On extensive passage of foot-and-mouth disease virus in baby hamster kidney-21 cells, the virus accumulated multiple point mutations and underwent a transition akin to genome segmentation. The standard single RNA genome molecule was replaced by genomes harboring internal in-frame deletions affecting the L- or capsid-coding region. These genomes were infectious and killed cells by complementation. Here we show that the point mutations in the nonstructural protein-coding region (P2, P3) that accumulated in the standard genome before segmentation increased the relative fitness of the segmented version relative to the standard genome. Fitness increase was documented by intracellular expression of virus-coded proteins and infectious progeny production by RNAs with the internal deletions placed in the sequence context of the parental and evolved genome. The complementation activity involved several viral proteins, one of them being the leader proteinase L. Thus, a history of genetic drift with accumulation of point mutations was needed to allow a major variation in the structure of a viral genome. Thus, exploration of sequence space by a viral genome (in this case an unsegmented RNA) can reach a point of the space in which a totally different genome structure (in this case, a segmented RNA) is favored over the form that performed the exploration.

  5. Satellite RNAs interfere with the function of viral RNA silencing suppressors

    Directory of Open Access Journals (Sweden)

    Wanxia eShen

    2015-04-01

    Full Text Available Viral satellite RNAs (satRNAs are small subviral RNAs and depend on the helper virus for replication and spread. satRNAs can attenuate helper virus-induced symptoms, the mechanism of which remains unclear. Here, we show that two virus-encoded suppressors of RNA silencing (VSRs, Cucumber mosaic virus (CMV 2b and Tombusvirus P19, suppress hairpin RNA (hpRNA-induced silencing of a β-glucuronidase (GUS gene in Nicotiana benthamiana. This suppression can be overcome by CMV Y-satellite RNA (Y-Sat via the Y-Sat-derived small interfering RNAs (siRNAs, which bind to the VSRs and displace the bound hpGUS-derived siRNAs. We also show that microRNA target gene expression in N. tabacum was elevated by CMV infection, presumably due to function of the 2b VSR, but this upregulation of microRNA target genes was reversed in the presence of Y-Sat. These results suggest that satRNA infection minimizes the effect of VSRs on host siRNA and microRNA-directed silencing. Our results suggest that the high abundance of satRNA-derived siRNAs contributes to symptom attenuation by binding helper virus-encoded VSRs, minimizing the capacity of the VSRs to bind host siRNA and miRNA and interfere with their function.

  6. Respiratory Syncytial Virus Induces Host RNA Stress Granules To Facilitate Viral Replication▿

    Science.gov (United States)

    Lindquist, Michael E.; Lifland, Aaron W.; Utley, Thomas J.; Santangelo, Philip J.; Crowe, James E.

    2010-01-01

    Mammalian cell cytoplasmic RNA stress granules are induced during various conditions of stress and are strongly associated with regulation of host mRNA translation. Several viruses induce stress granules during the course of infection, but the exact function of these structures during virus replication is not well understood. In this study, we showed that respiratory syncytial virus (RSV) induced host stress granules in epithelial cells during the course of infection. We also showed that stress granules are distinct from cytoplasmic viral inclusion bodies and that the RNA binding protein HuR, normally found in stress granules, also localized to viral inclusion bodies during infection. Interestingly, we demonstrated that infected cells containing stress granules also contained more RSV protein than infected cells that did not form inclusion bodies. To address the role of stress granule formation in RSV infection, we generated a stable epithelial cell line with reduced expression of the Ras-GAP SH3 domain-binding protein (G3BP) that displayed an inhibited stress granule response. Surprisingly, RSV replication was impaired in these cells compared to its replication in cells with intact G3BP expression. In contrast, knockdown of HuR by RNA interference did not affect stress granule formation or RSV replication. Finally, using RNA probes specific for RSV genomic RNA, we found that viral RNA predominantly localized to viral inclusion bodies but a small percentage also interacted with stress granules during infection. These results suggest that RSV induces a host stress granule response and preferentially replicates in host cells that have committed to a stress response. PMID:20844027

  7. Reinitiated viral RNA-dependent RNA polymerase resumes replication at a reduced rate

    NARCIS (Netherlands)

    Vilfan, I.D.; Candelli, A.; Hage, S.; Aalto, A.P.; Poranen, M.M.; Bamford, D.H.; Dekker, N.H.

    2008-01-01

    RNA-dependent RNA polymerases (RdRP) form an important class of enzymes that is responsible for genome replication and transcription in RNA viruses and involved in the regulation of RNA interference in plants and fungi. The RdRP kinetics have been extensively studied, but pausing, an important regul

  8. A SELEX-screened aptamer of human hepatitis B virus RNA encapsidation signal suppresses viral replication.

    Directory of Open Access Journals (Sweden)

    Hui Feng

    Full Text Available BACKGROUND: The specific interaction between hepatitis B virus (HBV polymerase (P protein and the ε RNA stem-loop on pregenomic (pg RNA is crucial for viral replication. It triggers both pgRNA packaging and reverse transcription and thus represents an attractive antiviral target. RNA decoys mimicking ε in P protein binding but not supporting replication might represent novel HBV inhibitors. However, because generation of recombinant enzymatically active HBV polymerase is notoriously difficult, such decoys have as yet not been identified. METHODOLOGY/PRINCIPAL FINDINGS: Here we used a SELEX approach, based on a new in vitro reconstitution system exploiting a recombinant truncated HBV P protein (miniP, to identify potential ε decoys in two large ε RNA pools with randomized upper stem. Selection of strongly P protein binding RNAs correlated with an unexpected strong enrichment of A residues. Two aptamers, S6 and S9, displayed particularly high affinity and specificity for miniP in vitro, yet did not support viral replication when part of a complete HBV genome. Introducing S9 RNA into transiently HBV producing HepG2 cells strongly suppressed pgRNA packaging and DNA synthesis, indicating the S9 RNA can indeed act as an ε decoy that competitively inhibits P protein binding to the authentic ε signal on pgRNA. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the first successful identification of human HBV ε aptamers by an in vitro SELEX approach. Effective suppression of HBV replication by the S9 aptamer provides proof-of-principle for the ability of ε decoy RNAs to interfere with viral P-ε complex formation and suggests that S9-like RNAs may further be developed into useful therapeutics against chronic hepatitis B.

  9. Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection

    OpenAIRE

    2015-01-01

    Viruses use alternate mechanisms to increase the coding capacity of their viral genomes. The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts an RNA structure that can direct translation in 0 and +1 reading frames to produce the viral structural proteins and an overlapping ORFx product. Here we provide structural and biochemical evidence that the PKI domain of the IRES mimics a complete tRNA-like structure to facilitate reading frame selection and allows the viral IR...

  10. HIV-1 tropism testing in subjects achieving undetectable HIV-1 RNA: diagnostic accuracy, viral evolution and compartmentalization.

    Directory of Open Access Journals (Sweden)

    Christian Pou

    Full Text Available BACKGROUND: Technically, HIV-1 tropism can be evaluated in plasma or peripheral blood mononuclear cells (PBMCs. However, only tropism testing of plasma HIV-1 has been validated as a tool to predict virological response to CCR5 antagonists in clinical trials. The preferable tropism testing strategy in subjects with undetectable HIV-1 viremia, in whom plasma tropism testing is not feasible, remains uncertain. METHODS & RESULTS: We designed a proof-of-concept study including 30 chronically HIV-1-infected individuals who achieved HIV-1 RNA <50 copies/mL during at least 2 years after first-line ART initiation. First, we determined the diagnostic accuracy of 454 and population sequencing of gp120 V3-loops in plasma and PBMCs, as well as of MT-2 assays before ART initiation. The Enhanced Sensitivity Trofile Assay (ESTA was used as the technical reference standard. 454 sequencing of plasma viruses provided the highest agreement with ESTA. The accuracy of 454 sequencing decreased in PBMCs due to reduced specificity. Population sequencing in plasma and PBMCs was slightly less accurate than plasma 454 sequencing, being less sensitive but more specific. MT-2 assays had low sensitivity but 100% specificity. Then, we used optimized 454 sequence data to investigate viral evolution in PBMCs during viremia suppression and only found evolution of R5 viruses in one subject. No de novo CXCR4-using HIV-1 production was observed over time. Finally, Slatkin-Maddison tests suggested that plasma and cell-associated V3 forms were sometimes compartmentalized. CONCLUSIONS: The absence of tropism shifts during viremia suppression suggests that, when available, testing of stored plasma samples is generally safe and informative, provided that HIV-1 suppression is maintained. Tropism testing in PBMCs may not necessarily produce equivalent biological results to plasma, because the structure of viral populations and the diagnostic performance of tropism assays may sometimes vary

  11. A viral microRNA down-regulates multiple cell cycle genes through mRNA 5'UTRs.

    Directory of Open Access Journals (Sweden)

    Finn Grey

    Full Text Available Global gene expression data combined with bioinformatic analysis provides strong evidence that mammalian miRNAs mediate repression of gene expression primarily through binding sites within the 3' untranslated region (UTR. Using RNA induced silencing complex immunoprecipitation (RISC-IP techniques we have identified multiple cellular targets for a human cytomegalovirus (HCMV miRNA, miR-US25-1. Strikingly, this miRNA binds target sites primarily within 5'UTRs, mediating significant reduction in gene expression. Intriguingly, many of the genes targeted by miR-US25-1 are associated with cell cycle control, including cyclin E2, BRCC3, EID1, MAPRE2, and CD147, suggesting that miR-US25-1 is targeting genes within a related pathway. Deletion of miR-US25-1 from HCMV results in over expression of cyclin E2 in the context of viral infection. Our studies demonstrate that a viral miRNA mediates translational repression of multiple cellular genes by targeting mRNA 5'UTRs.

  12. Visitor, an informatic pipeline for analysis of viral siRNA sequencing datasets.

    Science.gov (United States)

    Antoniewski, Christophe

    2011-01-01

    High-throughput sequencing emerged as a powerful approach to characterize siRNA populations -generated by hosts in response to viral infections. Here we described an informatic pipeline visitor to analyze in-house large sequencing datasets generated from Illumina sequencing of Drosophila small RNA libraries. The visitor perl script is designed to treat fastq sequence datasets from the Illumina sequencing platform, using a computer running under a UNIX compliant operating system (MacOS X, Linux, etc.). visitor first generates a detailed report of the sequence quality of the Illumina run. Then, using the Novoalign software, the script removes reads that match with the D. melanogaster genome from the sequencing data set. The remaining reads are aligned to a viral reference library, which can contain one or several virus genomes. visitor provides a hit table of identified viral siRNAs as well as graphics eps files of viral siRNA profiles. Unmatched small RNAs are also available in a fast format for de novo assembly and new virus discovery.

  13. Translational regulation of viral secretory proteins by the 5' coding regions and a viral RNA-binding protein.

    Science.gov (United States)

    Nordholm, Johan; Petitou, Jeanne; Östbye, Henrik; da Silva, Diogo V; Dou, Dan; Wang, Hao; Daniels, Robert

    2017-08-07

    A primary function of 5' regions in many secretory protein mRNAs is to encode an endoplasmic reticulum (ER) targeting sequence. In this study, we show how the regions coding for the ER-targeting sequences of the influenza glycoproteins NA and HA also function as translational regulatory elements that are controlled by the viral RNA-binding protein (RBP) NS1. The translational increase depends on the nucleotide composition and 5' positioning of the ER-targeting sequence coding regions and is facilitated by the RNA-binding domain of NS1, which can associate with ER membranes. Inserting the ER-targeting sequence coding region of NA into different 5' UTRs confirmed that NS1 can promote the translation of secretory protein mRNAs based on the nucleotides within this region rather than the resulting amino acids. By analyzing human protein mRNA sequences, we found evidence that this mechanism of using 5' coding regions and particular RBPs to achieve gene-specific regulation may extend to human-secreted proteins. © 2017 Nordholm et al.

  14. Repression of RNA polymerase by the archaeo-viral regulator ORF145/RIP

    DEFF Research Database (Denmark)

    Sheppard, Carol; Blombach, Fabian; Belsom, Adam

    2016-01-01

    initiation, as well as elongation. Both host and viral promoters are subjected to ORF145 repression. Thus, ORF145 has the properties of a global transcription repressor and its overexpression is toxic for Sulfolobus. On the basis of its properties, we have re-named ORF145 RNAP Inhibitory Protein (RIP).......Little is known about how archaeal viruses perturb the transcription machinery of their hosts. Here we provide the first example of an archaeo-viral transcription factor that directly targets the host RNA polymerase (RNAP) and efficiently represses its activity. ORF145 from the temperate Acidianus...

  15. Ebola Virus VP35 Interaction with Dynein LC8 Regulates Viral RNA Synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Luthra, Priya; Jordan, David S.; Leung, Daisy W.; Amarasinghe, Gaya K.; Basler, Christopher F.; Lyles, D. S.

    2015-03-04

    Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8.

  16. A viral suppressor protein inhibits host RNA silencing by hooking up with Argonautes

    KAUST Repository

    Jin, Hailing

    2010-05-01

    RNA viruses are particularly vulnerable to RNAi-based defenses in the host, and thus have evolved specific proteins, known as viral suppressors of RNA silencing (VSRs), as a counterdefense. In this issue of Genes & Development, Azevedo and colleagues (pp. 904-915) discovered that P38, the VSR of Turnip crinkle virus, uses its glycine/tryptophane (GW) motifs as an ARGONAUTE (AGO) hook to attract and disarm the host\\'s essential effector of RNA silencing. Several GW motif-containing cellular proteins are known to be important partners of AGOs in RNA silencing effector complexes in yeast, plants, and animals. The GW motif appears to be a versatile and effective tool for regulating the activities of RNA silencing pathways, and the use of GW mimicry to compete for and inhibit host AGOs may be a strategy used by many pathogens to counteract host RNAi-based defenses. © 2010 by Cold Spring Harbor Laboratory Press.

  17. Simple rules for efficient assembly predict the layout of a packaged viral RNA.

    Science.gov (United States)

    Dykeman, E C; Grayson, N E; Toropova, K; Ranson, N A; Stockley, P G; Twarock, R

    2011-05-06

    Single-stranded RNA (ssRNA) viruses, which include major human pathogens, package their genomes as they assemble their capsids. We show here that the organization of the viral genomes within the capsids provides intriguing insights into the highly cooperative nature of the assembly process. A recent cryo-electron microscopy structure of bacteriophage MS2, determined with only 5-fold symmetry averaging, has revealed the asymmetric distribution of its encapsidated genome. Here we show that this RNA distribution is consistent with an assembly mechanism that follows two simple rules derived from experiment: (1) the binding of the MS2 maturation protein to the RNA constrains its conformation into a loop, and (2) the capsid must be built in an energetically favorable way. These results provide a new level of insight into the factors that drive efficient assembly of ssRNA viruses in vivo. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. HCV-RNA positivity in peripheral blood mononuclear cells of patients with chronic HCV-infection: does it really mean viral replication?

    Institute of Scientific and Technical Information of China (English)

    Volker Meier; Sabine Mihm; Perdita Wietzke-Braun; Guliano Ramadori

    2001-01-01

    AIM To analyze the association of HCV-RNA with peripheral blood mononuclear cells (PBMC)and to answer the question whether HCV-RNA positivity in PBMC is due to viral replication,METHODS HCV-RNA was monitored in serumand PBMC preparations from 15 patients with chronic HCV infection before, during and after an IFN-α therapy using a nested RT/ PCRtechnique. In a second approach, PBMC from healthy donors were incubated in HCV positive plasma.RESULTS In the IFN-α responding patients,HCV-RNA disappeared first from total RNApreparations of PBMC and then from serum. In contrast, in relapsing patients, HCV-RNAreappeared first in serum and then in PBMC. A quantitative analysis of the HCV-RNAconcentration in serum was performed before and after transition from detectable to nondetectable HCV-RNA in PBMC-RNA and vice versa. When HCV-RNA was detectable in PBMCpreparations, the HCV concentration in serum was significantly higher than the serum HCV-RNA concentration when HCV-RNA in PBMC was not detectable. Furthermore, at no time during the observation period was HCV specific RNA observed in PBMC, if HCV-RNA in serum was under the detection limit. Incubation of PBMCfrom healthy donors with several dilutions of HCV positive plasma for two hours showed a concentration-dependent PCR-positivity for HCV-RNA in reisolated PBMC.CONCLUSION The detectability of HCV-RNA in total RNA from PBMC seems to depend on the HCV concentration in serum. Contamination or passive adsorption by circulating virus could be the reason for detection of HCV-RNA in PBMCpreparations of chronically infected patients.

  19. Antiretroviral-treated HIV-1 patients can harbour resistant viruses in CSF despite an undetectable viral load in plasma.

    Science.gov (United States)

    Soulie, Cathia; Grudé, Maxime; Descamps, Diane; Amiel, Corinne; Morand-Joubert, Laurence; Raymond, Stéphanie; Pallier, Coralie; Bellecave, Pantxika; Reigadas, Sandrine; Trabaud, Mary-Anne; Delaugerre, Constance; Montes, Brigitte; Barin, Francis; Ferré, Virginie; Jeulin, Hélène; Alloui, Chakib; Yerly, Sabine; Signori-Schmuck, Anne; Guigon, Aurélie; Fafi-Kremer, Samira; Haïm-Boukobza, Stéphanie; Mirand, Audrey; Maillard, Anne; Vallet, Sophie; Roussel, Catherine; Assoumou, Lambert; Calvez, Vincent; Flandre, Philippe; Marcelin, Anne-Geneviève

    2017-08-01

    HIV therapy reduces the CSF HIV RNA viral load (VL) and prevents disorders related to HIV encephalitis. However, these brain disorders may persist in some cases. A large population of antiretroviral-treated patients who had a VL > 1.7 log 10 copies/mL in CSF with detectable or undetectable VL in plasma associated with cognitive impairment was studied, in order to characterize discriminatory factors of these two patient populations. Blood and CSF samples were collected at the time of neurological disorders for 227 patients in 22 centres in France and 1 centre in Switzerland. Genotypic HIV resistance tests were performed on CSF. The genotypic susceptibility score was calculated according to the last Agence Nationale de Recherche sur le Sida et les hépatites virales Action Coordonnée 11 (ANRS AC11) genotype interpretation algorithm. Among the 227 studied patients with VL > 1.7 log 10 copies/mL in CSF, 195 had VL detectable in plasma [median (IQR) HIV RNA was 3.7 (2.7-4.7) log 10 copies/mL] and 32 had discordant VL in plasma (VL  1.7 log 10 copies/mL. Resistance to antiretrovirals was observed in CSF for the two groups of patients. Fourteen percent of this population of patients with cognitive impairment and detectable VL in CSF had well controlled VL in plasma. Thus, it is important to explore CSF HIV (VL and genotype) even if the HIV VL is controlled in plasma because HIV resistance may be observed.

  20. Multiple functions of DDX3 RNA helicase in gene regulation, tumorigenesis and viral infection

    Directory of Open Access Journals (Sweden)

    YASUO eARIUMI

    2014-12-01

    Full Text Available The DEAD-box RNA helicase DDX3 is a multifunctional protein involved in all aspects of RNA metabolism, including transcription, splicing, mRNA nuclear export, translation, RNA decay and ribosome biogenesis. In addition, DDX3 is also implicated in cell cycle regulation, apoptosis, Wnt-ß-catenin signaling, tumorigenesis, and viral infection. Notably, recent studies suggest that DDX3 is a component of anti-viral innate immune signaling pathways. Indeed, DDX3 contributes to enhance the induction of anti-viral mediators, interferon regulatory factor (IRF 3 and type I interferon (IFN. However, DDX3 seems to be an important target for several viruses, such as human immunodeficiency virus (HIV-1, hepatitis C virus (HCV, hepatitis B virus (HBV, and poxvirus. DDX3 interacts with HIV-1 Rev or HCV Core protein and modulates its function. At least, DDX3 is required for both HIV-1 and HCV replication. Therefore, DDX3 could be a novel therapeutic target for the development of drug against HIV-1 and HCV.

  1. Multiple functions of DDX3 RNA helicase in gene regulation, tumorigenesis, and viral infection.

    Science.gov (United States)

    Ariumi, Yasuo

    2014-01-01

    The DEAD-box RNA helicase DDX3 is a multifunctional protein involved in all aspects of RNA metabolism, including transcription, splicing, mRNA nuclear export, translation, RNA decay and ribosome biogenesis. In addition, DDX3 is also implicated in cell cycle regulation, apoptosis, Wnt-β-catenin signaling, tumorigenesis, and viral infection. Notably, recent studies suggest that DDX3 is a component of anti-viral innate immune signaling pathways. Indeed, DDX3 contributes to enhance the induction of anti-viral mediators, interferon (IFN) regulatory factor 3 and type I IFN. However, DDX3 seems to be an important target for several viruses, such as human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV), hepatitis B virus (HBV), and poxvirus. DDX3 interacts with HIV-1 Rev or HCV Core protein and modulates its function. At least, DDX3 is required for both HIV-1 and HCV replication. Therefore, DDX3 could be a novel therapeutic target for the development of drug against HIV-1 and HCV.

  2. Accessory Factors of Cytoplasmic Viral RNA Sensors Required for Antiviral Innate Immune Response

    Science.gov (United States)

    Oshiumi, Hiroyuki; Kouwaki, Takahisa; Seya, Tsukasa

    2016-01-01

    Type I interferon (IFN) induces many antiviral factors in host cells. RIG-I-like receptors (RLRs) are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs) and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and, thus, cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway. PMID:27252702

  3. Accessory Factors of Cytoplasmic Viral RNA Sensors Required for Antiviral Innate Immune Response.

    Science.gov (United States)

    Oshiumi, Hiroyuki; Kouwaki, Takahisa; Seya, Tsukasa

    2016-01-01

    Type I interferon (IFN) induces many antiviral factors in host cells. RIG-I-like receptors (RLRs) are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs) and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and, thus, cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway.

  4. Accessory factors of cytoplasmic viral RNA sensors required for antiviral innate immune response

    Directory of Open Access Journals (Sweden)

    Hiroyuki eOshiumi

    2016-05-01

    Full Text Available Type I interferon (IFN induces many antiviral factors in host cells. RIG-I-like receptors (RLRs are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and thus cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway.

  5. A Bayesian approach to analyse genetic variation within RNA viral populations.

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    Trevelyan J McKinley

    2011-03-01

    Full Text Available The development of modern and affordable sequencing technologies has allowed the study of viral populations to an unprecedented depth. This is of particular interest for the study of within-host RNA viral populations, where variation due to error-prone polymerases can lead to immune escape, antiviral resistance and adaptation to new host species. Methods to sequence RNA virus genomes include reverse transcription (RT and polymerase chain reaction (PCR. RT-PCR is a molecular biology technique widely used to amplify DNA from an RNA template. The method itself relies on the in vitro synthesis of copy DNA from RNA followed by multiple cycles of DNA amplification. However, this method introduces artefactual errors that can act as confounding factors when the sequence data are analysed. Although there are a growing number of published studies exploring the intra- and inter-host evolutionary dynamics of RNA viruses, the complexity of the methods used to generate sequences makes it difficult to produce probabilistic statements about the likely sources of observed sequence variants. This complexity is further compounded as both the depth of sequencing and the length of the genome segment of interest increase. Here we develop a bayesian method to characterise and differentiate between likely structures for the background viral population. This approach can then be used to identify nucleotide sites that show evidence of change in the within-host viral population structure, either over time or relative to a reference sequence (e.g. an inoculum or another source of infection, or both, without having to build complex evolutionary models. Identification of these sites can help to inform the design of more focussed experiments using molecular biology tools, such as site-directed mutagenesis, to assess the function of specific amino acids. We illustrate the method by applying to datasets from experimental transmission of equine influenza, and a pre

  6. A viral suppressor of RNA silencing inhibits ARGONAUTE 1 function by precluding target RNA binding to pre-assembled RISC.

    Science.gov (United States)

    Kenesi, Erzsébet; Carbonell, Alberto; Lózsa, Rita; Vértessy, Beáta; Lakatos, Lóránt

    2017-07-27

    In most eukaryotes, RNA silencing is an adaptive immune system regulating key biological processes including antiviral defense. To evade this response, viruses of plants, worms and insects have evolved viral suppressors of RNA silencing proteins (VSRs). Various VSRs, such as P1 from Sweet potato mild mottle virus (SPMMV), inhibit the activity of RNA-induced silencing complexes (RISCs) including an ARGONAUTE (AGO) protein loaded with a small RNA. However, the specific mechanisms explaining this class of inhibition are unknown. Here, we show that SPMMV P1 interacts with AGO1 and AGO2 from Arabidopsis thaliana, but solely interferes with AGO1 function. Moreover, a mutational analysis of a newly identified zinc finger domain in P1 revealed that this domain could represent an effector domain as it is required for P1 suppressor activity but not for AGO1 binding. Finally, a comparative analysis of the target RNA binding capacity of AGO1 in the presence of wild-type or suppressor-defective P1 forms revealed that P1 blocks target RNA binding to AGO1. Our results describe the negative regulation of RISC, the small RNA containing molecular machine. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Short hairpin RNA targeting NP mRNA inhibiting Newcastle disease virus production and other viral structural mRNA transcription.

    Science.gov (United States)

    Yue, Hua; Deng, Shu; Yang, Fa-Long; Li, Ding-Fei; Fu, An-Jing; Yang, Fan; Tang, Cheng

    2009-02-01

    Newcastle disease virus (NDV), formally recognized as avian paramyxovirus 1 (APMV-1), is the etiological agent of Newcastle disease (ND), an affliction which can cause severe losses in the poultry industry. Better understanding of the molecular basis of viral structural genes involved with production should contribute significantly toward the development of improved prophylactic and therapeutic reagents to control the infection. Here we show that a short hairpin RNA (shRNA) eukaryotic expression vector targeting nucleocapsid (NP) gene of NDV can potently inhibit NDV production in both primary cells and embryonated chicken eggs. Moreover, shRNA specific for NP abolished the accumulation of not only the corresponding mRNA but also P, HN, F, M gene mRNA. The findings reveal that newly synthesized NP mRNA is essential for NDV transcription and replication, and provide a basis for the development of shRNAs as a prophylaxis and therapy for NDV infection in poultry.

  8. Comparison of methods for miRNA extraction from plasma and quantitative recovery of RNA from plasma and cerebrospinal fluid

    Directory of Open Access Journals (Sweden)

    Melissa A McAlexander

    2013-05-01

    Full Text Available Interest in extracellular RNA has intensified as evidence accumulates that these molecules may be useful as indicators of a wide variety of biological conditions. To establish specific extracellular RNA molecules as clinically relevant biomarkers, reproducible recovery from biological samples and reliable measurements of the isolated RNA are paramount. Towards these ends, careful and rigorous comparisons of technical procedures are needed at all steps from sample handling to RNA isolation to RNA measurement protocols. In the investigations described in this methods paper, RT-qPCR was used to examine the apparent recovery of specific endogenous miRNAs and a spiked-in synthetic RNA from blood plasma samples. RNA was isolated using several widely used RNA isolation kits, with or without the addition of glycogen as a carrier. Kits examined included total RNA isolation systems that have been commercially available for several years and commonly adapted for extraction of biofluid RNA, as well as more recently introduced biofluids-specific RNA methods. Our conclusions include the following: some RNA isolation methods appear to be superior to others for the recovery of RNA from biological fluids; addition of a carrier molecule seems to be beneficial for some but not all isolation methods; and partially or fully quantitative recovery of RNA is observed from increasing volumes of plasma and cerebrospinal fluid.

  9. Viral factors reveal a role for REF/Aly in nuclear RNA stability.

    Science.gov (United States)

    Stubbs, Sarah H; Hunter, Olga V; Hoover, Ashley; Conrad, Nicholas K

    2012-04-01

    TREX is a conserved multiprotein complex that is necessary for efficient mRNA export to the cytoplasm. In Saccharomyces cerevisiae, the TREX complex is additionally implicated in RNA quality control pathways, but it is unclear whether this function is conserved in mammalian cells. The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein binds and recruits the TREX component REF/Aly to viral mRNAs. Here, we demonstrate that REF/Aly is recruited to the KSHV noncoding polyadenylated nuclear (PAN) RNA by ORF57. This recruitment correlates with ORF57-mediated stabilization of PAN RNA, suggesting that REF/Aly promotes nuclear RNA stability. Further supporting this idea, tethering REF/Aly to PAN RNA is sufficient to increase the nuclear abundance and half-life of PAN RNA but is not sufficient to promote its export. Interestingly, REF/Aly appears to protect the poly(A) tail from deadenylation, and REF/Aly-stabilized transcripts are further adenylated over time, consistent with previous reports linking poly(A) tail length with nuclear RNA surveillance. These studies show that REF/Aly can stabilize nuclear RNAs independently of their export and support a broader conservation of RNA quality control mechanisms from yeast to humans.

  10. [Efficacy of plasma exchange combined with fetal hepacyties on viral hepatitis gravis].

    Science.gov (United States)

    Zheng, X H; Tang, X P; Chen, J

    2001-10-28

    To evaluate the efficacy of mid-artificial liver support system (ALSS) on viral hepatitis gravis. One hundred and thirty eight patients with hepatitis gravis were treated with plasma exchange combined with fetal hepacyties, fifty six patients were treated with plasma exchange and other forty eight patients were treated with fetal hepacyties respectively. The liver function was examined in all patients before ALSS. The liver function, amino acid spectrum and cardiac muscle enzyme were examined before and after ALSS in patients treated with plasma exchange and fetal hepacyties. It showed that the survival rate of the patients treated with plasma exchange combined with fetal hepacyties was higher than that of the patients only treated with plasma exchange or fetal hepacyties (P viral hepatits gravis.

  11. Viral RNA levels and env variants in semen and tissues of mature male rhesus macaques infected with SIV by penile inoculation.

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    Francis Fieni

    Full Text Available HIV is shed in semen but the anatomic site of virus entry into the genital secretions is unknown. We determined viral RNA (vRNA levels and the envelope gene sequence in the SIVmac 251 viral populations in the genital tract and semen of 5 adult male rhesus monkeys (Macaca mulatta that were infected after experimental penile SIV infection. Paired blood and semen samples were collected from 1-9 weeks after infection and the monkeys were necropsied eleven weeks after infection. The axillary lymph nodes, testes, epididymis, prostate, and seminal vesicles were collected and vRNA levels and single-genome analysis of the SIVmac251 env variants was performed. At the time of semen collection, blood vRNA levels were between 3.09 and 7.85 log10 vRNA copies/ml plasma. SIV RNA was found in the axillary lymph nodes of all five monkeys and in 3 of 5 monkeys, all tissues examined were vRNA positive. In these 3 monkeys, vRNA levels (log10 SIVgag copies/ug of total tissue RNA in the axillary lymph node (6.48 ± 0.50 were significantly higher than in the genital tract tissues: testis (3.67 ± 2.16; p<0.05, epididymis (3.08 ± 1.19; p<0.0001, prostate (3.36 ± 1.30; p<0.01, and seminal vesicle (2.67 ± 1.50; p<0.0001. Comparison of the SIVmac251 env viral populations in blood plasma, systemic lymph node, and genital tract tissues was performed in two of the macaques. Visual inspection of the Neighbor-Joining phylograms revealed that in both animals, all the sequences were generally distributed evenly among all tissue compartments. Importantly, viral populations in the genital tissues were not distinct from those in the systemic tissues. Our findings demonstrate striking similarity in the viral populations in the blood and male genital tract tissues within 3 months of penile SIV transmission.

  12. SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5′UTR RNA

    Science.gov (United States)

    Han, Yang; Wang, Lvyin; Cui, Jin; Song, Yu; Luo, Zhen; Chen, Junbo; Xiong, Ying; Zhang, Qi; Liu, Fang; Ho, Wenzhe; Liu, Yingle; Wu, Jianguo

    2016-01-01

    ABSTRACT Enterovirus 71 (EV71) possesses a single-stranded positive RNA genome that contains a single open reading frame (ORF) flanked by a 5′ untranslated region (5′UTR) and a polyadenylated 3′UTR. Here, we demonstrated that EV71 activates the production of silent mating type information regulation 2 homolog 1 (SIRT1), a histone deacetylase (HDAC). EV71 further stimulates SIRT1 sumoylation and deacetylase activity, and enhances SIRT1 translocation from the nucleus to the cytoplasm. More interestingly, activated SIRT1 subsequently binds with the EV71 3Dpol protein (a viral RNA-dependent RNA polymerase, RdRp) to repress the acetylation and RdRp activity of 3Dpol, resulting in the attenuation of viral genome replication. Moreover, SIRT1 interacts with the cloverleaf structure of the EV71 RNA 5′UTR to inhibit viral RNA transcription, and binds to the internal ribosome entry site (IRES) of the EV71 5′UTR to attenuate viral RNA translation. Thus, EV71 stimulates SIRT1 production and activity, which in turn represses EV71 genome replication by inhibiting viral polymerase, and attenuates EV71 RNA transcription and translation by interfering with viral RNA. These results uncover a new function of SIRT1 and reveal a new mechanism underlying the regulation of EV71 replication. PMID:27875274

  13. Viral counterdefense on RNA silencing : analysis of RNA silencing suppressors from arthropod-borne negative strand RNA plant viruses

    NARCIS (Netherlands)

    Schnettler, E.

    2010-01-01

    This thesis describes that RNA silencing suppressor (RSS) proteins encoded by negative-stranded RNA plant viruses are able to interfere with different RNA silencing pathways in a variety of organisms by interacting with double stranded (ds)RNA molecules. These RSS proteins are able to counteract the

  14. Viral counterdefense on RNA silencing : analysis of RNA silencing suppressors from arthropod-borne negative strand RNA plant viruses

    NARCIS (Netherlands)

    Schnettler, E.

    2010-01-01

    This thesis describes that RNA silencing suppressor (RSS) proteins encoded by negative-stranded RNA plant viruses are able to interfere with different RNA silencing pathways in a variety of organisms by interacting with double stranded (ds)RNA molecules. These RSS proteins are able to counteract the

  15. Synthesis and Biological Evaluation of Brain-Specific Anti-RNA Viral Agents

    Science.gov (United States)

    1989-06-30

    TITLE (Include Securrty Clasification ) Synthesis and Biological Evaluation of Brain Specific Anti-RNA Viral Agents 12. PERSONAL. AUTHOR(S) Marcus E...are submicroscopic pathogens which depend on the cellular nucleic acid and protein synthesizing mechanisms of its host for propagation’𔃼 . In...general, viruses invade cells by first interacting at a recognizable surface protein , penetrating the cell membrane and subsequently releasing itself from a

  16. Role of the C terminus of Lassa virus L protein in viral mRNA synthesis.

    Science.gov (United States)

    Lehmann, Maria; Pahlmann, Meike; Jérôme, Hanna; Busch, Carola; Lelke, Michaela; Günther, Stephan

    2014-08-01

    The N terminus of arenavirus L protein contains an endonuclease presumably involved in "cap snatching." Here, we employed the Lassa virus replicon system to map other L protein sites that might be involved in this mechanism. Residues Phe-1979, Arg-2018, Phe-2071, Asp-2106, Trp-2173, Tyr-2179, Arg-2200, and Arg-2204 were important for viral mRNA synthesis but dispensable for genome replication. Thus, the C terminus of L protein is involved in the mRNA synthesis process, potentially by mediating cap binding.

  17. Characterization of viral siRNA populations in honey bee colony collapse disorder.

    Science.gov (United States)

    Chejanovsky, Nor; Ophir, Ron; Schwager, Michal Sharabi; Slabezki, Yossi; Grossman, Smadar; Cox-Foster, Diana

    2014-04-01

    Colony Collapse Disorder (CCD), a special case of collapse of honey bee colonies, has resulted in significant losses for beekeepers. CCD-colonies show abundance of pathogens which suggests that they have a weakened immune system. Since honey bee viruses are major players in colony collapse and given the important role of viral RNA interference (RNAi) in combating viral infections we investigated if CCD-colonies elicit an RNAi response. Deep-sequencing analysis of samples from CCD-colonies from US and Israel revealed abundant small interfering RNAs (siRNA) of 21-22 nucleotides perfectly matching the Israeli acute paralysis virus (IAPV), Kashmir virus and Deformed wing virus genomes. Israeli colonies showed high titers of IAPV and a conserved RNAi-pattern of matching the viral genome. That was also observed in sample analysis from colonies experimentally infected with IAPV. Our results suggest that CCD-colonies set out a siRNA response that is specific against predominant viruses associated with colony losses. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Global mRNA degradation during lytic gammaherpesvirus infection contributes to establishment of viral latency.

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    Justin M Richner

    2011-07-01

    Full Text Available During a lytic gammaherpesvirus infection, host gene expression is severely restricted by the global degradation and altered 3' end processing of mRNA. This host shutoff phenotype is orchestrated by the viral SOX protein, yet its functional significance to the viral lifecycle has not been elucidated, in part due to the multifunctional nature of SOX. Using an unbiased mutagenesis screen of the murine gammaherpesvirus 68 (MHV68 SOX homolog, we isolated a single amino acid point mutant that is selectively defective in host shutoff activity. Incorporation of this mutation into MHV68 yielded a virus with significantly reduced capacity for mRNA turnover. Unexpectedly, the MHV68 mutant showed little defect during the acute replication phase in the mouse lung. Instead, the virus exhibited attenuation at later stages of in vivo infections suggestive of defects in both trafficking and latency establishment. Specifically, mice intranasally infected with the host shutoff mutant accumulated to lower levels at 10 days post infection in the lymph nodes, failed to develop splenomegaly, and exhibited reduced viral DNA levels and a lower frequency of latently infected splenocytes. Decreased latency establishment was also observed upon infection via the intraperitoneal route. These results highlight for the first time the importance of global mRNA degradation during a gammaherpesvirus infection and link an exclusively lytic phenomenon with downstream latency establishment.

  19. Storage of cellular 5' mRNA caps in P bodies for viral cap-snatching.

    Science.gov (United States)

    Mir, M A; Duran, W A; Hjelle, B L; Ye, C; Panganiban, A T

    2008-12-09

    The minus strand and ambisense segmented RNA viruses include multiple important human pathogens and are divided into three families, the Orthomyxoviridae, the Bunyaviridae, and the Arenaviridae. These viruses all initiate viral transcription through the process of "cap-snatching," which involves the acquisition of capped 5' oligonucleotides from cellular mRNA. Hantaviruses are emerging pathogenic viruses of the Bunyaviridae family that replicate in the cytoplasm of infected cells. Cellular mRNAs can be actively translated in polysomes or physically sequestered in cytoplasmic processing bodies (P bodies) where they are degraded or stored for subsequent translation. Here we show that the hantavirus nucleocapsid protein binds with high affinity to the 5' cap of cellular mRNAs, protecting the 5' cap from degradation. We also show that the hantavirus nucleocapsid protein accumulates in P bodies, where it sequesters protected 5' caps. P bodies then serve as a pool of primers during the initiation of viral mRNA synthesis by the viral polymerase. We propose that minus strand segmented viruses replicating in the cytoplasm have co-opted the normal degradation machinery of P bodies for storage of cellular caps. Our data also indicate that modification of the cap-snatching model is warranted to include a role for the nucleocapsid protein in cap acquisition and storage.

  20. Systematic Review of the Performance of HIV Viral Load Technologies on Plasma Samples

    Science.gov (United States)

    Sollis, Kimberly A.; Smit, Pieter W.; Fiscus, Susan; Ford, Nathan; Vitoria, Marco; Essajee, Shaffiq; Barnett, David; Cheng, Ben; Crowe, Suzanne M.; Denny, Thomas; Landay, Alan; Stevens, Wendy; Habiyambere, Vincent; Perrins, Jos; Peeling, Rosanna W.

    2014-01-01

    Background Viral load (VL) monitoring is the standard of care in developing country settings for detecting HIV treatment failure. Since 2010 the World Health Organization has recommended a phase-in approach to VL monitoring in resource-limited settings. We conducted a systematic review of the accuracy and precision of HIV VL technologies for treatment monitoring. Methods and Findings A search of Medline and Embase was conducted for studies evaluating the accuracy or reproducibility of commercially available HIV VL assays. 37 studies were included for review including evaluations of the Amplicor Monitor HIV-1 v1.5 (n = 25), Cobas TaqMan v2.0 (n = 11), Abbott RealTime HIV-1 (n = 23), Versant HIV-1 RNA bDNA 3.0 (n = 15), Versant HIV-1 RNA kPCR 1.0 (n = 2), ExaVir Load v3 (n = 2), and NucliSens EasyQ v2.0 (n = 1). All currently available HIV VL assays are of sufficient sensitivity to detect plasma virus levels at a lower detection limit of 1,000 copies/mL. Bias data comparing the Abbott RealTime HIV-1, TaqMan v2.0 to the Amplicor Monitor v1.5 showed a tendency of the Abbott RealTime HIV-1 to under-estimate results while the TaqMan v2.0 overestimated VL counts. Compared to the Amplicor Monitor v1.5, 2–26% and 9–70% of results from the Versant bDNA 3.0 and Abbott RealTime HIV-1 differed by greater than 0.5log10. The average intra and inter-assay variation of the Abbott RealTime HIV-1 were 2.95% (range 2.0–5.1%) and 5.44% (range 1.17–30.00%) across the range of VL counts (2log10–7log10). Conclusions This review found that all currently available HIV VL assays are of sufficient sensitivity to detect plasma VL of 1,000 copies/mL as a threshold to initiate investigations of treatment adherence or possible treatment failure. Sources of variability between VL assays include differences in technology platform, plasma input volume, and ability to detect HIV-1 subtypes. Monitoring of individual patients should be performed on the same

  1. Systematic review of the performance of HIV viral load technologies on plasma samples.

    Directory of Open Access Journals (Sweden)

    Kimberly A Sollis

    Full Text Available BACKGROUND: Viral load (VL monitoring is the standard of care in developing country settings for detecting HIV treatment failure. Since 2010 the World Health Organization has recommended a phase-in approach to VL monitoring in resource-limited settings. We conducted a systematic review of the accuracy and precision of HIV VL technologies for treatment monitoring. METHODS AND FINDINGS: A search of Medline and Embase was conducted for studies evaluating the accuracy or reproducibility of commercially available HIV VL assays. 37 studies were included for review including evaluations of the Amplicor Monitor HIV-1 v1.5 (n = 25, Cobas TaqMan v2.0 (n = 11, Abbott RealTime HIV-1 (n = 23, Versant HIV-1 RNA bDNA 3.0 (n = 15, Versant HIV-1 RNA kPCR 1.0 (n = 2, ExaVir Load v3 (n = 2, and NucliSens EasyQ v2.0 (n = 1. All currently available HIV VL assays are of sufficient sensitivity to detect plasma virus levels at a lower detection limit of 1,000 copies/mL. Bias data comparing the Abbott RealTime HIV-1, TaqMan v2.0 to the Amplicor Monitor v1.5 showed a tendency of the Abbott RealTime HIV-1 to under-estimate results while the TaqMan v2.0 overestimated VL counts. Compared to the Amplicor Monitor v1.5, 2-26% and 9-70% of results from the Versant bDNA 3.0 and Abbott RealTime HIV-1 differed by greater than 0.5log10. The average intra and inter-assay variation of the Abbott RealTime HIV-1 were 2.95% (range 2.0-5.1% and 5.44% (range 1.17-30.00% across the range of VL counts (2log10-7log10. CONCLUSIONS: This review found that all currently available HIV VL assays are of sufficient sensitivity to detect plasma VL of 1,000 copies/mL as a threshold to initiate investigations of treatment adherence or possible treatment failure. Sources of variability between VL assays include differences in technology platform, plasma input volume, and ability to detect HIV-1 subtypes. Monitoring of individual patients should be performed on the same

  2. Prediction of viral microRNA precursors based on human microRNA precursor sequence and structural features.

    Science.gov (United States)

    Kumar, Shiva; Ansari, Faraz A; Scaria, Vinod

    2009-08-20

    MicroRNAs (small approximately 22 nucleotide long non-coding endogenous RNAs) have recently attracted immense attention as critical regulators of gene expression in multi-cellular eukaryotes, especially in humans. Recent studies have proved that viruses also express microRNAs, which are thought to contribute to the intricate mechanisms of host-pathogen interactions. Computational predictions have greatly accelerated the discovery of microRNAs. However, most of these widely used tools are dependent on structural features and sequence conservation which limits their use in discovering novel virus expressed microRNAs and non-conserved eukaryotic microRNAs. In this work an efficient prediction method is developed based on the hypothesis that sequence and structure features which discriminate between host microRNA precursor hairpins and pseudo microRNAs are shared by viral microRNA as they depend on host machinery for the processing of microRNA precursors. The proposed method has been found to be more efficient than recently reported ab-initio methods for predicting viral microRNAs and microRNAs expressed by mammals.

  3. Analysis of correlation between cerebrospinal fluid and plasma HIV-1 RNA levels in patients with neurological opportunistic diseases

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    Paulo Pereira Christo

    2011-08-01

    Full Text Available The question of whether HIV-1 RNA in cerebrospinal fluid (CSF is derived from viral replication in the central nervous system or simply reflects the transit of infected lymphocytes from the blood compartment has long been a matter of debate. Some studies found no correlation between CSF and plasma viral load, whereas others did. The lack of a correlation between the two compartments suggests that the presence of HIV-1 RNA is not simply due to the passive passage of the virus from blood to CSF but rather due to intrathecal replication. To evaluate the correlation between plasma and CSF HIV-1 RNA levels and to identify situations in which there is no correlation between the two compartments, seventy patients were prospectively studied. The association between CSF and plasma viral load was evaluated in the total population and in subgroups of patients with similar characteristics. A correlation between the CSF and plasma compartments was observed for patients undergoing highly active antiretroviral therapy (HAART, those with a CD4 T lymphocyte count lower than 200 cells/mm³, and those with increased CSF protein content. On the other hand, no correlation was observed for patients without adequate virological control, who had a CD4 count higher than 200 cells/mm³ and who did not use HAART. The correlation between the two compartments observed in some patients suggests that CSF HIV-1 RNA levels may reflect plasma levels in these subjects. In contrast, the lack of a correlation between the two compartments in patients who were not on HAART and who had normal CSF proteins and a poor virological control possibly indicates compartmentalization of the virus in CSF and, consequently, plasma-independent intrathecal viral replication.

  4. Anti-Japanese-encephalitis-viral effects of kaempferol and daidzin and their RNA-binding characteristics.

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    Ting Zhang

    Full Text Available BACKGROUND: New therapeutic tools and molecular targets are needed for treatment of Japanese encephalitis virus (JEV infections. JEV requires an α-1 translational frameshift to synthesize the NS1' protein required for viral neuroinvasiveness. Several flavonoids have been shown to possess antiviral activity in vitro against a wide spectrum of viruses. To date, the antiviral activities of flavonol kaempferol (Kae and isoflavonoid daidzin (Dai against JEV have not been described. METHODOLOGY/PRINCIPAL FINDINGS: The 50% cytotoxic concentration (CC(50 and 50% effective concentration (EC(50 against JEV were investigated in BHK21 cells by MTS reduction. Activity against viral genomic RNA and proteins was measured by real-time RT-PCR and western blotting. The frameshift site RNA-binding characterization was also determined by electrospray ionization mass spectrometry, isothermal titration calorimetry and autodocking analysis. EC(50 values of Kae and Dai were 12.6 and 25.9 µM against JEV in cells pretreated before infection, whereas in cells infected before treatment, EC(50 was 21.5 and 40.4 µM, respectively. Kae exhibited more potent activity against JEV and RNA binding in cells following internalization through direct inhibition of viral replication and protein expression, indicating that its antiviral activity was principally due to direct virucidal effects. The JEV frameshift site RNA (fsRNA was selected as a target for assaying Kae and Dai. ITC of fsRNA revealed an apparent K(b value for Kae that was nine fold stronger than that for Dai. This binding was confirmed and localized to the RNA using ESI-MS and autodock analysis. Kae could form non-covalent complexes with fsRNA more easily than Dai could. CONCLUSIONS/SIGNIFICANCE: Kae demonstrates more potent antiviral activity against JEV than does Dai. The mode of action of Kae as an anti-JEV agent seems to be related to its ability to inactivate virus by binding with JEV fsRNA.

  5. The proteasomal Rpn11 metalloprotease suppresses tombusvirus RNA recombination and promotes viral replication via facilitating assembly of the viral replicase complex.

    Science.gov (United States)

    Prasanth, K Reddisiva; Barajas, Daniel; Nagy, Peter D

    2015-03-01

    RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92(pol) replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role

  6. Strategies for ocular siRNA delivery: Potential and limitations of non-viral nanocarriers

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    Thakur Ajit

    2012-06-01

    Full Text Available Abstract Controlling gene expression via small interfering RNA (siRNA has opened the doors to a plethora of therapeutic possibilities, with many currently in the pipelines of drug development for various ocular diseases. Despite the potential of siRNA technologies, barriers to intracellular delivery significantly limit their clinical efficacy. However, recent progress in the field of drug delivery strongly suggests that targeted manipulation of gene expression via siRNA delivered through nanocarriers can have an enormous impact on improving therapeutic outcomes for ophthalmic applications. Particularly, synthetic nanocarriers have demonstrated their suitability as a customizable multifunctional platform for the targeted intracellular delivery of siRNA and other hydrophilic and hydrophobic drugs in ocular applications. We predict that synthetic nanocarriers will simultaneously increase drug bioavailability, while reducing side effects and the need for repeated intraocular injections. This review will discuss the recent advances in ocular siRNA delivery via non-viral nanocarriers and the potential and limitations of various strategies for the development of a ‘universal’ siRNA delivery system for clinical applications.

  7. Strategies for ocular siRNA delivery: Potential and limitations of non-viral nanocarriers.

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    Thakur, Ajit; Fitzpatrick, Scott; Zaman, Abeyat; Kugathasan, Kapilan; Muirhead, Ben; Hortelano, Gonzalo; Sheardown, Heather

    2012-06-11

    Controlling gene expression via small interfering RNA (siRNA) has opened the doors to a plethora of therapeutic possibilities, with many currently in the pipelines of drug development for various ocular diseases. Despite the potential of siRNA technologies, barriers to intracellular delivery significantly limit their clinical efficacy. However, recent progress in the field of drug delivery strongly suggests that targeted manipulation of gene expression via siRNA delivered through nanocarriers can have an enormous impact on improving therapeutic outcomes for ophthalmic applications. Particularly, synthetic nanocarriers have demonstrated their suitability as a customizable multifunctional platform for the targeted intracellular delivery of siRNA and other hydrophilic and hydrophobic drugs in ocular applications. We predict that synthetic nanocarriers will simultaneously increase drug bioavailability, while reducing side effects and the need for repeated intraocular injections. This review will discuss the recent advances in ocular siRNA delivery via non-viral nanocarriers and the potential and limitations of various strategies for the development of a 'universal' siRNA delivery system for clinical applications.

  8. RNA aptamers inhibit the growth of the fish pathogen viral hemorrhagic septicemia virus (VHSV).

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    Punnarak, Porntep; Santos, Mudjekeewis D; Hwang, Seong Don; Kondo, Hidehiro; Hirono, Ikuo; Kikuchi, Yo; Aoki, Takashi

    2012-12-01

    Viral hemorrhagic septicemia virus (VHSV) is a serious disease impacting wild and cultured fish worldwide. Hence, an effective therapeutic method against VHSV infection needs to be developed. Aptamer technology is a new and promising method for diagnostics and therapeutics. It revolves around the use of an aptamer molecule, an artificial ligand (nucleic acid or protein), which has the capacity to recognize target molecules with high affinity and specificity. Here, we aimed at selecting RNA aptamers that can specifically bind to and inhibit the growth of a strain of fish VHSV both in vitro and in vivo. Three VHSV-specific RNA aptamers (F1, F2, and C6) were selected from a pool of artificially and randomly produced oligonucleotides using systematic evolution of ligands by exponential enrichment. The three RNA aptamers showed obvious binding to VHSV in an electrophoretic mobility shift assay but not to other tested viruses. The RNA aptamers were tested for their ability to inhibit VHSV in vitro using hirame natural embryo (HINAE) cells. Cytopathic effect and plaque assays showed that all aptamers inhibited the growth of VHSV in HINAE cells. In vivo tests using RNA aptamers produced by Rhodovulum sulfidophilum showed that extracellular RNA aptamers inhibited VHSV infection in Japanese flounder. These results suggest that the RNA aptamers are a useful tool for protection against VHSV infection in Japanese flounder.

  9. Using double-stranded RNA to prevent in vitro and in vivo viral infections by recombinant baculovirus.

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    Valdes, Victor Julian; Sampieri, Alicia; Sepulveda, Jorge; Vaca, Luis

    2003-05-23

    Introduction of double-stranded RNA (dsRNA) into a wide variety of cells and organisms results in post-transcriptional depletion of the homologue endogenous mRNA. This well-preserved phenomenon known as RNA interference (RNAi) is present in evolutionarily diverse organisms such as plants, fungi, insects, metazoans, and mammals. Because the identification of the targeted mRNA by the RNAi machinery depends upon Watson-Crick base-pairing interactions, RNAi can be exquisitely specific. We took advantage of this powerful and flexible technique to demonstrate that selective silencing of genes essential for viral propagation prevents in vitro and in vivo viral infection. Using the baculovirus Autographa californica, a rapidly replicating and highly cytolytic double-stranded DNA virus that infects many different insect species, we show for the first time that introduction of dsRNA from gp64 and ie1, two genes essential for baculovirus propagation, results in prevention of viral infection in vitro and in vivo. This is the first report demonstrating the use of RNAi to inhibit a viral infection in animals. This inhibition was specific, because dsRNA from the polyhedrin promoter (used as control) or unrelated dsRNAs did not affect the time course of viral infection. The most relevant consequences from the present study are: 1) RNAi offers a rapid and efficient way to interfere with viral genes to assess the role of specific proteins in viral function and 2) using RNAi to interfere with viral genes essential for cell infection may provide a powerful therapeutic tool for the treatment of viral infections.

  10. microRNA control of interferons and interferon induced anti-viral activity.

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    Sedger, Lisa M

    2013-12-01

    Interferons (IFNs) are cytokines that are spontaneously produced in response to virus infection. They act by binding to IFN-receptors (IFN-R), which trigger JAK/STAT cell signalling and the subsequent induction of hundreds of IFN-inducible genes, including both protein-coding and microRNA genes. IFN-induced genes then act synergistically to prevent virus replication and create an anti-viral state. miRNA are therefore integral to the innate response to virus infection and are important components of IFN-mediated biology. On the other hand viruses also encode miRNAs that in some cases interfere directly with the IFN response to infection. This review summarizes the important roles of miRNAs in virus infection acting both as IFN-stimulated anti-viral molecules and as critical regulators of IFNs and IFN-stimulated genes. It also highlights how recent knowledge in RNA editing influence miRNA control of virus infection.

  11. Expanding access to HIV viral load testing: a systematic review of RNA stability in EDTA tubes and PPT beyond current time and temperature thresholds.

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    Kimberly Bonner

    Full Text Available BACKGROUND: HIV viral load (VL testing is the gold standard for antiretroviral treatment monitoring, but many barriers exist to VL testing in resource-limited settings, including storage and transport limitations for whole blood and plasma. Data from various studies indicate that HIV RNA is stable beyond current recommendations. We conducted a systematic review to assess stability data of HIV RNA in whole blood and plasma across times and temperatures. METHODS AND FINDINGS: Using a pre-defined protocol, five databases were searched for studies where blood samples from HIV patients were stored at time and temperature points that exceeded manufacturer recommendations. RNA stability, the primary outcome, was measured by the difference in means compared to samples stored within established thresholds. RNA stability was defined as ≤0.5 log degradation. The search identified 10,716 titles, of which nine full-text articles were included for review. HIV RNA maintained stability in EDTA whole blood and plasma at all measured time points up to 168 hours when stored at 4°C, while stability was detected at 72 hours (95% confidence in whole blood at 25°C, with data points before and beyond 72 hours suggesting stability but not reaching statistical significance. For EDTA plasma stored at 30°C, stability was maintained up to 48 hours (95% confidence, with OLS linear regression estimates up to 127 hours, suggesting stability. Overall, quality of studies was moderate. Limitations included small sample sizes, few studies meeting inclusion criteria, and no studies examining RNA stability in low viremia (<3,000 copies/mL environments. CONCLUSIONS: Whole blood and plasma samples in EDTA may remain stable under conditions exceeding current manufacturer recommendations for HIV VL testing. However, given the limited number of studies addressing this question, especially at low levels of viremia, additional evaluations on HIV RNA stability in EDTA tubes and PPT in

  12. Polymorphism of viral dsRNA in Xanthophyllomyces dendrorhous strains isolated from different geographic areas

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    Libkind Diego

    2009-10-01

    Full Text Available Abstract Background Strains of the astaxanthin producing yeast Xanthophyllomyces dendrorhous have been isolated from different cold regions around the earth, and the presence of double stranded RNA (dsRNA elements was described in some isolates. This kind of viruses is widely distributed among yeasts and filamentous fungi and, although generally are cryptic in function, their studies have been a key factor in the knowledge of important fungi. In this work, the characterization and genetic relationships among dsRNA elements were determined in strains representatives of almost all regions of the earth where X. dendrorhous have been isolated. Results Almost all strains of X. dendrorhous analyzed carry one, two or four dsRNA elements, of molecular sizes in the range from 0.8 to 5.0 kb. Different dsRNA-patterns were observed in strains with different geographic origin, being L1 (5.0 kb the common dsRNA element. By hybridization assays a high genomic polymorphism was observed among L1 dsRNAs of different X. dendrorhous strains. Contrary, hybridization was observed between L1 and L2 dsRNAs of strains from same or different regions, while the dsRNA elements of minor sizes (M, S1, and S2 present in several strains did not show hybridization with neither L1 or L2 dsRNAs. Along the growth curve of UCD 67-385 (harboring four dsRNAs an increase of L2 relative to L1 dsRNA was observed, whiles the S1/L1 ratio remains constant, as well as the M/L1 ratio of Patagonian strain. Strains cured of S2 dsRNA were obtained by treatment with anisomycin, and comparison of its dsRNA contents with uncured strain, revealed an increase of L1 dsRNA while the L2 and S1 dsRNA remain unaltered. Conclusion The dsRNA elements of X. dendrorhous are highly variable in size and sequence, and the dsRNA pattern is specific to the geographic region of isolation. Each L1 and L2 dsRNA are viral elements able to self replicate and to coexist into a cell, and L1 and S2 dsRNAs elements could

  13. Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export.

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    Behrens, Ryan T; Aligeti, Mounavya; Pocock, Ginger M; Higgins, Christina A; Sherer, Nathan M

    2017-02-01

    HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding.

  14. Which adherence measure - self-report, clinician recorded or pharmacy refill - is best able to predict detectable viral load in a public ART programme without routine plasma viral load monitoring?

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    Mekuria, Legese A; Prins, Jan M; Yalew, Alemayehu W; Sprangers, Mirjam A G; Nieuwkerk, Pythia T

    2016-07-01

    Combination antiretroviral therapy (cART) suppresses viral replication to an undetectable level if a sufficiently high level of adherence is achieved. We investigated which adherence measurement best distinguishes between patients with and without detectable viral load in a public ART programme without routine plasma viral load monitoring. We randomly selected 870 patients who started cART between May 2009 and April 2012 in 10 healthcare facilities in Addis Ababa, Ethiopia. Six hundred and sixty-four (76.3%) patients who were retained in HIV care and were receiving cART for at least 6 months were included and 642 had their plasma HIV-1 RNA concentration measured. Patients' adherence to cART was assessed according to self-report, clinician recorded and pharmacy refill measures. Multivariate logistic regression model was fitted to identify the predictors of detectable viremia. Model accuracy was evaluated by computing the area under the receiver operating characteristic (ROC) curve. A total of 9.2% and 5.5% of the 642 patients had a detectable viral load of ≥40 and ≥400 RNA copies/ml, respectively. In the multivariate analyses, younger age, lower CD4 cell count at cART initiation, being illiterate and widowed, and each of the adherence measures were significantly and independently predictive of having ≥400 RNA copies/ml. The ROC curve showed that these variables altogether had a likelihood of more than 80% to distinguish patients with a plasma viral load of ≥400 RNA copies/ml from those without. Adherence to cART was remarkably high. Self-report, clinician recorded and pharmacy refill non-adherence were all significantly predictive of detectable viremia. The choice for one of these methods to detect non-adherence and predict a detectable viral load can therefore be based on what is most practical in a particular setting. © 2016 John Wiley & Sons Ltd.

  15. Cyclophilin A binds to the viral RNA and replication proteins, resulting in inhibition of tombusviral replicase assembly.

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    Kovalev, Nikolay; Nagy, Peter D

    2013-12-01

    Replication of plus-stranded RNA viruses is greatly affected by numerous host-encoded proteins that act as restriction factors. Cyclophilins, which are a large family of cellular prolyl isomerases, have been found to inhibit Tomato bushy stunt tombusvirus (TBSV) replication in a Saccharomyces cerevisiae model based on genome-wide screens and global proteomics approaches. In this report, we further characterize single-domain cyclophilins, including the mammalian cyclophilin A and plant Roc1 and Roc2, which are orthologs of the yeast Cpr1p cyclophilin, a known inhibitor of TBSV replication in yeast. We found that recombinant CypA, Roc1, and Roc2 strongly inhibited TBSV replication in a cell-free replication assay. Additional in vitro studies revealed that CypA, Roc1, and Roc2 cyclophilins bound to the viral replication proteins, and CypA and Roc1 also bound to the viral RNA. These interactions led to inhibition of viral RNA recruitment, the assembly of the viral replicase complex, and viral RNA synthesis. A catalytically inactive mutant of CypA was also able to inhibit TBSV replication in vitro due to binding to the replication proteins and the viral RNA. Overexpression of CypA and its mutant in yeast or plant leaves led to inhibition of tombusvirus replication, confirming that CypA is a restriction factor for TBSV. Overall, the current work has revealed a regulatory role for the cytosolic single-domain Cpr1-like cyclophilins in RNA virus replication.

  16. Comparing the MicroRNA spectrum between serum and plasma.

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    Kai Wang

    Full Text Available MicroRNAs (miRNAs are small, non-coding RNAs that regulate various biological processes, primarily through interaction with messenger RNAs. The levels of specific, circulating miRNAs in blood have been shown to associate with various pathological conditions including cancers. These miRNAs have great potential as biomarkers for various pathophysiological conditions. In this study we focused on different sample types' effects on the spectrum of circulating miRNA in blood. Using serum and corresponding plasma samples from the same individuals, we observed higher miRNA concentrations in serum samples compared to the corresponding plasma samples. The difference between serum and plasma miRNA concentration showed some associations with miRNA from platelets, which may indicate that the coagulation process may affect the spectrum of extracellular miRNA in blood. Several miRNAs also showed platform dependent variations in measurements. Our results suggest that there are a number of factors that might affect the measurement of circulating miRNA concentration. Caution must be taken when comparing miRNA data generated from different sample types or measurement platforms.

  17. RNA helicase DDX3: at the crossroad of viral replication and antiviral immunity.

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    Valiente-Echeverría, Fernando; Hermoso, Marcela A; Soto-Rifo, Ricardo

    2015-09-01

    Asp-Glu-Ala-Asp (DEAD)-box polypeptide 3, or DDX3, belongs to the DEAD-box family of ATP-dependent RNA helicases and is known to play different roles in RNA metabolism ranging from transcription to nuclear export, translation, and assembly of stress granules. In addition, there is growing evidence that DDX3 is a component of the innate immune response against viral infections. As such, DDX3 has been shown to play roles both upstream and downstream of I-kappa beta kinase ε (IKKε)/TANK-binding kinase 1, leading to IFN-β production. Interestingly, several RNA viruses, including human threats such as HIV-1 and hepatitis C virus, hijack DDX3 to accomplish various steps of their replication cycles. Thus, it seems that viruses have evolved to exploit DDX3's functions while threatening the innate immune response. Understanding this interesting dichotomy in DDX3 function will help us not only to improve our knowledge of virus-host interactions but also to develop novel antiviral drugs targeting the multifaceted roles of DDX3 in viral replication.

  18. Cooler temperatures destabilize RNA interference and increase susceptibility of disease vector mosquitoes to viral infection.

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    Zach N Adelman

    Full Text Available BACKGROUND: The impact of global climate change on the transmission dynamics of infectious diseases is the subject of extensive debate. The transmission of mosquito-borne viral diseases is particularly complex, with climatic variables directly affecting many parameters associated with the prevalence of disease vectors. While evidence shows that warmer temperatures often decrease the extrinsic incubation period of an arthropod-borne virus (arbovirus, exposure to cooler temperatures often predisposes disease vector mosquitoes to higher infection rates. RNA interference (RNAi pathways are essential to antiviral immunity in the mosquito; however, few experiments have explored the effects of temperature on the RNAi machinery. METHODOLOGY/PRINCIPAL FINDINGS: We utilized transgenic "sensor" strains of Aedes aegypti to examine the role of temperature on RNA silencing. These "sensor" strains express EGFP only when RNAi is inhibited; for example, after knockdown of the effector proteins Dicer-2 (DCR-2 or Argonaute-2 (AGO-2. We observed an increase in EGFP expression in transgenic sensor mosquitoes reared at 18°C as compared with 28°C. Changes in expression were dependent on the presence of an inverted repeat with homology to a portion of the EGFP sequence, as transgenic strains lacking this sequence, the double stranded RNA (dsRNA trigger for RNAi, showed no change in EGFP expression when reared at 18°C. Sequencing small RNAs in sensor mosquitoes reared at low temperature revealed normal processing of dsRNA substrates, suggesting the observed deficiency in RNAi occurs downstream of DCR-2. Rearing at cooler temperatures also predisposed mosquitoes to higher levels of infection with both chikungunya and yellow fever viruses. CONCLUSIONS/SIGNIFICANCE: This data suggest that microclimates, such as those present in mosquito breeding sites, as well as more general climactic variables may influence the dynamics of mosquito-borne viral diseases by affecting

  19. Discovery of novel dsRNA viral sequences by in silico cloning and implications for viral diversity, host range and evolution.

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    Huiquan Liu

    Full Text Available Genome sequence of viruses can contribute greatly to the study of viral evolution, diversity and the interaction between viruses and hosts. Traditional molecular cloning methods for obtaining RNA viral genomes are time-consuming and often difficult because many viruses occur in extremely low titers. DsRNA viruses in the families, Partitiviridae, Totiviridae, Endornaviridae, Chrysoviridae, and other related unclassified dsRNA viruses are generally associated with symptomless or persistent infections of their hosts. These characteristics indicate that samples or materials derived from eukaryotic organisms used to construct cDNA libraries and EST sequencing might carry these viruses, which were not easily detected by the researchers. Therefore, the EST databases may include numerous unknown viral sequences. In this study, we performed in silico cloning, a procedure for obtaining full or partial cDNA sequence of a gene by bioinformatics analysis, using known dsRNA viral sequences as queries to search against NCBI Expressed Sequence Tag (EST database. From this analysis, we obtained 119 novel virus-like sequences related to members of the families, Endornaviridae, Chrysoviridae, Partitiviridae, and Totiviridae. Many of them were identified in cDNA libraries of eukaryotic lineages, which were not known to be hosts for these viruses. Furthermore, comprehensive phylogenetic analysis of these newly discovered virus-like sequences with known dsRNA viruses revealed that these dsRNA viruses may have co-evolved with respective host supergroups over a long evolutionary time while potential horizontal transmissions of viruses between different host supergroups also is possible. We also found that some of the plant partitiviruses may have originated from fungal viruses by horizontal transmissions. These findings extend our knowledge of the diversity and possible host range of dsRNA viruses and offer insight into the origin and evolution of relevant viruses with

  20. Assessing an improved protocol for plasma microRNA extraction.

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    Inés Moret

    Full Text Available The first step in biomarkers discovery is to identify the best protocols for their purification and analysis. This issue is critical when considering peripheral blood samples (plasma and serum that are clinically interesting but meet several methodological problems, mainly complexity and low biomarker concentration. Analysis of small molecules, such as circulating microRNAs, should overcome these disadvantages. The present study describes an optimal RNA extraction method of microRNAs from human plasma samples. Different reagents and commercially available kits have been analyzed, identifying also the best pre-analytical conditions for plasma isolation. Between all of them, the column-based approaches were shown to be the most effective. In this context, miRNeasy Serum/Plasma Kit (from Qiagen rendered more concentrated RNA, that was better suited for microarrays studies and did not require extra purification steps for sample concentration and purification than phenol based extraction methods. We also present evidences that the addition of low doses of an RNA carrier before starting the extraction process improves microRNA purification while an already published carrier dose can result in significant bias over microRNA profiles. Quality controls for best protocol selection were developed by spectrophotometry measurement of contaminants and microfluidics electrophoresis (Agilent 2100 Bioanalyzer for RNA integrity. Selected donor and patient plasma samples and matched biopsies were tested by Affymetrix microarray technology to compare differentially expressed microRNAs. In summary, this study defines an optimized protocol for microRNA purification from human blood samples, increasing the performance of assays and shedding light over the best way to discover and use these biomarkers in clinical practice.

  1. Assessing an improved protocol for plasma microRNA extraction.

    Science.gov (United States)

    Moret, Inés; Sánchez-Izquierdo, Dolors; Iborra, Marisa; Tortosa, Luis; Navarro-Puche, Ana; Nos, Pilar; Cervera, José; Beltrán, Belén

    2013-01-01

    The first step in biomarkers discovery is to identify the best protocols for their purification and analysis. This issue is critical when considering peripheral blood samples (plasma and serum) that are clinically interesting but meet several methodological problems, mainly complexity and low biomarker concentration. Analysis of small molecules, such as circulating microRNAs, should overcome these disadvantages. The present study describes an optimal RNA extraction method of microRNAs from human plasma samples. Different reagents and commercially available kits have been analyzed, identifying also the best pre-analytical conditions for plasma isolation. Between all of them, the column-based approaches were shown to be the most effective. In this context, miRNeasy Serum/Plasma Kit (from Qiagen) rendered more concentrated RNA, that was better suited for microarrays studies and did not require extra purification steps for sample concentration and purification than phenol based extraction methods. We also present evidences that the addition of low doses of an RNA carrier before starting the extraction process improves microRNA purification while an already published carrier dose can result in significant bias over microRNA profiles. Quality controls for best protocol selection were developed by spectrophotometry measurement of contaminants and microfluidics electrophoresis (Agilent 2100 Bioanalyzer) for RNA integrity. Selected donor and patient plasma samples and matched biopsies were tested by Affymetrix microarray technology to compare differentially expressed microRNAs. In summary, this study defines an optimized protocol for microRNA purification from human blood samples, increasing the performance of assays and shedding light over the best way to discover and use these biomarkers in clinical practice.

  2. Differential expression of miRNA-423-5p in serum from cattle challenged with bovine viral diarrhea virus

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    Bovine viral diarrhea virus (BVDV) is an RNA virus that causes respiratory disease in cattle. MicroRNAs have been proposed as indicators of exposure to respiratory pathogens. However, microRNA profiles in cattle exposed to BVDV are currently nonexistent and few studies have been reported; therefore,...

  3. Significant impact of non-B HIV-1 variants genetic diversity in Gabon on plasma HIV-1 RNA quantitation.

    Science.gov (United States)

    Mouinga-Ondémé, Augustin; Mabika-Mabika, Arsène; Alalade, Patrick; Mongo, Arnaud Delis; Sica, Jeanne; Liégeois, Florian; Rouet, François

    2014-01-01

    Evaluations of HIV-1 RNA viral load assays are lacking in Central Africa. The main objective of our study was to assess the reliability of HIV-1 RNA results obtained with three different assays for samples collected in Gabon. A total of 137 plasma specimens were assessed for HIV-1 RNA using the Abbott RealTime HIV-1® and Nuclisens HIV-1 EasyQ® version 2.0 assays. It included HIV-1 non-B samples (n = 113) representing six subtypes, 10 CRFs and 18 URFs from patients infected with HIV-1 and treated with antiretrovirals that were found HIV-1 RNA positive (≥300 copies/ml) with the Generic HIV viral load® assay; and samples (n = 24) from untreated individuals infected with HIV-1 but showing undetectable (<300 copies/ml) results with the Biocentric kit. For samples found positive with the Generic HIV viral load® test, correlation coefficients obtained between the three techniques were relatively low (R = 0.65 between Generic HIV viral load® and Abbott RealTime HIV-1®, 0.50 between Generic HIV viral load® and Nuclisens HIV-1 EasyQ®, and 0.66 between Abbott RealTime HIV-1® and Nuclisens HIV-1 EasyQ®). Discrepancies by at least one log10 were obtained for 19.6%, 33.7%, and 20% of samples, respectively, irrespective of genotype. Most of samples (22/24) from untreated study patients, found negative with the Biocentric kit, were also found negative with the two other techniques. In Central Africa, HIV-1 genetic diversity remains challenging for viral load testing. Further studies are required in the same area to confirm the presence of HIV-1 strains that are not amplified with at least two different viral load assays.

  4. Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus Replication

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    Stacia L. Phillips

    2016-01-01

    Full Text Available Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches.

  5. Effects of medical male circumcision (MC on plasma HIV viral load in HIV+ HAART naive men; Rakai, Uganda.

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    Godfrey Kigozi

    Full Text Available BACKGROUND: Medical male circumcision (MC of HIV-infected men may increase plasma HIV viral load and place female partners at risk of infection. We assessed the effect of MC on plasma HIV viral load in HIV-infected men in Rakai, Uganda. METHODS: 195 consenting HIV-positive, HAART naïve men aged 12 and above provided blood for plasma HIV viral load testing before surgery and weekly for six weeks and at 2 and 3 months post surgery. Data were also collected on baseline social demographic characteristics and CD4 counts. Change in log10 plasma viral load between baseline and follow-up visits was estimated using paired t tests and multivariate generalized estimating equation (GEE. RESULTS: Of the 195 men, 129 had a CD4 count ≧ 350 and 66 had CD4 <350 cells/mm3. Men with CD4 counts <350 had higher baseline mean log10 plasma viral load than those with CD4 counts ≧ 350 cells/mm3 (4.715 vs 4.217 cps/mL, respectively, p = 0.0005. Compared to baseline, there was no statistically significant increase in post-MC HIV plasma viral loads irrespective of CD4. Multivariate analysis showed that higher baseline log10 plasma viral load was significantly associated with reduction in mean log10 plasma viral load following MC (coef.  = -0.134, p<0.001. CONCLUSION: We observed no increase in plasma HIV viral load following MC in HIV-infected, HAART naïve men.

  6. A cucumber mosaic virus (CMV) RNA 1 transgene mediates suppression of the homologous viral RNA 1 constitutively and prevents CMV entry into the phloem.

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    Canto, T; Palukaitis, P

    2001-10-01

    Resistance to Cucumber mosaic virus (CMV) in tobacco lines transformed with CMV RNA 1 is characterized by reduced virus accumulation in the inoculated leaf, with specific suppression of accumulation of the homologous viral RNA 1, and by the absence of systemic infection. We show that the suppression of viral RNA 1 occurs in protoplasts from resistant transgenic plants and therefore is not due to a host response activated by the cell-to-cell spread of virus. In contrast, suppression of Tobacco rattle virus vectors carrying CMV RNA 1 sequences did not occur in protoplasts from resistant plants. Furthermore, steady-state levels of transgene mRNA 1 were higher in resistant than in susceptible lines. Thus, the data indicate that sequence homology is not sufficient to induce suppression. Grafting experiments using transgenic resistant or susceptible rootstocks and scions demonstrated that the resistance mechanism exhibited an additional barrier to phloem entry, preventing CMV from moving a long distance in resistant plants. On the other hand, virus from susceptible rootstocks could systemically infect grafted resistant scions via the phloem. Analysis of viral RNA accumulation in the infected scions showed that the mechanism that suppresses the accumulation of viral RNA 1 at the single-cell level was overcome. The data indicate that this transgene-mediated systemic resistance probably is not based on a posttranscriptional gene-silencing mechanism.

  7. Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region

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    Bienz, K.; Egger, D.; Troxler, M.; Pasamontes, L. (Univ. of Basel (Switzerland))

    1990-03-01

    Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but did not change their association with viral RNA. The findings allow the conclusion that the protein 2C or 2C-containing precursor(s) is responsible for the attachment of the viral RNA to the vesicular membrane and for the spatial organization of the replication complex necessary for its proper functioning in viral transcription. A model for the structure of the viral replication complex and for the function of the 2C-containing P2 protein(s) and the vesicular membranes is proposed.

  8. The Lsm1-7-Pat1 complex promotes viral RNA translation and replication by differential mechanisms.

    Science.gov (United States)

    Jungfleisch, Jennifer; Chowdhury, Ashis; Alves-Rodrigues, Isabel; Tharun, Sundaresan; Díez, Juana

    2015-08-01

    The Lsm1-7-Pat1 complex binds to the 3' end of cellular mRNAs and promotes 3' end protection and 5'-3' decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes.

  9. Viral evasion of a bacterial suicide system by RNA-based molecular mimicry enables infectious altruism.

    Science.gov (United States)

    Blower, Tim R; Evans, Terry J; Przybilski, Rita; Fineran, Peter C; Salmond, George P C

    2012-01-01

    Abortive infection, during which an infected bacterial cell commits altruistic suicide to destroy the replicating bacteriophage and protect the clonal population, can be mediated by toxin-antitoxin systems such as the Type III protein-RNA toxin-antitoxin system, ToxIN. A flagellum-dependent bacteriophage of the Myoviridae, ΦTE, evolved rare mutants that "escaped" ToxIN-mediated abortive infection within Pectobacterium atrosepticum. Wild-type ΦTE encoded a short sequence similar to the repetitive nucleotide sequence of the RNA antitoxin, ToxI, from ToxIN. The ΦTE escape mutants had expanded the number of these "pseudo-ToxI" genetic repeats and, in one case, an escape phage had "hijacked" ToxI from the plasmid-borne toxIN locus, through recombination. Expression of the pseudo-ToxI repeats during ΦTE infection allowed the phage to replicate, unaffected by ToxIN, through RNA-based molecular mimicry. This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system. Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.

  10. Viral evasion of a bacterial suicide system by RNA-based molecular mimicry enables infectious altruism.

    Directory of Open Access Journals (Sweden)

    Tim R Blower

    Full Text Available Abortive infection, during which an infected bacterial cell commits altruistic suicide to destroy the replicating bacteriophage and protect the clonal population, can be mediated by toxin-antitoxin systems such as the Type III protein-RNA toxin-antitoxin system, ToxIN. A flagellum-dependent bacteriophage of the Myoviridae, ΦTE, evolved rare mutants that "escaped" ToxIN-mediated abortive infection within Pectobacterium atrosepticum. Wild-type ΦTE encoded a short sequence similar to the repetitive nucleotide sequence of the RNA antitoxin, ToxI, from ToxIN. The ΦTE escape mutants had expanded the number of these "pseudo-ToxI" genetic repeats and, in one case, an escape phage had "hijacked" ToxI from the plasmid-borne toxIN locus, through recombination. Expression of the pseudo-ToxI repeats during ΦTE infection allowed the phage to replicate, unaffected by ToxIN, through RNA-based molecular mimicry. This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system. Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.

  11. Bovine viral diarrhea virus: molecular cloning of genomic RNA and its diagnostic application

    Energy Technology Data Exchange (ETDEWEB)

    Brock, K.V.

    1987-01-01

    Molecular cloning of a field isolate of bovine viral diarrhea virus (BVDV) strain 72 RNA was done in this study. The sensitivity and specificity of cloned cDNA sequences in hybridization assays with various BVDV strains were determined. cDNA was synthesized from polyadenylated BVDV RNA templates with oligo-dT primers, reverse transcriptase, and DNA polymerase I. The newly synthesized double-stranded BVDV cDNA was C-tailed with terminal deoxytransferase and annealed into G-tailed, Pst-1-cut pUC9 plasmid. Escherichia coli was transformed with the recombinant plasmids and a library of approximately 200 BVDV specific cDNA clones varying in length from 0.5 to 2.6 kilobases were isolated. The sensitivity and specificity of hybridization between the labelled cDNA and BVDV target sequences were determined. Cloned BVDV sequences were isolated from pUC9 plasmid DNA and labelled with /sup 32/P by nick translation. The detection limit by dot blot hybridization assay was 20 pg of purified genomic BVDV RNA. cDNA hybridization probes were specific for all strains of BVDV tested, regardless of whether they were noncytopathic and cytopathic, but did not hybridize with heterologous bovine viruses tested. Probes did not hybridize with uninfected cell culture or cellular RNA. Hybridization probes were at least as sensitive as infectivity assays in detecting homologous virus.

  12. Expanding access to HIV viral load testing: a systematic review of RNA stability in EDTA tubes and PPT beyond current time and temperature thresholds.

    Science.gov (United States)

    Bonner, Kimberly; Siemieniuk, Reed A; Boozary, Andrew; Roberts, Teri; Fajardo, Emmanuel; Cohn, Jennifer

    2014-01-01

    HIV viral load (VL) testing is the gold standard for antiretroviral treatment monitoring, but many barriers exist to VL testing in resource-limited settings, including storage and transport limitations for whole blood and plasma. Data from various studies indicate that HIV RNA is stable beyond current recommendations. We conducted a systematic review to assess stability data of HIV RNA in whole blood and plasma across times and temperatures. Using a pre-defined protocol, five databases were searched for studies where blood samples from HIV patients were stored at time and temperature points that exceeded manufacturer recommendations. RNA stability, the primary outcome, was measured by the difference in means compared to samples stored within established thresholds. RNA stability was defined as ≤0.5 log degradation. The search identified 10,716 titles, of which nine full-text articles were included for review. HIV RNA maintained stability in EDTA whole blood and plasma at all measured time points up to 168 hours when stored at 4°C, while stability was detected at 72 hours (95% confidence) in whole blood at 25°C, with data points before and beyond 72 hours suggesting stability but not reaching statistical significance. For EDTA plasma stored at 30°C, stability was maintained up to 48 hours (95% confidence), with OLS linear regression estimates up to 127 hours, suggesting stability. Overall, quality of studies was moderate. Limitations included small sample sizes, few studies meeting inclusion criteria, and no studies examining RNA stability in low viremia (PPT in field conditions are needed.

  13. HIV-1 Viral RNA Dynamics at the Plasma Membrane May Provide Insight into Viral Assembly | Poster

    Science.gov (United States)

    Many aspects of how infectious viruses assemble in cells have yet to be completely deciphered. However, as reported in a recent Journal of Virology paper, researchers may be one step closer to understanding how HIV-1, the virus that causes AIDS, assembles and replicates.

  14. Water isotope effect on the thermostability of a polio viral RNA hairpin: A metadynamics study

    Science.gov (United States)

    Pathak, Arup K.; Bandyopadhyay, Tusar

    2017-04-01

    Oral polio vaccine is considered to be the most thermolabile of all the common childhood vaccines. Despite heavy water (D2O) having been known for a long time to stabilise attenuated viral RNA against thermodegradation, the molecular underpinnings of its mechanism of action are still lacking. Whereas, understanding the basis of D2O action is an important step that might reform the way other thermolabile drugs are stored and could possibly minimize the cold chain problem. Here using a combination of parallel tempering and well-tempered metadynamics simulation in light water (H2O) and in D2O, we have fully described the free energy surface associated with the folding/unfolding of a RNA hairpin containing a non-canonical basepair motif, which is conserved within the 3'-untranslated region of poliovirus-like enteroviruses. Simulations reveal that in heavy water (D2O) there is a considerable increase of the stability of the folded basin as monitored through an intramolecular hydrogen bond (HB), size, shape, and flexibility of RNA structures. This translates into a higher melting temperature in D2O by 41 K when compared with light water (H2O). We have explored the hydration dynamics of the RNA, hydration shell around the RNA surface, and spatial dependence of RNA-solvent collective HB dynamics in the two water systems. Simulation in heavy water clearly showed that D2O strengthens the HB network in the solvent, lengthens inter-residue water-bridge lifetime, and weakens dynamical coupling of the hairpin to its solvation environment, which enhances the rigidity of solvent exposed sites of the native configurations. The results might suggest that like other added osmoprotectants, D2O can act as a thermostabilizer when used as a solvent.

  15. Structure-function relationship of viral cis-acting RNA elements : the role of the OriI and OriR in enterovirus replication

    NARCIS (Netherlands)

    Ooij, Martinus Johannes Maria van

    2007-01-01

    The genus Enterovirus belongs to Picornaviridae, a family of small, non-enveloped, lytic RNA viruses. They contain a single-stranded RNA genome of positive polarity of approximately 7,500 nucleotides. A viral protein VPg is specifically linked to the 5'terminus of the viral RNA. IRES-mediated initi

  16. Inhibition of post-transcriptional RNA processing by CDK inhibitors and its implication in anti-viral therapy.

    Directory of Open Access Journals (Sweden)

    Jitka Holcakova

    Full Text Available Cyclin-dependent kinases (CDKs are key regulators of the cell cycle and RNA polymerase II mediated transcription. Several pharmacological CDK inhibitors are currently in clinical trials as potential cancer therapeutics and some of them also exhibit antiviral effects. Olomoucine II and roscovitine, purine-based inhibitors of CDKs, were described as effective antiviral agents that inhibit replication of a broad range of wild type human viruses. Olomoucine II and roscovitine show high selectivity for CDK7 and CDK9, with important functions in the regulation of RNA polymerase II transcription. RNA polymerase II is necessary for viral transcription and following replication in cells. We analyzed the effect of inhibition of CDKs by olomoucine II on gene expression from viral promoters and compared its effect to widely-used roscovitine. We found that both roscovitine and olomoucine II blocked the phosphorylation of RNA polymerase II C-terminal domain. However the repression of genes regulated by viral promoters was strongly dependent on gene localization. Both roscovitine and olomoucine II inhibited expression only when the viral promoter was not integrated into chromosomal DNA. In contrast, treatment of cells with genome-integrated viral promoters increased their expression even though there was decreased phosphorylation of the C-terminal domain of RNA polymerase II. To define the mechanism responsible for decreased gene expression after pharmacological CDK inhibitor treatment, the level of mRNA transcription from extrachromosomal DNA was determined. Interestingly, our results showed that inhibition of RNA polymerase II C-terminal domain phosphorylation increased the number of transcribed mRNAs. However, some of these mRNAs were truncated and lacked polyadenylation, which resulted in decreased translation. These results suggest that phosphorylation of RNA polymerase II C-terminal domain is critical for linking transcription and posttrancriptional

  17. Prognostic value of single measurements of beta-2-microglobulin, immunoglobulin A in HIV disease after controlling for CD4 lymphocyte counts and plasma HIV RNA levels

    DEFF Research Database (Denmark)

    Ullum, H; Lepri, A Cozzi; Katzenstein, T L

    2000-01-01

    The interrelationships between the CD4 lymphocyte count, plasma viral load [human immunodeficiency virus (HIV) RNA], beta-2-microglobulin (beta2-M) and immunoglobulin A (IgA) and the mortality risk was explored in 234 HIV-infected individuals (median CD4 count 230 cells/mm3, range 1-1,247). Produ...

  18. Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA.

    Science.gov (United States)

    Pereira, Carina F; Read, Eliot K C; Wise, Helen M; Amorim, Maria J; Digard, Paul

    2017-08-01

    Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export.IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as

  19. Apigenin inhibits enterovirus-71 infection by disrupting viral RNA association with trans-acting factors.

    Science.gov (United States)

    Zhang, Wei; Qiao, Haishi; Lv, Yuanzi; Wang, Jingjing; Chen, Xiaoqing; Hou, Yayi; Tan, Renxiang; Li, Erguang

    2014-01-01

    Flavonoids are widely distributed natural products with broad biological activities. Apigenin is a dietary flavonoid that has recently been demonstrated to interact with heterogeneous nuclear ribonucleoproteins (hnRNPs) and interferes with their RNA editing activity. We investigated whether apigenin possessed antiviral activity against enterovirus-71 (EV71) infection since EV71 infection requires of hnRNP proteins. We found that apigenin selectively blocks EV71 infection by disrupting viral RNA association with hnRNP A1 and A2 proteins. The estimated EC50 value for apigenin to block EV71 infection was determined at 10.3 µM, while the CC50 was estimated at 79.0 µM. The anti-EV71 activity was selective since no activity was detected against several DNA and RNA viruses. Although flavonoids in general share similar structural features, apigenin and kaempferol were among tested compounds with significant activity against EV71 infection. hnRNP proteins function as trans-acting factors regulating EV71 translation. We found that apigenin treatment did not affect EV71-induced nucleocytoplasmic redistribution of hnRNP A1 and A2 proteins. Instead, it prevented EV71 RNA association with hnRNP A1 and A2 proteins. Accordingly, suppression of hnRNP A1 and A2 expression markedly reduced EV71 infection. As a positive sense, single strand RNA virus, EV71 has a type I internal ribosome entry site (IRES) that cooperates with host factors and regulates EV71 translation. The effect of apigenin on EV71 infection was further demonstrated using a bicistronic vector that has the expression of a GFP protein under the control of EV71 5'-UTR. We found that apigenin treatment selectively suppressed the expression of GFP, but not a control gene. In addition to identification of apigenin as an antiviral agent against EV71 infection, this study also exemplifies the significance in antiviral agent discovery by targeting host factors essential for viral replication.

  20. Interaction between viral RNA silencing suppressors and host factors in plant immunity.

    Science.gov (United States)

    Nakahara, Kenji S; Masuta, Chikara

    2014-08-01

    To elucidate events in the molecular arms race between the host and pathogen in evaluating plant immunity, a zigzag model is useful for uncovering aspects common to different host-pathogen interactions. By analogy of the steps in virus-host interactions with the steps in the standard zigzag model outlined in recent papers, we may regard RNA silencing as pattern-triggered immunity (PTI) against viruses, RNA silencing suppressors (RSSs) as effectors to overcome host RNA silencing and resistance gene (R-gene)-mediated defense as effector-triggered immunity (ETI) recognizing RSSs as avirulence proteins. However, because the standard zigzag model does not fully apply to some unique aspects in the interactions between a plant host and virus, we here defined a model especially designed for viruses. Although we simplified the phenomena involved in the virus-host interactions in the model, certain specific interactive steps can be explained by integrating additional host factors into the model. These host factors are thought to play an important role in maintaining the efficacy of the various steps in the main pathway of defense against viruses in this model for virus-plant interactions. For example, we propose candidates that may interact with viral RSSs to induce the resistance response. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Leeches as a source of mammalian viral DNA and RNA - a study in medicinal leeches

    DEFF Research Database (Denmark)

    Kampmann, Marie-Louise; Schnell, Ida Bærholm; Jensen, Randi Holm

    2017-01-01

    leeches suggest that host viruses may also be detectable. To systematically test this hypothesis, we performed a proof of concept study using quantitative PCR (qPCR) to detect DNA viruses (bovine herpesvirus [BHV], human adenovirus [HAdV]) and RNA viruses (influenza A [InfA] and measles morbillivirus [Me......V]) from nucleic acids extracted from medicinal leeches fed with blood spiked with each virus. All viruses except BHV showed a gradual decline in concentration from day 1 to 50, and all except BHV were detectable in at least half of the samples even after 50 days. BHV exhibited a rapid decline at day 27...... and was undetectable at day 50. Our findings in medicinal leeches indicate that leeches collected in the wild might be an untapped resource for detecting vertebrate viruses and could provide new opportunities to study wildlife viral diseases of rare species in challenging environments, where capturing and handling...

  2. Wolbachia Blocks Viral Genome Replication Early in Infection without a Transcriptional Response by the Endosymbiont or Host Small RNA Pathways.

    Directory of Open Access Journals (Sweden)

    Stephanie M Rainey

    2016-04-01

    Full Text Available The intracellular endosymbiotic bacterium Wolbachia can protect insects against viral infection, and is being introduced into mosquito populations in the wild to block the transmission of arboviruses that infect humans and are a major public health concern. To investigate the mechanisms underlying this antiviral protection, we have developed a new model system combining Wolbachia-infected Drosophila melanogaster cell culture with the model mosquito-borne Semliki Forest virus (SFV; Togaviridae, Alphavirus. Wolbachia provides strong antiviral protection rapidly after infection, suggesting that an early stage post-infection is being blocked. Wolbachia does appear to have major effects on events distinct from entry, assembly or exit as it inhibits the replication of an SFV replicon transfected into the cells. Furthermore, it causes a far greater reduction in the expression of proteins from the 3' open reading frame than the 5' non-structural protein open reading frame, indicating that it is blocking the replication of viral RNA. Further to this separation of the replicase proteins and viral RNA in transreplication assays shows that uncoupling of viral RNA and replicase proteins does not overcome Wolbachia's antiviral activity. This further suggests that replicative processes are disrupted, such as translation or replication, by Wolbachia infection. This may occur by Wolbachia mounting an active antiviral response, but the virus did not cause any transcriptional response by the bacterium, suggesting that this is not the case. Host microRNAs (miRNAs have been implicated in protection, but again we found that host cell miRNA expression was unaffected by the bacterium and neither do our findings suggest any involvement of the antiviral siRNA pathway. We conclude that Wolbachia may directly interfere with early events in virus replication such as translation of incoming viral RNA or RNA transcription, and this likely involves an intrinsic (as opposed to

  3. Wolbachia Blocks Viral Genome Replication Early in Infection without a Transcriptional Response by the Endosymbiont or Host Small RNA Pathways.

    Science.gov (United States)

    Rainey, Stephanie M; Martinez, Julien; McFarlane, Melanie; Juneja, Punita; Sarkies, Peter; Lulla, Aleksei; Schnettler, Esther; Varjak, Margus; Merits, Andres; Miska, Eric A; Jiggins, Francis M; Kohl, Alain

    2016-04-01

    The intracellular endosymbiotic bacterium Wolbachia can protect insects against viral infection, and is being introduced into mosquito populations in the wild to block the transmission of arboviruses that infect humans and are a major public health concern. To investigate the mechanisms underlying this antiviral protection, we have developed a new model system combining Wolbachia-infected Drosophila melanogaster cell culture with the model mosquito-borne Semliki Forest virus (SFV; Togaviridae, Alphavirus). Wolbachia provides strong antiviral protection rapidly after infection, suggesting that an early stage post-infection is being blocked. Wolbachia does appear to have major effects on events distinct from entry, assembly or exit as it inhibits the replication of an SFV replicon transfected into the cells. Furthermore, it causes a far greater reduction in the expression of proteins from the 3' open reading frame than the 5' non-structural protein open reading frame, indicating that it is blocking the replication of viral RNA. Further to this separation of the replicase proteins and viral RNA in transreplication assays shows that uncoupling of viral RNA and replicase proteins does not overcome Wolbachia's antiviral activity. This further suggests that replicative processes are disrupted, such as translation or replication, by Wolbachia infection. This may occur by Wolbachia mounting an active antiviral response, but the virus did not cause any transcriptional response by the bacterium, suggesting that this is not the case. Host microRNAs (miRNAs) have been implicated in protection, but again we found that host cell miRNA expression was unaffected by the bacterium and neither do our findings suggest any involvement of the antiviral siRNA pathway. We conclude that Wolbachia may directly interfere with early events in virus replication such as translation of incoming viral RNA or RNA transcription, and this likely involves an intrinsic (as opposed to an induced

  4. High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.

    Directory of Open Access Journals (Sweden)

    Yanwei Bi

    2014-05-01

    Full Text Available A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR-associated (Cas RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ and homology-directed repair (HDR pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

  5. Using plasma viral load to guide antiretroviral therapy initiation to prevent HIV-1 transmission.

    Directory of Open Access Journals (Sweden)

    Pamela M Murnane

    Full Text Available BACKGROUND: Current WHO guidelines recommend antiretroviral therapy (ART initiation at CD4 counts ≤350 cells/µL. Increasing this threshold has been proposed, with a primary goal of reducing HIV-1 infectiousness. Because the quantity of HIV-1 in plasma is the primary predictor of HIV-1 transmission, consideration of plasma viral load in ART initiation guidelines is warranted. METHODS: Using per-sex-act infectivity estimates and cross-sectional sexual behavior data from 2,484 HIV-1 infected persons with CD4 counts >350 enrolled in a study of African heterosexual HIV-1 serodiscordant couples, we calculated the number of transmissions expected and the number potentially averted under selected scenarios for ART initiation: i CD4 count 350 while averting 40.5% of expected transmissions (ratio 2.0; treating at viral load ≥10,0000 copies/mL had a ratio of 1.5. In contrast, initiation at CD4 count <500 would require treating 41.8%, while averting 48.4% (ratio 1.1. CONCLUSION: Inclusion of viral load in ART initiation guidelines could permit targeting ART resources to HIV-1 infected persons who have a higher risk of transmitting HIV-1. Further work is needed to estimate costs and feasibility.

  6. Relationship between serum HBV RNA levels and intrahepatic viral as well as histologic activity markers in entecavir-treated patients.

    Science.gov (United States)

    Wang, Jing; Yu, Yiqi; Li, Guojun; Shen, Chuan; Meng, Zhefeng; Zheng, Jianming; Jia, Yanhong; Chen, Shaolong; Zhang, Xiao; Zhu, MengQi; Zheng, Jiangjiang; Song, Zhangzhang; Wu, Jing; Shao, Lingyun; Qian, Peiyu; Mao, Xiaona; Wang, Xuanyi; Huang, Yuxian; Zhao, Caiyan; Zhang, Jiming; Qiu, Chao; Zhang, Wenhong

    2017-09-01

    In diagnostics, serum hepatitis B virus (HBV)-RNA levels are valuable when the HBV-DNA load in circulation is effectively suppressed by nucleos(t)ide analogue (NUC) therapy. This study aimed to determine the intrahepatic viral replication activity reflected in serum HBV-RNA and whether HBV-RNA contributes to liver histological changes in NUC-treated patients. A cross-sectional set of serum and liver biopsy samples was obtained from entecavir-treated patients with undetectable levels of serum HBV-DNA. The correlations between HBV-RNA concentration in serum and levels of peripheral and intrahepatic viral replicative forms and histological scores were analyzed. Quasispecies of serum HBV-RNA and intrahepatic viral replicative forms were examined by deep sequencing. HBV-RNA-positive hepatocytes were visualized by in situ hybridization. Serum HBV-RNA was detected in 35 of 47 patients (74.47%, 2.33-4.80 log10 copies/mL). These levels correlated not only with the intrahepatic HBV-RNA level and the ratio of intrahepatic HBV-RNA to covalently closed circular DNA (cccDNA), but also with the histological scores for grading and staging. From the view of quasispecies, serum HBV-RNA was more genetically homogenous with contemporaneously sampled intrahepatic HBV-RNA relative to cccDNA pool and dynamically changed over time in consecutive samples. In situ histology study revealed that HBV-RNA-positive hepatocytes were clustered in foci, sporadically distributed across the lobules, and co-localized with hepatitis B surface antigen. Serum HBV-RNA levels reflect intrahepatic viral transcriptional activity and are associated with liver histopathology in patients receiving NUC therapy. Our study sheds light on the nature of HBV-RNA in the pathogenesis of chronic HBV infection and has implications for the management of chronic hepatitis B during NUC therapy. Serum HBV-RNA levels are indicative of the intrahepatic transcriptional activity of covalently closed circular DNA and are

  7. A viral genome landscape of RNA polyadenylation from KSHV latent to lytic infection.

    Directory of Open Access Journals (Sweden)

    Vladimir Majerciak

    Full Text Available RNA polyadenylation (pA is one of the major steps in regulation of gene expression at the posttranscriptional level. In this report, a genome landscape of pA sites of viral transcripts in B lymphocytes with Kaposi sarcoma-associated herpesvirus (KSHV infection was constructed using a modified PA-seq strategy. We identified 67 unique pA sites, of which 55 could be assigned for expression of annotated ~90 KSHV genes. Among the assigned pA sites, twenty are for expression of individual single genes and the rest for multiple genes (average 2.7 genes per pA site in cluster-gene loci of the genome. A few novel viral pA sites that could not be assigned to any known KSHV genes are often positioned in the antisense strand to ORF8, ORF21, ORF34, K8 and ORF50, and their associated antisense mRNAs to ORF21, ORF34 and K8 could be verified by 3'RACE. The usage of each mapped pA site correlates to its peak size, the larger (broad and wide peak size, the more usage and thus, the higher expression of the pA site-associated gene(s. Similar to mammalian transcripts, KSHV RNA polyadenylation employs two major poly(A signals, AAUAAA and AUUAAA, and is regulated by conservation of cis-elements flanking the mapped pA sites. Moreover, we found two or more alternative pA sites downstream of ORF54, K2 (vIL6, K9 (vIRF1, K10.5 (vIRF3, K11 (vIRF2, K12 (Kaposin A, T1.5, and PAN genes and experimentally validated the alternative polyadenylation for the expression of KSHV ORF54, K11, and T1.5 transcripts. Together, our data provide not only a comprehensive pA site landscape for understanding KSHV genome structure and gene expression, but also the first evidence of alternative polyadenylation as another layer of posttranscriptional regulation in viral gene expression.

  8. The interplay between MDV and HVT affects viral miRNa expression.

    Science.gov (United States)

    Goher, Mohamed; Hicks, Julie A; Liu, Hsiao-Ching

    2013-06-01

    It is well established that herpesviruses encode numerous microRNAs (miRNAs) and that these virally encoded small RNAs play multiple roles in infection. The present study was undertaken to determine how co-infection of a pathogenic MDV serotype one (MDV1) strain (MD5) and a vaccine strain (herpesvirus of turkeys [HVT]) alters viral miRNA expression in vivo. We first used small RNA deep sequencing to identify MDV1-encoded miRNAs that are expressed in tumorigenic spleens of MDV1-infected birds. The expression patterns of these miRNAs were then further assessed at an early time point (7 days postinfection [dpi]) and a late time point (42 dpi) in birds with and without HVT vaccination using real-time PCR (RT-PCR). Additionally, the effect of MDV1 co-infection on HVT-encoded miRNAs was determined using RT-PCR. A diverse population of miRNAs was expressed in MDV-induced tumorigenic spleens at 42 dpi, with 18 of the 26 known mature miRNAs represented. Of these, both mdv1-miR-M4-5p and mdv1-miR-M2-3p were the most highly expressed miRNAs. RT-PCR analysis further revealed that nine MDV miRNAs were differentially expressed between 7 dpi and 42 dpi infected spleens. At 7 dpi, three miRNAs were differentially expressed between the spleens of birds co-infected with HVT and MD5 compared with birds singly infected with MD5, whereas at 42 dpi, nine miRNAs were differentially expressed. At 7 dpi, the expression of seven HVT-encoded miRNAs was affected in the spleens of co-infected birds compared with birds only receiving the HVT vaccine. At 42 dpi, six HVT-encoded miRNAs were differentially expressed between the two groups. Target prediction analysis suggests that these differentially expressed viral miRNAs are involved in regulating several cellular processes, including cell proliferation and the adaptive immune response.

  9. Interactions between p27 and p88 replicase proteins of Red clover necrotic mosaic virus play an essential role in viral RNA replication and suppression of RNA silencing via the 480-kDa viral replicase complex assembly.

    Science.gov (United States)

    Mine, Akira; Hyodo, Kiwamu; Takeda, Atsushi; Kaido, Masanori; Mise, Kazuyuki; Okuno, Tetsuro

    2010-11-25

    Red clover necrotic mosaic virus (RCNMV), a positive-sense RNA virus with a bipartite genome, encodes p27 and p88 replicase proteins that are required for viral RNA replication and suppression of RNA silencing. In this study, we identified domains in p27 and p88 responsible for their protein-protein interactions using in vitro pull-down assays with the purified recombinant proteins. Coimmunoprecipitation analysis in combination with blue-native polyacrylamide gel electrophoresis using mutated p27 proteins showed that both p27-p27 and p27-p88 interactions are essential for the formation of the 480-kDa complex, which has RCNMV-specific RNA-dependent RNA polymerase activity. Furthermore, we found a good correlation between the accumulated levels of the 480-kDa complex and replication levels and the suppression of RNA silencing activity. Our results indicate that interactions between RCNMV replicase proteins play an essential role in viral RNA replication and in suppressing RNA silencing via the 480-kDa replicase complex assembly.

  10. rgs-CaM Detects and Counteracts Viral RNA Silencing Suppressors in Plant Immune Priming.

    Science.gov (United States)

    Jeon, Eun Jin; Tadamura, Kazuki; Murakami, Taiki; Inaba, Jun-Ichi; Kim, Bo Min; Sato, Masako; Atsumi, Go; Kuchitsu, Kazuyuki; Masuta, Chikara; Nakahara, Kenji S

    2017-10-01

    Primary infection of a plant with a pathogen that causes high accumulation of salicylic acid in the plant typically via a hypersensitive response confers enhanced resistance against secondary infection with a broad spectrum of pathogens, including viruses. This phenomenon is called systemic acquired resistance (SAR), which is a plant priming for adaption to repeated biotic stress. However, the molecular mechanisms of SAR-mediated enhanced inhibition, especially of virus infection, remain unclear. Here, we show that SAR against cucumber mosaic virus (CMV) in tobacco plants (Nicotiana tabacum) involves a calmodulin-like protein, rgs-CaM. We previously reported the antiviral function of rgs-CaM, which binds to and directs degradation of viral RNA silencing suppressors (RSSs), including CMV 2b, via autophagy. We found that rgs-CaM-mediated immunity is ineffective against CMV infection in normally growing tobacco plants but is activated as a result of SAR induction via salicylic acid signaling. We then analyzed the effect of overexpression of rgs-CaM on salicylic acid signaling. Overexpressed and ectopically expressed rgs-CaM induced defense reactions, including cell death, generation of reactive oxygen species, and salicylic acid signaling. Further analysis using a combination of the salicylic acid analogue benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and the Ca(2+) ionophore A23187 revealed that rgs-CaM functions as an immune receptor that induces salicylic acid signaling by simultaneously perceiving both viral RSS and Ca(2+) influx as infection cues, implying its autoactivation. Thus, secondary infection of SAR-induced tobacco plants with CMV seems to be effectively inhibited through 2b recognition and degradation by rgs-CaM, leading to reinforcement of antiviral RNA silencing and other salicylic acid-mediated antiviral responses.IMPORTANCE Even without an acquired immune system like that in vertebrates, plants show enhanced whole

  11. In vitro neutralization of viral hemorrhagic septicemia virus by plasma from immunized zebrafish.

    Science.gov (United States)

    Chinchilla, Blanca; Gomez-Casado, Eduardo; Encinas, Paloma; Falco, Alberto; Estepa, Amparo; Coll, Julio

    2013-03-01

    We studied humoral long-term adaptive viral neutralization responses in zebrafish (Danio rerio), an increasingly useful vertebrate model for viral diseases actually limited by the absence of standardized anti-zebrafish immunoglobulin M (IgM) antibodies. We established an alternative method, similar to those used in other fish, to achieve a first estimation of zebrafish anti-viral antibody-like responses. We used the viral hemorrhagic septicemia virus (VHSV) model because, although protection after this non-natural infection was demonstrated in cold-acclimatized zebrafish, little is known about their induced anti-VHSV antibody-like responses. Therefore, we first optimized a micro-neutralization method based on immunostaining VHSV-infected fish cell monolayers to detect zebrafish neutralizing activity in plasma samples in one day. We then used the method to measure the specific anti-VHSV neutralization in plasma obtained from individual zebrafish under various VHSV challenges or immunization protocols. The neutralizing activity was inhibited by protein A-sepharose and rabbit anti-zebrafish IgM antibodies, suggesting the implication of IgM zebrafish antibodies in such responses. To our knowledge, this is the first report to demonstrate detectable and significant VHSV neutralization titers in zebrafish surviving VHSV infections. This micro-method might be useful, not only for the follow-up of infection/vaccine development in the zebrafish/VHSV model in particular, but also for similar work involving other in vitro neutralizable zebrafish pathogens. This technique might also further the development of alternative ELISA assay methods to measure specific immunoglobulins in zebrafish.

  12. Phosphorylation of hepatitis C virus RNA polymerases ser29 and ser42 by protein kinase C-related kinase 2 regulates viral RNA replication.

    Science.gov (United States)

    Han, Song-Hee; Kim, Seong-Jun; Kim, Eun-Jung; Kim, Tae-Eun; Moon, Jae-Su; Kim, Geon-Woo; Lee, Seung-Hoon; Cho, Kun; Yoo, Jong Shin; Son, Woo Sung; Rhee, Jin-Kyu; Han, Seung Hyun; Oh, Jong-Won

    2014-10-01

    Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse-genetics approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo. Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, in the Δ1 finger loop region that interacts with the thumb subdomain of NS5B. Colony-forming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse-genetics studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular-modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its Δ1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication. While the role of RNA-dependent RNA polymerases (RdRps) in viral RNA replication is clear, little is known about their functional regulation by phosphorylation. In this study, we addressed several important questions about the function and structure of phosphorylated hepatitis C virus (HCV) nonstructural protein 5B (NS5B). Reverse-genetics studies with HCV replicons encoding phosphorylation-defective NS5B mutants and analysis of their RdRp activities revealed previously unidentified

  13. Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method

    Directory of Open Access Journals (Sweden)

    Dan Dou

    2017-07-01

    Full Text Available Genome delivery to the proper cellular compartment for transcription and replication is a primary goal of viruses. However, methods for analyzing viral genome localization and differentiating genomes with high identity are lacking, making it difficult to investigate entry-related processes and co-examine heterogeneous RNA viral populations. Here, we present an RNA labeling approach for single-cell analysis of RNA viral replication and co-infection dynamics in situ, which uses the versatility of padlock probes. We applied this method to identify influenza A virus (IAV infections in cells and lung tissue with single-nucleotide specificity and to classify entry and replication stages by gene segment localization. Extending the classification strategy to co-infections of IAVs with single-nucleotide variations, we found that the dependence on intracellular trafficking places a time restriction on secondary co-infections necessary for genome reassortment. Altogether, these data demonstrate how RNA viral genome labeling can help dissect entry and co-infections.

  14. Longitudinal comparison between plasma and seminal HIV-1 viral loads during antiretroviral treatment Comparação longitudinal entre cargas virais seminais e plasmáticas do HIV-1 durante terapia anti-retroviral

    Directory of Open Access Journals (Sweden)

    Lauro Ferreira da Silva Pinto Neto

    2003-12-01

    Full Text Available This study was designed to investigate the impact of anti-retroviral therapy on both plasma and seminal HIV-1 viral loads and the correlation between viral loads in these compartments after treatment. Viral load, CD4+ and CD8+ T-cell counts were evaluated in paired plasma and semen samples from 36 antiretroviral therapy-naïve patients at baseline and on days 45, 90, and 180 of treatment. Slopes for blood and seminal viral loads in all treated patients were similar (p = 0.21. Median HIV-1 RNA titers in plasma and semen at baseline were 4.95 log10 and 4.48 log10 copies/ml, respectively. After 180 days of therapy, the median viral load declined to 3.15 log10 copies/ml (plasma and 3.2 log10 copies/ml (semen. At this timepoint 22 patients presented HIV-1 viral load below 400 copies/ml in either plasma or semen, but only 9 had viral loads below 400 copies/ml in both compartments.Este estudo foi desenhado para investigar o impacto do tratamento com anti-retrovirais na evolução das cargas virais plasmáticas e seminais do HIV-1. A carga viral do HIV-1 e a contagem de linfócitos T CD4+ e CD8+ foi determinada em amostras pareadas de sangue e sêmen de 36 pacientes virgem de tratamento nos dias 0, 45, 90 e 180 após o início da terapia. As curvas de declínio das cargas virais plasmática e seminal foram semelhantes (p= 0.21. As medianas da carga viral plasmática e seminal no pré-tratamento (dia 0 foram 4.95 e 4.48 log10 cópias/ml, respectivamente. Seis meses após o início da terapia, a mediana da carga viral plasmática era 3.15 log10 cópias/ml e a seminal 3.2 log10 cópias/ml. Neste mesmo periodo, 22 pacientes apresentavam carga viral abaixo de 400 cópias/ml no plasma e/ou sêmen, enquanto apenas 9 pacientes apresentavam carga viral abaixo do limite de detecção nos dois compartimentos.

  15. Newly identified phosphorylation site in the vesicular stomatitis virus P protein is required for viral RNA synthesis.

    Science.gov (United States)

    Mondal, Arindam; Victor, Ken G; Pudupakam, R S; Lyons, Charles E; Wertz, Gail W

    2014-02-01

    The vesicular stomatitis virus (VSV) RNA-dependent RNA polymerase consists of two viral proteins; the large (L) protein is the main catalytic subunit, and the phosphoprotein (P) is an essential cofactor for polymerase function. The P protein interacts with the L protein and the N-RNA template, thus connecting the polymerase to the template. P protein also binds to free N protein to maintain it in a soluble, encapsidation-competent form. Previously, five sites of phosphorylation were identified on the P protein and these sites were reported to be differentially important for mRNA synthesis or genomic replication. The previous studies were carried out by biochemical analysis of portions of the authentic viral P protein or by analysis of bacterium-expressed, exogenously phosphorylated P protein by mutagenesis. However, there has been no systematic biochemical search for phosphorylation sites on authentic, virus-expressed P protein. In this study, we analyzed the P protein isolated from VSV-infected cells for sites of phosphorylation by mass spectrometry. We report the identification of Tyr14 as a previously unidentified phosphorylation site of VSV P and show that it is essential for viral transcription and replication. However, our mass spectral analysis failed to observe the phosphorylation of previously reported C-terminal residues Ser226 and Ser227 and mutagenic analyses did not demonstrate a role for these sites in RNA synthesis.

  16. Acute hepatitis A virus infection is associated with a limited type I interferon response and persistence of intrahepatic viral RNA.

    Science.gov (United States)

    Lanford, Robert E; Feng, Zongdi; Chavez, Deborah; Guerra, Bernadette; Brasky, Kathleen M; Zhou, Yan; Yamane, Daisuke; Perelson, Alan S; Walker, Christopher M; Lemon, Stanley M

    2011-07-05

    Hepatitis A virus (HAV) is an hepatotropic human picornavirus that is associated only with acute infection. Its pathogenesis is not well understood because there are few studies in animal models using modern methodologies. We characterized HAV infections in three chimpanzees, quantifying viral RNA by quantitative RT-PCR and examining critical aspects of the innate immune response including intrahepatic IFN-stimulated gene expression. We compared these infection profiles with similar studies of chimpanzees infected with hepatitis C virus (HCV), an hepatotropic flavivirus that frequently causes persistent infection. Surprisingly, HAV-infected animals exhibited very limited induction of type I IFN-stimulated genes in the liver compared with chimpanzees with acute resolving HCV infection, despite similar levels of viremia and 100-fold greater quantities of viral RNA in the liver. Minimal IFN-stimulated gene 15 and IFIT1 responses peaked 1-2 wk after HAV challenge and then subsided despite continuing high hepatic viral RNA. An acute inflammatory response at 3-4 wk correlated with the appearance of virus-specific antibodies and apoptosis and proliferation of hepatocytes. Despite this, HAV RNA persisted in the liver for months, remaining present long after clearance from serum and feces and revealing dramatic differences in the kinetics of clearance in the three compartments. Viral RNA was detected in the liver for significantly longer (35 to >48 wk) than HCV RNA in animals with acute resolving HCV infection (10-20 wk). Collectively, these findings indicate that HAV is far stealthier than HCV early in the course of acute resolving infection. HAV infections represent a distinctly different paradigm in virus-host interactions within the liver.

  17. The potato mop-top virus TGB2 protein and viral RNA associate with chloroplasts and viral infection induces inclusions in the plastids

    Directory of Open Access Journals (Sweden)

    Graham H Cowan

    2012-12-01

    Full Text Available The potato mop-top virus (PMTV triple gene block 2 (TGB2 movement protein fused to monomeric red fluorescent protein (mRFP-TGB2 was expressed under the control of the PMTV subgenomic promoter from a PMTV vector. The subcellular localisations and interactions of mRFP-TGB2 were investigated using confocal imaging (CLSM and biochemical analysis. The results revealed associations with membranes of the endoplasmic reticulum, mobile granules, small round structures (1-2 µm in diameter and chloroplasts. Expression of mRFP-TGB2 in epidermal cells enabled cell-to-cell movement of a TGB2 defective PMTV reporter clone, indicating that the mRFP-TGB2 fusion protein was functional and required for cell-to-cell movement. Protein-lipid interaction assays revealed an association between TGB2 and lipids present in chloroplasts, consistent with microscopical observations where the plastid envelope was labelled later in infection. To further investigate the association of PMTV infection with chloroplasts, ultrastructural studies of thin sections of PMTV-infected potato and Nicotiana benthamiana leaves by electron microscopy revealed abnormal chloroplasts with cytoplasmic inclusions and terminal projections. Viral coat protein, genomic RNA and fluorescently-labelled TGB2 were detected in plastid preparations isolated from the infected leaves, and viral RNA was localised to chloroplasts in infected tissues. The results reveal a novel association of TGB2 and vRNA with chloroplasts, and suggest viral replication is associated with chloroplast membranes, and that TGB2 plays a novel role in targeting the virus to chloroplasts.

  18. Intracerebral Borna disease virus infection of bank voles leading to peripheral spread and reverse transcription of viral RNA.

    Directory of Open Access Journals (Sweden)

    Paula Maria Kinnunen

    Full Text Available Bornaviruses, which chronically infect many species, can cause severe neurological diseases in some animal species; their association with human neuropsychiatric disorders is, however, debatable. The epidemiology of Borna disease virus (BDV, as for other members of the family Bornaviridae, is largely unknown, although evidence exists for a reservoir in small mammals, for example bank voles (Myodes glareolus. In addition to the current exogenous infections and despite the fact that bornaviruses have an RNA genome, bornavirus sequences integrated into the genomes of several vertebrates millions of years ago. Our hypothesis is that the bank vole, a common wild rodent species in traditional BDV-endemic areas, can serve as a viral host; we therefore explored whether this species can be infected with BDV, and if so, how the virus spreads and whether viral RNA is transcribed into DNA in vivo.We infected neonate bank voles intracerebrally with BDV and euthanized them 2 to 8 weeks post-infection. Specific Ig antibodies were detectable in 41%. Histological evaluation revealed no significant pathological alterations, but BDV RNA and antigen were detectable in all infected brains. Immunohistology demonstrated centrifugal spread throughout the nervous tissue, because viral antigen was widespread in peripheral nerves and ganglia, including the mediastinum, esophagus, and urinary bladder. This was associated with viral shedding in feces, of which 54% were BDV RNA-positive, and urine at 17%. BDV nucleocapsid gene DNA occurred in 66% of the infected voles, and, surprisingly, occasionally also phosphoprotein DNA. Thus, intracerebral BDV infection of bank vole led to systemic infection of the nervous tissue and viral excretion, as well as frequent reverse transcription of the BDV genome, enabling genomic integration. This first experimental bornavirus infection in wild mammals confirms the recent findings regarding bornavirus DNA, and suggests that bank voles are

  19. Intracerebral Borna disease virus infection of bank voles leading to peripheral spread and reverse transcription of viral RNA.

    Science.gov (United States)

    Kinnunen, Paula Maria; Inkeroinen, Hanna; Ilander, Mette; Kallio, Eva Riikka; Heikkilä, Henna Pauliina; Koskela, Esa; Mappes, Tapio; Palva, Airi; Vaheri, Antti; Kipar, Anja; Vapalahti, Olli

    2011-01-01

    Bornaviruses, which chronically infect many species, can cause severe neurological diseases in some animal species; their association with human neuropsychiatric disorders is, however, debatable. The epidemiology of Borna disease virus (BDV), as for other members of the family Bornaviridae, is largely unknown, although evidence exists for a reservoir in small mammals, for example bank voles (Myodes glareolus). In addition to the current exogenous infections and despite the fact that bornaviruses have an RNA genome, bornavirus sequences integrated into the genomes of several vertebrates millions of years ago. Our hypothesis is that the bank vole, a common wild rodent species in traditional BDV-endemic areas, can serve as a viral host; we therefore explored whether this species can be infected with BDV, and if so, how the virus spreads and whether viral RNA is transcribed into DNA in vivo.We infected neonate bank voles intracerebrally with BDV and euthanized them 2 to 8 weeks post-infection. Specific Ig antibodies were detectable in 41%. Histological evaluation revealed no significant pathological alterations, but BDV RNA and antigen were detectable in all infected brains. Immunohistology demonstrated centrifugal spread throughout the nervous tissue, because viral antigen was widespread in peripheral nerves and ganglia, including the mediastinum, esophagus, and urinary bladder. This was associated with viral shedding in feces, of which 54% were BDV RNA-positive, and urine at 17%. BDV nucleocapsid gene DNA occurred in 66% of the infected voles, and, surprisingly, occasionally also phosphoprotein DNA. Thus, intracerebral BDV infection of bank vole led to systemic infection of the nervous tissue and viral excretion, as well as frequent reverse transcription of the BDV genome, enabling genomic integration. This first experimental bornavirus infection in wild mammals confirms the recent findings regarding bornavirus DNA, and suggests that bank voles are capable of

  20. SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication.

    OpenAIRE

    Maarit Neuvonen; Arunas Kazlauskas; Miika Martikainen; Ari Hinkkanen; Tero Ahola; Kalle Saksela

    2011-01-01

    Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homolo...

  1. Viral RNA testing and automation on the bead-based CBNE detection microsystem.

    Energy Technology Data Exchange (ETDEWEB)

    Galambos, Paul C.; Bourdon, Christopher Jay; Farrell, Cara M.; Rossito, Paul (University of California at Davis); McClain, Jaime L.; Derzon, Mark Steven; Cullor, James Sterling (University of California at Davis); Rahimian, Kamayar

    2008-09-01

    We developed prototype chemistry for nucleic acid hybridization on our bead-based diagnostics platform and we established an automatable bead handling protocol capable of 50 part-per-billion (ppb) sensitivity. We are working towards a platform capable of parallel, rapid (10 minute), raw sample testing for orthogonal (in this case nucleic acid and immunoassays) identification of biological (and other) threats in a single sensor microsystem. In this LDRD we developed the nucleic acid chemistry required for nucleic acid hybridization. Our goal is to place a non-cell associated RNA virus (Bovine Viral Diarrhea, BVD) on the beads for raw sample testing. This key pre-requisite to showing orthogonality (nucleic acid measurements can be performed in parallel with immunoassay measurements). Orthogonal detection dramatically reduces false positives. We chose BVD because our collaborators (UC-Davis) can supply samples from persistently infected animals; and because proof-of-concept field testing can be performed with modification of the current technology platform at the UC Davis research station. Since BVD is a cattle-prone disease this research dovetails with earlier immunoassay work on Botulinum toxin simulant testing in raw milk samples. Demonstration of BVD RNA detection expands the repertoire of biological macromolecules that can be adapted to our bead-based detection. The resources of this late start LDRD were adequate to partially demonstrate the conjugation of the beads to the nucleic acids. It was never expected to be adequate for a full live virus test but to motivate that additional investment. In addition, we were able to reduce the LOD (Limit of Detection) for the botulinum toxin stimulant to 50 ppb from the earlier LOD of 1 ppm. A low LOD combined with orthogonal detection provides both low false negatives and low false positives. The logical follow-on steps to this LDRD research are to perform live virus identification as well as concurrent nucleic acid and

  2. Expression of IMP1 enhances production of murine leukemia virus vector by facilitating viral genomic RNA packaging.

    Directory of Open Access Journals (Sweden)

    Yun Mai

    Full Text Available Murine leukemia virus (MLV-based retroviral vector is widely used for gene transfer. Efficient packaging of the genomic RNA is critical for production of high-titer virus. Here, we report that expression of the insulin-like growth factor II mRNA binding protein 1 (IMP1 enhanced the production of infectious MLV vector. Overexpression of IMP1 increased the stability of viral genomic RNA in virus producer cells and packaging of the RNA into progeny virus in a dose-dependent manner. Downregulation of IMP1 in virus producer cells resulted in reduced production of the retroviral vector. These results indicate that IMP1 plays a role in regulating the packaging of MLV genomic RNA and can be used for improving production of retroviral vectors.

  3. Expression of IMP1 enhances production of murine leukemia virus vector by facilitating viral genomic RNA packaging.

    Science.gov (United States)

    Mai, Yun; Gao, Guangxia

    2010-12-29

    Murine leukemia virus (MLV)-based retroviral vector is widely used for gene transfer. Efficient packaging of the genomic RNA is critical for production of high-titer virus. Here, we report that expression of the insulin-like growth factor II mRNA binding protein 1 (IMP1) enhanced the production of infectious MLV vector. Overexpression of IMP1 increased the stability of viral genomic RNA in virus producer cells and packaging of the RNA into progeny virus in a dose-dependent manner. Downregulation of IMP1 in virus producer cells resulted in reduced production of the retroviral vector. These results indicate that IMP1 plays a role in regulating the packaging of MLV genomic RNA and can be used for improving production of retroviral vectors.

  4. The rhinovirus type 14 genome contains an internally located RNA structure that is required for viral replication.

    Science.gov (United States)

    McKnight, K L; Lemon, S M

    1998-12-01

    Cis-acting RNA signals are required for replication of positive-strand viruses such as the picornaviruses. Although these generally have been mapped to the 5' and/or 3' termini of the viral genome, RNAs derived from human rhinovirus type 14 are unable to replicate unless they contain an internal cis-acting replication element (cre) located within the genome segment encoding the capsid proteins. Here, we show that the essential cre sequence is 83-96 nt in length and located between nt 2318-2413 of the genome. Using dicistronic RNAs in which translation of the P1 and P2-P3 segments of the polyprotein were functionally dissociated, we further demonstrate that translation of the cre sequence is not required for RNA replication. Thus, although it is located within a protein-coding segment of the genome, the cre functions as an RNA entity. Computer folds suggested that cre sequences could form a stable structure in either positive- or minus-strand RNA. However, an analysis of mutant RNAs containing multiple covariant and non-covariant nucleotide substitutions within these putative structures demonstrated that only the predicted positive-strand structure is essential for efficient RNA replication. The absence of detectable minus-strand synthesis from RNAs that lack the cre suggests that the cre is required for initiation of minus-strand RNA synthesis. Since a lethal 3' noncoding region mutation could be partially rescued by a compensating mutation within the cre, the cre appears to participate in a long-range RNA-RNA interaction required for this process. These data provide novel insight into the mechanisms of replication of a positive-strand RNA virus, as they define the involvement of an internally located RNA structure in the recognition of viral RNA by the viral replicase complex. Since internally located RNA replication signals have been shown to exist in several other positive-strand RNA virus families, these observations are potentially relevant to a wide array of

  5. Sphingosine kinase 1 serves as a pro-viral factor by regulating viral RNA synthesis and nuclear export of viral ribonucleoprotein complex upon influenza virus infection.

    Directory of Open Access Journals (Sweden)

    Young-Jin Seo

    Full Text Available Influenza continues to pose a threat to humans by causing significant morbidity and mortality. Thus, it is imperative to investigate mechanisms by which influenza virus manipulates the function of host factors and cellular signal pathways. In this study, we demonstrate that influenza virus increases the expression and activation of sphingosine kinase (SK 1, which in turn regulates diverse cellular signaling pathways. Inhibition of SK suppressed virus-induced NF-κB activation and markedly reduced the synthesis of viral RNAs and proteins. Further, SK blockade interfered with activation of Ran-binding protein 3 (RanBP3, a cofactor of chromosome region maintenance 1 (CRM1, to inhibit CRM1-mediated nuclear export of the influenza viral ribonucleoprotein complex. In support of this observation, SK inhibition altered the phosphorylation of ERK, p90RSK, and AKT, which is the upstream signal of RanBP3/CRM1 activation. Collectively, these results indicate that SK is a key pro-viral factor regulating multiple cellular signal pathways triggered by influenza virus infection.

  6. Detecting respiratory viral RNA using expanded genetic alphabets and self-avoiding DNA.

    Science.gov (United States)

    Glushakova, Lyudmyla G; Sharma, Nidhi; Hoshika, Shuichi; Bradley, Andrea C; Bradley, Kevin M; Yang, Zunyi; Benner, Steven A

    2015-11-15

    Nucleic acid (NA)-targeted tests detect and quantify viral DNA and RNA (collectively xNA) to support epidemiological surveillance and, in individual patients, to guide therapy. They commonly use polymerase chain reaction (PCR) and reverse transcription PCR. Although these all have rapid turnaround, they are expensive to run. Multiplexing would allow their cost to be spread over multiple targets, but often only with lower sensitivity and accuracy, noise, false positives, and false negatives; these arise by interactions between the multiple nucleic acid primers and probes in a multiplexed kit. Here we offer a multiplexed assay for a panel of respiratory viruses that mitigates these problems by combining several nucleic acid analogs from the emerging field of synthetic biology: (i) self-avoiding molecular recognition systems (SAMRSs), which facilitate multiplexing, and (ii) artificially expanded genetic information systems (AEGISs), which enable low-noise PCR. These are supplemented by "transliteration" technology, which converts standard nucleotides in a target to AEGIS nucleotides in a product, improving hybridization. The combination supports a multiplexed Luminex-based respiratory panel that potentially differentiates influenza viruses A and B, respiratory syncytial virus, severe acute respiratory syndrome coronavirus (SARS), and Middle East respiratory syndrome (MERS) coronavirus, detecting as few as 10 MERS virions in a 20-μl sample. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Nevirapine, sodium concentration and HIV-1 RNA in breast milk and plasma among HIV-infected women receiving short-course antiretroviral prophylaxis

    DEFF Research Database (Denmark)

    Salado-Rasmussen, Kirsten; Theilgaard, Zahra Persson; Chiduo, Mercy G.

    2015-01-01

    Introduction Risk factors for breast milk transmission of HIV-1 from mother to child include high plasma and breast milk viral load, low maternal CD4 count and breast pathology such as mastitis. Objective To determine the impact of nevirapine and subclinical mastitis on HIV-1 RNA in maternal plasma...... and breast milk after intrapartum single-dose nevirapine combined with either 1-week tail of Combivir (zidovudine/lamivudine) or single-dose Truvada (tenofovir/emtricitabine). Methods Maternal plasma and bilateral breast milk samples were collected between April 2008 and April 2011 at 1, 4 and 6 weeks...... postpartum from HIV-infected Tanzanian women. Moreover, plasma samples were collected at delivery from mother and infant. Results HIV-1 RNA was quantified in 1,212 breast milk samples from 273 women. At delivery, 96% of the women and 99% of the infants had detectable nevirapine in plasma with a median...

  8. Effects of hemoperfusion adsorption and/or plasma exchange in treatment of severe viral hepatitis:A comparative study

    Institute of Scientific and Technical Information of China (English)

    Nian-Hai He; Ying-Jie Wang; Ze-Wen Wang; Jun Liu; Jia-Jia Li; Guo-Dong Liu; Yu-Ming Wang

    2004-01-01

    AIM: Non-bioartificial liver has been applied to clinic for quite a long time, but the reported efficacy has been very different. The aim of this study was to compare the efficacy and safety of hemoperfusion adsorption, plasma exchange and plasma exchange plus hemoperfusion adsorption in treatment of severe viral hepatitis.METHODS: Seventy-five patients with severe viral hepatitis were treated with hemoperfusion adsorption therapy (24cases), plasma exchange therapy (17 cases) and plasma exchange plus hemoperfusion adsorption therapy (34 cases).The data of liver function, renal function, blood routine test,prothrombin time (PT) and prothrombin activity (PTa) preand post-therapy were analyzed.RESULTS: Clinical symptoms of patients improved after treatment. The levels of aminotransferase, total bilirubin,direct bilirubin decreased significantly after 3 therapies (P<0.05 or P<0.01). PT, the level of total serum protein decreased significantly and PTa increased significantly after plasma exchange therapy and plasma exchange plus hemoperfusion adsorption therapy (P<0.05 or P<0.01). The side effects were few and mild in all patients.CONCLUSION: Three therapies were effective in the treatment of severe viral hepatitis. Plasma exchange therapy and plasma exchange plus hemoperfusion adsorption therapy are better than hemoperfusion adsorption therapy.

  9. Effects of hemoperfusion adsorption and/or plasma exchange in treatment of severe viral hepatitis: A comparative study

    Science.gov (United States)

    He, Nian-Hai; Wang, Ying-Jie; Wang, Ze-Wen; Liu, Jun; Li, Jia-Jia; Liu, Guo-Dong; Wang, Yu-Ming

    2004-01-01

    AIM: Non-bioartificial liver has been applied to clinic for quite a long time, but the reported efficacy has been very different. The aim of this study was to compare the efficacy and safety of hemoperfusion adsorption, plasma exchange and plasma exchange plus hemoperfusion adsorption in treatment of severe viral hepatitis. METHODS: Seventy-five patients with severe viral hepatitis were treated with hemoperfusion adsorption therapy (24 cases), plasma exchange therapy (17 cases) and plasma exchange plus hemoperfusion adsorption therapy (34 cases). The data of liver function, renal function, blood routine test, prothrombin time (PT) and prothrombin activity (PTa) pre-and post-therapy were analyzed. RESULTS: Clinical symptoms of patients improved after treatment. The levels of aminotransferase, total bilirubin, direct bilirubin decreased significantly after 3 therapies (P < 0.05 or P < 0.01). PT, the level of total serum protein decreased significantly and PTa increased significantly after plasma exchange therapy and plasma exchange plus hemoperfusion adsorption therapy (P < 0.05 or P < 0.01). The side effects were few and mild in all patients. CONCLUSION: Three therapies were effective in the treatment of severe viral hepatitis. Plasma exchange therapy and plasma exchange plus hemoperfusion adsorption therapy are better than hemoperfusion adsorption therapy. PMID:15069730

  10. Poliovirus temperature-sensitive mutant containing a single nucleotide deletion in the 5'-noncoding region of the viral RNA.

    Science.gov (United States)

    Racaniello, V R; Meriam, C

    1986-12-01

    The effect on viral replication of deleting nucleotide 10 of the poliovirus RNA genome was determined. This deletion, which removes a base pair from a predicted hairpin structure in the viral RNA, was introduced into full-length cDNA. Virus recovered after transfection of HeLa cells with the mutated cDNA contained the expected deletion and was temperature sensitive for plaque formation. Analysis of viral replication by one-step growth experiments indicated that mutant virus production at the nonpermissive temperature was at least 100 times less than that of wild type virus, and release of virus from mutant-infected cells was delayed. The synthesis of positive- and negative-strand viral RNA in mutant virus-infected cells was temperature sensitive. Virus-specific protein synthesis in mutant virus-infected cells was not temperature sensitive but occurred at a slower rate than that of wild type virus at permissive and nonpermissive temperatures. Replication of the mutant virus was sensitive to actinomycin D, in contrast to the wild type parent virus, which was resistant to the drug. Mutant virus stocks contained a small percentage of ts+ viruses that were able to form plaques at the nonpermissive temperature. Nucleotide sequence analysis of genomic RNA from these ts+ viruses revealed a single base change at position 34 from a G to U. In the positive RNA strand, the effect of this mutation is to restore to the hairpin structure the single base pair whose formation was prevented by the original deletion. The ts+ pseudorevertants replicated to similar titers as wild type virus at 33 and 38.5 degrees and were partially sensitive to actinomycin D.

  11. In vivo evolution of the gp90 gene and consistently low plasma viral load during transient immune suppression demonstrate the safety of an attenuated equine infectious anemia virus (EIAV) vaccine.

    Science.gov (United States)

    Ma, Jian; Jiang, Chenggang; Lin, Yuezhi; Wang, Xuefeng; Zhao, Liping; Xiang, Wenhua; Shao, Yiming; Shen, Rongxian; Kong, Xiangang; Zhou, Jianhua

    2009-01-01

    To study the in vivo evolution of the attenuated Chinese equine infectious anemia virus (EIAV) vaccine, viral gp90 gene variation and virus replication in immunosuppressed hosts were investigated. The results showed that after vaccination, the gp90 gene followed an evolutionary trend of declining diversity. The trend coincided with the maturation of immunity to EIAV, and eventually, the gp90 gene became highly homologous. The sequences of these predominant quasispecies were consistently detected up to 18 months after vaccination. Furthermore, after transient immune suppression with dexamethasone, the plasma viral RNA copy number of the vaccine strain in three vaccinated ponies remained consistently below the "pathogenic threshold" level, while the viral load increased by 25,000-fold in the positive control of an inapparent carrier of the parental virulent strain. This study is the first to provide evidence for the safety of an attenuated lentiviral vaccine with decreased genomic diversity and consistently low viral replication under suppressed immunity.

  12. Factors influencing cerebrospinal fluid and plasma HIV-1 RNA detection rate in patients with and without opportunistic neurological disease during the HAART era

    Directory of Open Access Journals (Sweden)

    Aleixo Agdemir W

    2007-12-01

    Full Text Available Abstract Background In the central nervous system, HIV replication can occur relatively independent of systemic infection, and intrathecal replication of HIV-1 has been observed in patients with HIV-related and opportunistic neurological diseases. The clinical usefulness of HIV-1 RNA detection in the cerebrospinal fluid (CSF of patients with opportunistic neurological diseases, or the effect of opportunistic diseases on CSF HIV levels in patients under HAART has not been well defined. We quantified CSF and plasma viral load in HIV-infected patients with and without different active opportunistic neurological diseases, determined the characteristics that led to a higher detection rate of HIV RNA in CSF, and compared these two compartments. Methods A prospective study was conducted on 90 HIV-infected patients submitted to lumbar puncture as part of a work-up for suspected neurological disease. Seventy-one patients had active neurological diseases while the remaining 19 did not. Results HIV-1 RNA was quantified in 90 CSF and 70 plasma samples. The HIV-1 RNA detection rate in CSF was higher in patients with neurological diseases, in those with a CD4 count lower than 200 cells/mm3, and in those not receiving antiretroviral therapy, as well as in patients with detectable plasma HIV-1 RNA. Median viral load was lower in CSF than in plasma in the total population, in patients without neurological diseases, and in patients with toxoplasmic encephalitis, while no significant difference between the two compartments was observed for patients with cryptococcal meningitis and HIV-associated dementia. CSF viral load was lower in patients with cryptococcal meningitis and neurotoxoplasmosis under HAART than in those not receiving HAART. Conclusion Detection of HIV-1 RNA in CSF was more frequent in patients with neurological disease, a CD4 count lower than 200 cells/mm3 and detectable plasma HIV-1. Median HIV-1 RNA levels were generally lower in CSF than in

  13. Selective translational repression of HIV-1 RNA by Sam68DeltaC occurs by altering PABP1 binding to unspliced viral RNA

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    Soros Vanessa

    2008-10-01

    Full Text Available Abstract HIV-1 structural proteins are translated from incompletely spliced 9 kb and 4 kb mRNAs, which are transported to the cytoplasm by Crm1. It has been assumed that once in the cytoplasm, translation of incompletely spliced HIV-1 mRNAs occurs in the same manner as host mRNAs. Previous analyses have demonstrated that Sam68 and a mutant thereof, Sam68ΔC, have dramatic effects on HIV gene expression, strongly enhancing and inhibiting viral structural protein synthesis, respectively. While investigating the inhibition of incompletely spliced HIV-1 mRNAs by Sam68ΔC, we determined that the effect was independent of the perinuclear bundling of the viral RNA. Inhibition was dependent upon the nuclear export pathway used, as translation of viral RNA exported via the Tap/CTE export pathway was not blocked by Sam68ΔC. We demonstrate that inhibition of HIV expression by Sam68ΔC is correlated with a loss of PABP1 binding with no attendant change in polyadenosine tail length of the affected RNAs. The capacity of Sam68ΔC to selectively inhibit translation of HIV-1 RNAs exported by Crm1 suggests that it is able to recognize unique characteristics of these viral RNPs, a property that could lead to new therapeutic approaches to controlling HIV-1 replication.

  14. Stimulation of poliovirus RNA synthesis and virus maturation in a HeLa cell-free in vitro translation-RNA replication system by viral protein 3CDpro

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    Wimmer Eckard

    2005-11-01

    Full Text Available Abstract Poliovirus protein 3CDpro possesses both proteinase and RNA binding activities, which are located in the 3Cpro domain of the protein. The RNA polymerase (3Dpol domain of 3CDpro modulates these activities of the protein. We have recently shown that the level of 3CDpro in HeLa cell-free in vitro translation-RNA replication reactions is suboptimal for efficient virus production. However, the addition of either 3CDpro mRNA or of purified 3CDpro protein to in vitro reactions, programmed with viral RNA, results in a 100-fold increase in virus yield. Mutational analyses of 3CDpro indicated that RNA binding by the 3Cpro domain and the integrity of interface I in the 3Dpol domain of the protein are both required for function. The aim of these studies was to determine the exact step or steps at which 3CDpro enhances virus yield and to determine the mechanism by which this occurs. Our results suggest that the addition of extra 3CDpro to in vitro translation RNA-replication reactions results in a mild enhancement of both minus and plus strand RNA synthesis. By examining the viral particles formed in the in vitro reactions on sucrose gradients we determined that 3CDpro has only a slight stimulating effect on the synthesis of capsid precursors but it strikingly enhances the maturation of virus particles. Both the stimulation of RNA synthesis and the maturation of the virus particles are dependent on the presence of an intact RNA binding site within the 3Cpro domain of 3CDpro. In addition, the integrity of interface I in the 3Dpol domain of 3CDpro is required for efficient production of mature virus. Surprisingly, plus strand RNA synthesis and virus production in in vitro reactions, programmed with full-length transcript RNA, are not enhanced by the addition of extra 3CDpro. Our results indicate that the stimulation of RNA synthesis and virus maturation by 3CDpro in vitro is dependent on the presence of a VPg-linked RNA template.

  15. Sexually-Transmitted/Founder HIV-1 Cannot Be Directly Predicted from Plasma or PBMC-Derived Viral Quasispecies in the Transmitting Partner

    Science.gov (United States)

    Frange, Pierre; Meyer, Laurence; Jung, Matthieu; Goujard, Cecile; Zucman, David; Abel, Sylvie; Hochedez, Patrick; Gousset, Marine; Gascuel, Olivier; Rouzioux, Christine; Chaix, Marie-Laure

    2013-01-01

    Objective Characterization of HIV-1 sequences in newly infected individuals is important for elucidating the mechanisms of viral sexual transmission. We report the identification of transmitted/founder viruses in eight pairs of HIV-1 sexually-infected patients enrolled at the time of primary infection (“recipients”) and their transmitting partners (“donors”). Methods Using a single genome-amplification approach, we compared quasispecies in donors and recipients on the basis of 316 and 376 C2V5 env sequences amplified from plasma viral RNA and PBMC-associated DNA, respectively. Results Both DNA and RNA sequences indicated very homogeneous viral populations in all recipients, suggesting transmission of a single variant, even in cases of recent sexually transmitted infections (STIs) in donors (n = 2) or recipients (n = 3). In all pairs, the transmitted/founder virus was derived from an infrequent variant population within the blood of the donor. The donor variant sequences most closely related to the recipient sequences were found in plasma samples in 3/8 cases and/or in PBMC samples in 6/8 cases. Although donors were exclusively (n = 4) or predominantly (n = 4) infected by CCR5-tropic (R5) strains, two recipients were infected with highly homogeneous CXCR4/dual-mixed-tropic (X4/DM) viral populations, identified in both DNA and RNA. The proportion of X4/DM quasispecies in donors was higher in cases of X4/DM than R5 HIV transmission (16.7–22.0% versus 0–2.6%), suggesting that X4/DM transmission may be associated with a threshold population of X4/DM circulating quasispecies in donors. Conclusions These suggest that a severe genetic bottleneck occurs during subtype B HIV-1 heterosexual and homosexual transmission. Sexually-transmitted/founder virus cannot be directly predicted by analysis of the donor’s quasispecies in plasma and/or PBMC. Additional studies are required to fully understand the traits that confer the capacity to transmit and

  16. Double-stranded RNA viral infection of Trichomonas vaginalis infecting patients attending a sexually transmitted diseases clinic.

    Science.gov (United States)

    Wendel, Karen A; Rompalo, Anne M; Erbelding, Emily J; Chang, T-H; Alderete, John F

    2002-08-15

    Trichomonas vaginalis (TV) can be infected with double-stranded RNA (dsRNA) viruses that may have important implications for trichomonal virulence and disease pathogenesis. A cross-sectional study was conducted in a sexually transmitted diseases clinic to determine the prevalence and clinical significance of dsRNA viral infection of TV infecting men and women. Overall, dsRNA virus was present in 21 (75%) of 28 TV isolates (95% confidence interval [CI], 55%-89%). dsRNA viral infection of TV was not associated with the presence of discharge, dysuria, genital pruritus, or genital irritation or odor. However, patients with virus-positive isolates were significantly older than patients with virus-negative isolates (median age, 38 vs. 23 years; P=.003), and virus-positive isolates were more prevalent among women (19 [86%] of 22 isolates; 95% CI, 65%-97%) than among men (2 [33%] of 6 isolates; P=.02). The age and sex specificity of virus-positive isolates may aid in understanding the differences in chronicity and clinical presentation of TV in men and women.

  17. Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection.

    Science.gov (United States)

    Au, Hilda H; Cornilescu, Gabriel; Mouzakis, Kathryn D; Ren, Qian; Burke, Jordan E; Lee, Seonghoon; Butcher, Samuel E; Jan, Eric

    2015-11-24

    The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the three-helical junction of the honey bee dicistrovirus Israeli acute paralysis virus (IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. The tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame.

  18. RNA viral metagenome of whiteflies leads to the discovery and characterization of a whitefly-transmitted carlavirus in North America.

    Directory of Open Access Journals (Sweden)

    Karyna Rosario

    Full Text Available Whiteflies from the Bemisia tabaci species complex have the ability to transmit a large number of plant viruses and are some of the most detrimental pests in agriculture. Although whiteflies are known to transmit both DNA and RNA viruses, most of the diversity has been recorded for the former, specifically for the Begomovirus genus. This study investigated the total diversity of DNA and RNA viruses found in whiteflies collected from a single site in Florida to evaluate if there are additional, previously undetected viral types within the B. tabaci vector. Metagenomic analysis of viral DNA extracted from the whiteflies only resulted in the detection of begomoviruses. In contrast, whiteflies contained sequences similar to RNA viruses from divergent groups, with a diversity that extends beyond currently described viruses. The metagenomic analysis of whiteflies also led to the first report of a whitefly-transmitted RNA virus similar to Cowpea mild mottle virus (CpMMV Florida (genus Carlavirus in North America. Further investigation resulted in the detection of CpMMV Florida in native and cultivated plants growing near the original field site of whitefly collection and determination of its experimental host range. Analysis of complete CpMMV Florida genomes recovered from whiteflies and plants suggests that the current classification criteria for carlaviruses need to be reevaluated. Overall, metagenomic analysis supports that DNA plant viruses carried by B. tabaci are dominated by begomoviruses, whereas significantly less is known about RNA viruses present in this damaging insect vector.

  19. RNA viral metagenome of whiteflies leads to the discovery and characterization of a whitefly-transmitted carlavirus in North America.

    Science.gov (United States)

    Rosario, Karyna; Capobianco, Heather; Ng, Terry Fei Fan; Breitbart, Mya; Polston, Jane E

    2014-01-01

    Whiteflies from the Bemisia tabaci species complex have the ability to transmit a large number of plant viruses and are some of the most detrimental pests in agriculture. Although whiteflies are known to transmit both DNA and RNA viruses, most of the diversity has been recorded for the former, specifically for the Begomovirus genus. This study investigated the total diversity of DNA and RNA viruses found in whiteflies collected from a single site in Florida to evaluate if there are additional, previously undetected viral types within the B. tabaci vector. Metagenomic analysis of viral DNA extracted from the whiteflies only resulted in the detection of begomoviruses. In contrast, whiteflies contained sequences similar to RNA viruses from divergent groups, with a diversity that extends beyond currently described viruses. The metagenomic analysis of whiteflies also led to the first report of a whitefly-transmitted RNA virus similar to Cowpea mild mottle virus (CpMMV Florida) (genus Carlavirus) in North America. Further investigation resulted in the detection of CpMMV Florida in native and cultivated plants growing near the original field site of whitefly collection and determination of its experimental host range. Analysis of complete CpMMV Florida genomes recovered from whiteflies and plants suggests that the current classification criteria for carlaviruses need to be reevaluated. Overall, metagenomic analysis supports that DNA plant viruses carried by B. tabaci are dominated by begomoviruses, whereas significantly less is known about RNA viruses present in this damaging insect vector.

  20. Herpes simplex virus VP16 rescues viral mRNA from destruction by the virion host shutoff function.

    Science.gov (United States)

    Lam, Q; Smibert, C A; Koop, K E; Lavery, C; Capone, J P; Weinheimer, S P; Smiley, J R

    1996-05-15

    Herpes simplex virus (HSV) virions contain two regulatory proteins that facilitate the onset of the lytic cycle: VP16 activates transcription of the viral immediate-early genes, and vhs triggers shutoff of host protein synthesis and accelerated turnover of cellular and viral mRNAs. VP16 and vhs form a complex in infected cells, raising the possibility of a regulatory link between them. Here we show that viral protein synthesis and mRNA levels undergo a severe decline at intermediate times after infection with a VP16 null mutant, culminating in virtually complete translational arrest. This phenotype was rescued by a transcriptionally incompetent derivative of VP16 that retains vhs binding activity, and was eliminated by inactivating the vhs gene. These results indicate that VP16 dampens vhs activity, allowing HSV mRNAs to persist in infected cells. Further evidence supporting this hypothesis came from the demonstration that a stably transfected cell line expressing VP16 was resistant to host shutoff induced by superinfecting HSV virions. Thus, in addition to its well known function as a transcriptional activator, VP16 stimulates viral gene expression at a post-transcriptional level, by sparing viral mRNAs from degradation by one of the virus-induced host shutoff mechanisms.

  1. ASSASYING THE NEED OF COMMERCIAL PLASMA VIRAL LOAD TESTING IN RESOURCE LIMITED SETTINGS

    Directory of Open Access Journals (Sweden)

    Arnaw

    2015-09-01

    Full Text Available Around nine million Human Immunodeficiency Virus (HIV infected individuals are on antiretroviral therapy (ART. People living with HIV/AIDS in resource - limited settings where HIV burden is usually high, there is an urgent need of affordable, accessible and inexpensive tests to monitor response to treatment. Quite a few commercially available assay has been introduced to measure Plasma Viral Load (PVL as testing can increase adherence to ART and facilitate timely switching of failing regimens and thus minimizing the development of resistance. We analyzed Nucleic Acid Test (NAT based assay and Non Nucleic Acid Test based assay for PVL testing. Though both the assay has its own advantage and disadvantages, but the use of Non Nucleic Acid Test has an upper hand in resource limited settings. It is the duty of administration, clinicians, microbiologist and health care personnel to introduce appropriate laboratory monitoring assays in resource - limited settings.

  2. HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors.

    Science.gov (United States)

    Pocock, Ginger M; Becker, Jordan T; Swanson, Chad M; Ahlquist, Paul; Sherer, Nathan M

    2016-04-01

    Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent "burst-like" transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm.

  3. Viral Delivery of dsRNA for Control of Insect Agricultural Pests and Vectors of Human Disease: Prospects and Challenges

    Science.gov (United States)

    Kolliopoulou, Anna; Taning, Clauvis N. T.; Smagghe, Guy; Swevers, Luc

    2017-01-01

    RNAi is applied as a new and safe method for pest control in agriculture but efficiency and specificity of delivery of dsRNA trigger remains a critical issue. Various agents have been proposed to augment dsRNA delivery, such as engineered micro-organisms and synthetic nanoparticles, but the use of viruses has received relatively little attention. Here we present a critical view of the potential of the use of recombinant viruses for efficient and specific delivery of dsRNA. First of all, it requires the availability of plasmid-based reverse genetics systems for virus production, of which an overview is presented. For RNA viruses, their application seems to be straightforward since dsRNA is produced as an intermediate molecule during viral replication, but DNA viruses also have potential through the production of RNA hairpins after transcription. However, application of recombinant virus for dsRNA delivery may not be straightforward in many cases, since viruses can encode RNAi suppressors, and virus-induced silencing effects can be determined by the properties of the encoded RNAi suppressor. An alternative is virus-like particles that retain the efficiency and specificity determinants of natural virions but have encapsidated non-replicating RNA. Finally, the use of viruses raises important safety issues which need to be addressed before application can proceed. PMID:28659820

  4. eIF2A mediates translation of hepatitis C viral mRNA under stress conditions.

    Science.gov (United States)

    Kim, Joon Hyun; Park, Sung Mi; Park, Ji Hoon; Keum, Sun Ju; Jang, Sung Key

    2011-05-10

    Translation of most mRNAs is suppressed under stress conditions. Phosphorylation of the α-subunit of eukaryotic translation initiation factor 2 (eIF2), which delivers initiator tRNA (Met-tRNA(i)) to the P site of the 40S ribosomal subunit, is responsible for such translational suppression. However, translation of hepatitis C viral (HCV) mRNA is refractory to the inhibitory effects of eIF2α phosphorylation, which prevents translation by disrupting formation of the eIF2-GTP-Met-tRNA(i) ternary complex. Here, we report that eIF2A, an alternative initiator tRNA-binding protein, has a key role in the translation of HCV mRNA during HCV infection, in turn promoting eIF2α phosphorylation by activating the eIF2α kinase PKR. Direct interaction of eIF2A with the IIId domain of the HCV internal ribosome entry site (IRES) is required for eIF2A-dependent translation. These data indicate that stress-independent translation of HCV mRNA occurs by recruitment of eIF2A to the HCV IRES via direct interaction with the IIId domain and subsequent loading of Met-tRNA(i) to the P site of the 40S ribosomal subunit.

  5. Residual viraemia in HIV-1-infected patients with plasma viral load

    DEFF Research Database (Denmark)

    Ostrowski, S.R.; Katzenstein, T.L.; Pedersen, Bente Klarlund

    2008-01-01

    antiretroviral therapy (HAART)-treated HIV-1-infected patients with plasma HIV-1 RNA or=1 episode with TMA-RV whereas 9 patients had undetectable TMA-RV throughout the study-period. Time-points with TMA-RV and PCR-RV were associated with higher circulating sTNFrII (+0.234 ng/ml, P = 0.030) and beta(2......)-microglobulin (+22 nmol/l, P = 0.016) and time-points with PCR-RV were also associated with higher IgA (+0.82 micromol/l, P = 0.035) and CD8-count (+1.18-fold, P = 0.001). Patients with TMA-RV in the study-period had higher HIV-1 RNA pre-HAART (P = 0.032). RV was not associated with proviral-HIV-1-DNA, CD4...

  6. Evaluation of the Whole-Blood Alere Q NAT Point-of-Care RNA Assay for HIV-1 Viral Load Monitoring in a Primary Health Care Setting in Mozambique.

    Science.gov (United States)

    Jani, Ilesh V; Meggi, Bindiya; Vubil, Adolfo; Sitoe, Nádia E; Bhatt, Nilesh; Tobaiwa, Ocean; Quevedo, Jorge I; Loquiha, Osvaldo; Lehe, Jonathan D; Vojnov, Lara; Peter, Trevor F

    2016-08-01

    Viral load testing is the WHO-recommended monitoring assay for patients on HIV antiretroviral therapy (ART). Point-of-care (POC) assays may help improve access to viral load testing in resource-limited settings. We compared the performance of the Alere Q NAT POC viral load technology (Alere Technologies, Jena, Germany), measuring total HIV RNA using finger prick capillary whole-blood samples collected in a periurban health center, with that of a laboratory-based plasma RNA test (Roche Cobas Ampliprep/Cobas TaqMan v2) conducted on matched venous blood samples. The whole-blood Alere Q NAT POC assay produced results with a bias of 0.8593 log copy/ml compared to the laboratory-based plasma assay. However, at above 10,000 copies/ml, the bias was 0.07 log copy/ml. Using the WHO-recommended threshold to determine ART failure of 1,000 copies/ml, the sensitivity and specificity of the whole-blood Alere Q NAT POC assay were 96.83% and 47.80%, respectively. A cutoff of 10,000 copies/ml of whole blood with the Alere Q NAT POC assay appears to be a better predictor of ART failure threshold (1,000 copies/ml of plasma), with a sensitivity of 84.0% and specificity of 90.3%. The precision of the whole-blood Alere Q NAT POC assay was comparable to that observed with the laboratory technology (5.4% versus 7.5%) between detectable paired samples. HIV POC viral load testing is feasible at the primary health care level. Further research on the value of whole-blood viral load to monitor antiretroviral therapy is warranted.

  7. Evaluation of the Whole-Blood Alere Q NAT Point-of-Care RNA Assay for HIV-1 Viral Load Monitoring in a Primary Health Care Setting in Mozambique

    Science.gov (United States)

    Meggi, Bindiya; Vubil, Adolfo; Sitoe, Nádia E.; Bhatt, Nilesh; Tobaiwa, Ocean; Quevedo, Jorge I.; Loquiha, Osvaldo; Lehe, Jonathan D.; Vojnov, Lara; Peter, Trevor F.

    2016-01-01

    Viral load testing is the WHO-recommended monitoring assay for patients on HIV antiretroviral therapy (ART). Point-of-care (POC) assays may help improve access to viral load testing in resource-limited settings. We compared the performance of the Alere Q NAT POC viral load technology (Alere Technologies, Jena, Germany), measuring total HIV RNA using finger prick capillary whole-blood samples collected in a periurban health center, with that of a laboratory-based plasma RNA test (Roche Cobas Ampliprep/Cobas TaqMan v2) conducted on matched venous blood samples. The whole-blood Alere Q NAT POC assay produced results with a bias of 0.8593 log copy/ml compared to the laboratory-based plasma assay. However, at above 10,000 copies/ml, the bias was 0.07 log copy/ml. Using the WHO-recommended threshold to determine ART failure of 1,000 copies/ml, the sensitivity and specificity of the whole-blood Alere Q NAT POC assay were 96.83% and 47.80%, respectively. A cutoff of 10,000 copies/ml of whole blood with the Alere Q NAT POC assay appears to be a better predictor of ART failure threshold (1,000 copies/ml of plasma), with a sensitivity of 84.0% and specificity of 90.3%. The precision of the whole-blood Alere Q NAT POC assay was comparable to that observed with the laboratory technology (5.4% versus 7.5%) between detectable paired samples. HIV POC viral load testing is feasible at the primary health care level. Further research on the value of whole-blood viral load to monitor antiretroviral therapy is warranted. PMID:27252459

  8. Replication protein of tobacco mosaic virus cotranslationally binds the 5′ untranslated region of genomic RNA to enable viral replication

    Science.gov (United States)

    Kawamura-Nagaya, Kazue; Ishibashi, Kazuhiro; Huang, Ying-Ping; Miyashita, Shuhei; Ishikawa, Masayuki

    2014-01-01

    Genomic RNA of positive-strand RNA viruses replicate via complementary (i.e., negative-strand) RNA in membrane-bound replication complexes. Before replication complex formation, virus-encoded replication proteins specifically recognize genomic RNA molecules and recruit them to sites of replication. Moreover, in many of these viruses, selection of replication templates by the replication proteins occurs preferentially in cis. This property is advantageous to the viruses in several aspects of viral replication and evolution, but the underlying molecular mechanisms have not been characterized. Here, we used an in vitro translation system to show that a 126-kDa replication protein of tobacco mosaic virus (TMV), a positive-strand RNA virus, binds a 5′-terminal ∼70-nucleotide region of TMV RNA cotranslationally, but not posttranslationally. TMV mutants that carried nucleotide changes in the 5′-terminal region and showed a defect in the binding were unable to synthesize negative-strand RNA, indicating that this binding is essential for template selection. A C-terminally truncated 126-kDa protein, but not the full-length 126-kDa protein, was able to posttranslationally bind TMV RNA in vitro, suggesting that binding of the 126-kDa protein to the 70-nucleotide region occurs during translation and before synthesis of the C-terminal inhibitory domain. We also show that binding of the 126-kDa protein prevents further translation of the bound TMV RNA. These data provide a mechanistic explanation of how the 126-kDa protein selects replication templates in cis and how fatal collision between translating ribosomes and negative-strand RNA-synthesizing polymerases on the genomic RNA is avoided. PMID:24711385

  9. Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle.

    Directory of Open Access Journals (Sweden)

    Benjamin G Kopek

    2007-09-01

    Full Text Available Positive-strand RNA viruses are the largest genetic class of viruses and include many serious human pathogens. All positive-strand RNA viruses replicate their genomes in association with intracellular membrane rearrangements such as single- or double-membrane vesicles. However, the exact sites of RNA synthesis and crucial topological relationships between relevant membranes, vesicle interiors, surrounding lumens, and cytoplasm generally are poorly defined. We applied electron microscope tomography and complementary approaches to flock house virus (FHV-infected Drosophila cells to provide the first 3-D analysis of such replication complexes. The sole FHV RNA replication factor, protein A, and FHV-specific 5-bromouridine 5'-triphosphate incorporation localized between inner and outer mitochondrial membranes inside approximately 50-nm vesicles (spherules, which thus are FHV-induced compartments for viral RNA synthesis. All such FHV spherules were outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked channel of approximately 10-nm diameter, which is sufficient for ribonucleotide import and product RNA export. Tomographic, biochemical, and other results imply that FHV spherules contain, on average, three RNA replication intermediates and an interior shell of approximately 100 membrane-spanning, self-interacting protein As. The results identify spherules as the site of protein A and nascent RNA accumulation and define spherule topology, dimensions, and stoichiometry to reveal the nature and many details of the organization and function of the FHV RNA replication complex. The resulting insights appear relevant to many other positive-strand RNA viruses and support recently proposed structural and likely evolutionary parallels with retrovirus and double-stranded RNA virus virions.

  10. Differences in HIV type 1 RNA plasma load profile of closely related cocirculating Ethiopian subtype C strains: C and C'.

    Science.gov (United States)

    Ayele, Workenesh; Mekonnen, Yared; Messele, Tsehaynesh; Mengistu, Yohannes; Tsegaye, Aster; Bakker, Margreet; Berkhout, Ben; Dorigo-Zetsma, Wendelien; Wolday, Dawit; Goudsmit, Jaap; Coutinho, Roel; de Baar, Michel; Paxton, William A; Pollakis, Georgios

    2010-07-01

    Two HIV-1 subtype C subclusters have been identified in Ethiopia (C and C') with little knowledge regarding their biological or clinical differences. We longitudinally monitored HIV-1 viral loads and CD4(+) T cell counts for 130 subtype C-infected individuals from Ethiopia over 5 years. The genetic subclusters C and C' were determined and comparisons were made between the groups. None of the study individuals received antiretroviral therapy. Subcluster C' was found to be the more prevalent (72.3%) genotype circulating. Individuals infected with subcluster C' harbored higher viral loads in comparison to subcluster C-infected individuals when the CD4(+) T cell counts were high (500-900 cells/mm(3)), whereas at low CD4(+) T cell counts (0-150 cells/mm(3)) individuals infected with subcluster C viruses showed higher viral loads. We identified a greater number of deaths among individuals infected with subcluster C viruses in comparison to C'. Our results indicate that infection with subcluster C viruses leads to a more rapid onset of disease, despite the initial lower HIV-1 RNA plasma loads. Additionally, the higher viral loads seen for HIV-1 subcluster C' infections at higher CD4(+) T cell counts can help explain the higher prevalence of this subtype in Ethiopia.

  11. Expression of the late cytomegalovirus (CMV) pp150 transcript in leukocytes of AIDS patients is associated with a high viral DNA load in leukocytes and presence of CMV DNA in plasma.

    Science.gov (United States)

    Boivin, G; Handfield, J; Toma, E; Lalonde, R; Bergeron, M G

    1999-05-01

    The expression of a late cytomegalovirus (CMV) transcript (pp150) was sought in peripheral blood leukocytes (PBL) of subjects with AIDS and correlated with the amounts of CMV DNA in PBL and plasma, by means of quantitative polymerase chain reaction (PCR). The detection of the late CMV transcript was associated with a high number of CMV DNA copies in PBL (P=.0015) and with a positive CMV PCR assay in plasma (P<.001). Expression of CMV pp150 mRNA was best predicted by viral DNA thresholds corresponding to 7058 and 30 copies in PBL and plasma, respectively. The detection of CMV pp150 mRNA was associated with the presence of CMV disease in a univariate analysis but not in a multivariate analysis after controlling for the viral DNA load in PBL. Thus, active viral replication as determined by a high CMV DNA load in PBL is reflected by expression of the late CMV transcript in the same cells and by the presence of CMV DNA in plasma.

  12. HIV-1 Rev protein specifies the viral RNA export pathway by suppressing TAP/NXF1 recruitment.

    Science.gov (United States)

    Taniguchi, Ichiro; Mabuchi, Naoto; Ohno, Mutsuhito

    2014-06-01

    Nuclear RNA export pathways in eukaryotes are often linked to the fate of a given RNA. Therefore, the choice of export pathway should be well-controlled to avoid an unfavorable effect on gene expression. Although some RNAs could be exported by more than one pathway, little is known about how the choice is regulated. This issue is highlighted when the human immunodeficiency virus type 1 (HIV-1) Rev protein induces the export of singly spliced and unspliced HIV-1 transcripts. How these RNAs are exported is not well understood because such transcripts should have the possibility of utilizing CRM1-dependent export via Rev or cellular TAP/NXF1-dependent export via the transcription/export (TREX) complex, or both. Here we found that Rev suppressed TAP/NXF1-dependent export of model RNA substrates that recapitulated viral transcripts. In this effect, Rev interacted with the cap-binding complex and inhibited the recruitment of the TREX complex. Thus, Rev controls the identity of the factor occupying the cap-proximal region that determines the RNA export pathway. This ribonucleoprotein remodeling activity of Rev may favor viral gene expression.

  13. Separation and quantification of viral double-stranded RNA fragments by capillary electrophoresis in hydroxyethylcellulose polymer solutions.

    Science.gov (United States)

    Shambaugh, C L; Bodmer, J L; Hsu, D; Ranucci, C S

    2004-10-01

    Capillary electrophoresis (CE) is an analytical technique widely utilized to resolve complex mixtures of nucleic acids. CE uses a variety of polymers in solution that act as a molecular sieve to separate nucleic acid fragments according to size. It has been shown previously that purified dsDNA can be resolved efficiently by solutions of hydroxyethylcellulose (HEC) polymer, providing a rapid and high resolution method of separation. We have applied this separation technique to viral double-stranded (ds) RNA segments derived from rotavirus process samples. HEC polymers of various molecular masses and concentrations were identified and compared for their ability to separate dsRNA based on the extent of expected polymer network formation. The HEC polymer exhibiting the most desirable separation characteristics was then used for subsequent optimization of various method parameters, such as, injection time, electric field strength, dye concentration and capillary equilibration. The optimized method was then applied to the quantification of genome concentration based on a representative segment of the rotavirus genome. This study demonstrated that purified viral dsRNA material of known concentration could be used to generate an external standard curve relating concentration to peak area. This standard curve was used to determine the concentration of unknown samples by interpolation. This novel RNA quantification assay is likely to be applicable to other types of virus, including those containing dsDNA.

  14. Response of porcine hepatocytes in primary culture to plasma from severe viral hepatitis patients

    Institute of Scientific and Technical Information of China (English)

    Yong-Bo Cheng; Ying-Jie Wang; Shi-Chang Zhang; Jun Liu; Zhi Chen; Jia-Jia Li

    2005-01-01

    AIM: To observe the effects of plasma from patients with severe viral hepatitis (SVHP) on the growth and metabolism of porcine hepatocytes and the clinical efficiency of bioartificial liver device.METHODS: Hepatocytes were isolated from male porcines by collagenase perfusion. The synthesis of DNA and total protein, leakages of AST and LDH, changes in glutathione (GSH), catalase and morphology of porcine hepatocytes exposed to SVHP were investigated to indicate the effect of plasma from patients with severe hepatitis on the growth, injury, detoxification, and morphology of porcine hepatocytes.RESULTS: The synthesis of DNA and protein was inhibited in the medium containing 100% SVHP compared to the controls. The leakages of LDH and AST increased in porcine hepatocytes following exposure to 100% SVHP for 5 h. The difference between 100% SVHP and 10% newborn calf serum (NCS) was significant in t-test (LDH: t = 24.552, P = 0.001; AST: t = 4.169, P =0.014). After exposure to SVHP for 24 h, alterations in GSH status were significant (F = 2.746, P<0.05) between porcine hepatocytes in 100% SVHP and 10% NCS, but no alteration occurred in the culture medium after 48 h (F = 4.378, P<0.05). A similar profile was observed in catalase activity. Many round vacuoles were observed in porcine hepatocytes cultured in SVHP. The membranes of these cells became indistinct and almost all the cells died on d 5.CONCLUSION: Plasma from patients with severe hepatitis inhibits the growth, injures membrane, disturbs GSH homeostasis and induces morphological changes of porcine hepatocytes. It is suggested that SVHP should be pretreated to reduce the toxin load and improve the performance of porcine hepatocytes in extracorporeal liver-support devices.

  15. SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication.

    Science.gov (United States)

    Neuvonen, Maarit; Kazlauskas, Arunas; Martikainen, Miika; Hinkkanen, Ari; Ahola, Tero; Saksela, Kalle

    2011-11-01

    Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV), Sindbis (SINV), and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication.

  16. SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication.

    Directory of Open Access Journals (Sweden)

    Maarit Neuvonen

    2011-11-01

    Full Text Available Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3 domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV, Sindbis (SINV, and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication.

  17. Residues in human respiratory syncytial virus P protein that are essential for its activity on RNA viral synthesis.

    Science.gov (United States)

    Asenjo, Ana; Mendieta, Jesús; Gómez-Puertas, Paulino; Villanueva, Nieves

    2008-03-01

    Human respiratory syncytial virus (HRSV) P protein, 241 amino acid long, is a structural homotetrameric phosphoprotein. Viral transcription and replication processes are dependent on functional P protein interactions inside viral ribonucleoprotein complexes (RNPs). Binding capacity to RNPs proteins and transcription and replication complementation analyses, using inactive P protein variants, have identified residues essential for functional interactions with itself, L, N and M2-1 proteins. P protein may establish some of these interactions as monomer, but efficient viral transcription and replication requires P protein oligomerization through the central region of the molecule. A structurally stable three-dimensional model has been generated in silico for this region (residues 98-158). Our analysis has indicated that P protein residues L135, D139, E140 and L142 are involved in homotetramerization. Additionally, the residues D136, S156, T160 and E179 appear to be essential for P protein activity on viral RNA synthesis and very high turnover phosphorylation at S143, T160 and T210 could regulate it. Thus, compounds targeted to those of these residues, located in the modeled three-dimensional structure, could have specific anti-HRSV effect.

  18. The miRNA Plasma Signature in Response to Acute Aerobic Exercise and Endurance Training

    DEFF Research Database (Denmark)

    Nielsen, Søren; Åkerström, Thorbjörn; Rinnov, Anders

    2014-01-01

    the miRNA signature in human plasma in response to acute exercise and chronic endurance training by utilizing a novel methodological approach. RNA was isolated from human plasma collected from young healthy men before and after an acute endurance exercise bout and following 12 weeks of endurance training...

  19. Hsp90 interacts specifically with viral RNA and differentially regulates replication initiation of Bamboo mosaic virus and associated satellite RNA.

    Directory of Open Access Journals (Sweden)

    Ying Wen Huang

    Full Text Available Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3' untranslated region (3' UTR of BaMV genomic RNA, but not with the 3' UTR of BaMV-associated satellite RNA (satBaMV RNA or that of genomic RNA of other viruses, such as Potato virus X (PVX or Cucumber mosaic virus (CMV. Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3' UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3' UTR of BaMV RNA during the initiation of BaMV RNA replication.

  20. Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase.

    Science.gov (United States)

    Lee, M T Michael; Bishop, Konrad; Medcalf, Liz; Elton, Debra; Digard, Paul; Tiley, Laurence

    2002-01-15

    The first 11 nt at the 5' end of influenza virus genomic RNA were shown to be both necessary and sufficient for specific binding by the influenza virus polymerase. A novel in vitro transcription assay, in which the polymerase was bound to paramagnetic beads via a biotinylated 5'-vRNA oligonucleotide, was used to study the activities of different forms of the polymerase. Complexes composed of co-expressed PB1/PB2/PA proteins and a sub-complex composed of PB1/PA bound to the 5'-vRNA oligonucleotide, whereas PB1 expressed alone did not. The enriched 5'-vRNA/PB1/PB2/PA complex was highly active for ApG and globin mRNA primed transcription on a model 3'-vRNA template. RNA synthesis in the absence of added primers produced products with 5'-terminal tri- or diphosphate groups, indicating that genuine unprimed initiation of transcription also occurred. No transcriptase activity was detected for the PB1/PA complex. These results demonstrate a role for PA in the enhancement of 5' end binding activity of PB1, a role for PB2 in the assembly of a polymerase complex able to perform both cap-dependent and -independent synthesis and that NP is not required for the initiation of replicative transcription.

  1. Silencing MicroRNA-155 Attenuates Cardiac Injury and Dysfunction in Viral Myocarditis via Promotion of M2 Phenotype Polarization of Macrophages.

    Science.gov (United States)

    Zhang, Yingying; Zhang, Mengying; Li, Xueqin; Tang, Zongsheng; Wang, Xiangmin; Zhong, Min; Suo, Qifeng; Zhang, Yao; Lv, Kun

    2016-03-02

    Macrophage infiltration is a hallmark feature of viral myocarditis. As studies have shown that microRNA-155 regulates the differentiation of macrophages, we aimed to investigate the role of microRNA-155 in VM. We report that silencing microRNA-155 protects mice from coxsackievirus B3 induced myocarditis. We found that microRNA-155 expression was upregulated and localized primarily in heart-infiltrating macrophages and CD4(+) T lymphocytes during acute myocarditis. In contrast with wildtype (WT) mice, microRNA-155(-/-) mice developed attenuated viral myocarditis, which was characterized by decreased cardiac inflammation and decreased intracardiac CD45(+) leukocytes. Hearts of microRNA-155(-/-) mice expressed decreased levels of the IFN-γ and increased levels of the cytokines IL-4 and IL-13. Although total CD4(+) and regulatory T cells were unchanged in miR-155(-/-) spleen proportionally, the activation of T cells and CD4(+) T cell proliferation in miR-155(-/-) mice were significantly decreased. Beyond the acute phase, microRNA-15(5-/-) mice had reduced mortality and improved cardiac function during 5 weeks of follow-up. Moreover, silencing microRNA-155 led to increased levels of alternatively-activated macrophages (M2) and decreased levels of classically-activated macrophages (M1) in the heart. Combined, our studies suggest that microRNA-155 confers susceptibility to viral myocarditis by affecting macrophage polarization, and thus may be a potential therapeutic target for viral myocarditis.

  2. Crystal Structure of Poliovirus 3CD Protein: Virally Encoded Protease and Precursor to the RNA-Dependent RNA Polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Marcotte,L.; Wass, A.; Gohara, D.; Pathak, H.; Arnold, J.; Filman, D.; Cameron, C.; Hogle, J.

    2007-01-01

    Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3Cpro and the viral polymerase 3Dpol and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4- Angstroms resolution and the G64S fidelity mutant of 3Dpol at a 3.0- Angstroms resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active site is intact in both the 3CD and the 3Dpol G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domain's N terminus to the VPg binding site suggest how 3Dpol makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.

  3. Structure and assembly of the essential RNA ring component of a viral DNA packaging motor

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Fang; Lu, Changrui; Zhao, Wei; Rajashankar, Kanagalaghatta R.; Anderson, Dwight L.; Jardine, Paul J.; Grimes, Shelley; Ke, Ailong (Cornell); (UMM)

    2011-07-25

    Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage {psi}29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 {angstrom} resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNA cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of {psi}29 DNA.

  4. Factors influencing a low rate of hepatitis C viral RNA clearance in heroin users from Southern China

    Institute of Scientific and Technical Information of China (English)

    ShengHan Lai; Jin-Bing Zhang; Wei Liu; Jie Chen; Xiao-Fang Yu

    2008-01-01

    AIM: To study the virological and host factors influencing hepatitis C infection outcomes in heroin users in southern China.METHODS: HCV RNA and associated factors were analyzed among 347 heroin users from Guangxi Zhuang Autonomous Region, southern China who were hepatitis C virus (HCV) EIA positive for two or more consecutive visits.RESULTS: Using the COBAS AMPLICOR HCV TEST, a remarkably low HCV RNA negative rate of 8.6% was detected. After multivariate logistic regression analysis, HCV RNA clearance was significantly associated with the presence of HBsAg (OR = 8.436, P < 0.0001), the lack of HIV-1 infection (OR = 0.256, P = 0.038) and age younger than 25 (OR = 0.400, P = 0.029).CONCLUSION: Our study suggests HCV infection among Chinese heroin users results in high levels of viral persistence even amidst factors previously found to enhance viral clearance. Prospective studies of a possible genetic component within the Chinese population and the pathogenicity of non-genotype 1 HCV infections are needed.

  5. Bovine Mx1 enables resistance against foot-and-mouth disease virus in naturally susceptible cells by inhibiting the replication of viral RNA.

    Science.gov (United States)

    Wang, H-M; Xia, X-Z; Hu, G-X; Yu, L; He, H-B

    2016-03-01

    Innate immunity, especially the anti-viral genes, exerts an important barrier function in preventing viral infections. Myxovirus-resistant (Mx) gene take an anti-viral role, whereas its effects on foot-and-mouth disease virus (FMDV) in naturally susceptible cells are still unclear. The bovine primary fetal tracheal epithelial cell line BPTE-siMx1, in which bovine Mx1 gene was silenced, was established and treated with IFN alpha for 6 hr before FMDV infection. The copy numbers of the negative and positive strand viral RNA were determined by strand-specific real-time fluorescence quantitative RT-PCR. The TCID50 of BPTE-siMx1 cells increased at least 17-fold as compared to control cells BPTE-LacZ at 8 hr post infection, thus silencing of bovine Mx1 could promote the replication of FMDV. The amount of both the negative and positive strand viral RNA in BPTE-siMx1 cells significantly increased as compared to BPTE-LacZ cells, indicating that the replication levels of viral RNA were promoted by silencing bovine Mx1. The bovine Mx1 gene could provide resistance against FMDV in the bovine primary fetal tracheal epithelial cells via suppressing the replication of viral RNA.

  6. Modeling Zika plasma viral dynamics in non-human primates: insights into viral pathogenesis and antiviral strategies

    Energy Technology Data Exchange (ETDEWEB)

    Best, Katharine [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Guedj, Jeremie [Univ. of Paris (France). IAME; Madelain, Vincent [Univ. of Paris (France); de Lamballerie, Xavier [Aix-Marseille Univ. (France); L, So-Yonim [Harvard Univ., Cambridge, MA (United States). Center for Virology and Vaccine Research; Osuna, Christa E [Harvard Univ., Cambridge, MA (United States). Center for Virology and Vaccine Research; Whitney, James [Harvard Univ., Cambridge, MA (United States). Center for Virology and Vaccine Research; Perelson, Alan S. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2016-10-24

    The recent outbreak of Zika virus (ZIKV) has been associated with fetal abnormalities and neurological complications, prompting global concern. Here we present the first mathematical analysis of the within-host dynamics of plasma ZiKV burden in a non-human primate model, allowing for characterization of the growth and clearance of ZIKV within an individual macaque.

  7. Platelets confound the measurement of extracellular miRNA in archived plasma

    OpenAIRE

    Mitchell, Adam J.; Gray, Warren D; Hayek, Salim S.; Yi-An Ko; Sheena Thomas; Kim Rooney; Mosaab Awad; John D. Roback; Arshed Quyyumi; Searles, Charles D.

    2016-01-01

    Extracellular miRNAs are detectable in biofluids and represent a novel class of disease biomarker. Although many studies have utilized archived plasma for miRNA biomarker discovery, the effects of processing and storage have not been rigorously studied. Previous reports have suggested plasma samples are commonly contaminated by platelets, significantly confounding the measurement of extracellular miRNA, which was thought to be easily addressed by additional post-thaw plasma processing. In a c...

  8. Ultrasensitive HCV RNA Quantification in Antiviral Triple Therapy: New Insight on Viral Clearance Dynamics and Treatment Outcome Predictors

    Science.gov (United States)

    Garbuglia, Anna Rosa; Visco-Comandini, Ubaldo; Lionetti, Raffaella; Lapa, Daniele; Castiglione, Filippo; D’Offizi, Gianpiero; Taibi, Chiara; Montalbano, Marzia; Capobianchi, Maria Rosaria; Paci, Paola

    2016-01-01

    Objectives Identifying the predictive factors of Sustained Virological Response (SVR) represents an important challenge in new interferon-based DAA therapies. Here, we analyzed the kinetics of antiviral response associated with a triple drug regimen, and the association between negative residual viral load at different time points during treatment. Methods Twenty-three HCV genotype 1 (GT 1a n = 11; GT1b n = 12) infected patients were included in the study. Linear Discriminant Analysis (LDA) was used to establish possible association between HCV RNA values at days 1 and 4 from start of therapy and SVR. Principal component analysis (PCA) was applied to analyze the correlation between HCV RNA slope and SVR. A ultrasensitive (US) method was established to measure the residual HCV viral load in those samples which resulted “detected <12IU/ml” or undetectable with ABBOTT standard assay, and was retrospectively used on samples collected at different time points to establish its predictive power for SVR. Results According to LDA, there was no association between SVR and viral kinetics neither at time points earlier than 1 week (days 1 and 4) after therapy initiation nor later. The slopes were not relevant for classifying patients as SVR or no-SVR. No significant differences were observed in the median HCV RNA values at T0 among SVR and no-SVR patients. HCV RNA values with US protocol (LOD 1.2 IU/ml) after 1 month of therapy were considered; the area under the ROC curve was 0.70. Overall, PPV and NPV of undetectable HCV RNA with the US method for SVR was 100% and 46.7%, respectively; sensitivity and specificity were 38.4% and 100% respectively. Conclusion HCV RNA “not detected” by the US method after 1 month of treatment is predictive of SVR in first generation Protease inhibitor (PI)-based triple therapy. The US method could have clinical utility for advanced monitoring of virological response in new interferon based DAA combination regimens. PMID:27560794

  9. An MRI-visible non-viral vector for targeted Bcl-2 siRNA delivery to neuroblastoma

    Directory of Open Access Journals (Sweden)

    Shen M

    2012-07-01

    Full Text Available Min Shen,1,* Faming Gong,3,* Pengfei Pang,1,* Kangshun Zhu,1 Xiaochun Meng,1 Chun Wu,1 Jin Wang,1 Hong Shan,1,2 Xintao Shuai3,41Molecular Imaging Lab, Department of Radiology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China; 2Institute of Intervention Radiology, Sun Yat-sen University, Guangzhou, China; 3PCFM Lab of Ministry of Education, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, China; 4Center of Biomedical Engineering, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China*These authors contributed equally to this workAbstract: Polyethylene glycol-grafted polyethylenimine (PEG-g-PEI which was functionalized with a neuroblastoma cell-specific ligand, the GD2 single chain antibody (scAbGD2, was synthesized in order to effectively deliver Bcl-2 siRNA into neuroblastoma cells. This polymer was complexed first with superparamagnetic iron oxide nanoparticle (SPION to get a MRI-visible targeted non-viral vector (scAbGD2-PEG-g-PEI-SPION and then with Bcl-2 siRNA to form nanoparticles showing low cytotoxicity. The targeting capacity of scAbGD2-PEG-g-PEI-SPION was successfully verified in vivo and in vitro by magnetic resonance imaging. The single chain antibody encoded targeted polyplex was more effective in transferring Bcl-2 siRNA than the nontargeting one in SK-N-SH cells, a human neuroblastoma cell line, resulting in a 46.34% inhibition in the expression of Bcl-2 mRNA. Consequently, a high level of cell apoptosis up to 50.76% and a significant suppression of tumor growth were achieved, which indicates that scAbGD2-PEG-g-PEI-SPION is a promising magnetic resonance imaging-visible non-viral vector for targeted neuroblastoma siRNA therapy and diagnosis.Keywords: tumor targeting, GD2, non-viral vector, Bcl-2 small interfering RNA, magnetic resonance imaging

  10. Cell-free plasma microRNA in pancreatic ductal adenocarcinoma and disease controls

    DEFF Research Database (Denmark)

    Carlsen, Anting Liu; Joergensen, Maiken Thyregod; Knudsen, Steen;

    2013-01-01

    There are no tumor-specific biochemical markers for pancreatic ductal adenocarcinoma (PDAC). Tissue-specific gene expression including microRNA (miRNA) profiling, however, identifies specific PDAC signatures. This study evaluates associations between circulating, cell-free plasma-miRNA profiles...

  11. Efavirenz Plasma Concentrations and HIV Viral Load in HIV/AIDS-tuberculosis Infection Patients Treated with Rifampicin.

    Science.gov (United States)

    Mariana, Nina; Purwantyastuti; Instiaty; Rusli, Adria

    2016-01-01

    to determine the effect of a rifampicin-containing tuberculosis regimen on efavirenz plasma concentrations and viral load in HIV/AIDS-Tuberculosis infection patients who received efavirenz-based antiretroviral therapy. plasma efavirenz concentrations and HIV viral load were measured in HIV/AIDS patients treated with 600 mg efavirenz-based antiretroviral for 3 to 6 months and in HIV/AIDS-Tuberculosis infection patients treated with similar antiretroviral regimen plus rifampicin-containing antituberculosis in Sulianti Saroso Infectious disease Hospital, Jakarta. Plasma efavirenz concentration in both groups were compared using Mann-Whitney test, while proportion of patients with viral load >40 copy/mL were analyzed with chi-square test. forty five patients (27 with HIV/AIDS and 18 with HIV/AIDS-Tuberculosis infections) were recruited during the period of February to May 2015. The median efavirenz plasma concentration obtained from HIV/AIDS group was 0,680 mg/L(range 0,24 to 5,67 mg/L and that obtained from HIV/AIDS-Tuberculosis group was 0.685 mg/L (0.12 -2.23 mg/L) which was not significantly different statistically. The proportion of patients with viral load 40 copies/mL after 3-6 months of ARV treatment in the HIV/AIDS group was 51.9%, and in the HIV/AIDS-Tuberculosis group was 72.2%, which was not significantly different statistically (Chi Square test, p=0.291). plasma efavirenz concentration in HIV/AIDS-tuberculosis patients receiving antiretroviral and rifampicin is not significantly different from that on HIV/AIDS patients without tuberculosis. Proportion of patients with viral load of >40 copy/mL is higher in HIV/AIDS-tuberculosis patients receiving rifampicin compared to HIV/AIDS patients that not receive rifampicin. However, this difference did not reach statistical significance. Confirmatory studies with bigger sample size are needed to clarify the influence of rifampicin on plasma level of efavirenzand and on viral load.

  12. Discovery of Potent Non-Nucleoside Inhibitors of Dengue Viral RNA-Dependent RNA Polymerase from a Fragment Hit Using Structure-Based Drug Design.

    Science.gov (United States)

    Yokokawa, Fumiaki; Nilar, Shahul; Noble, Christian G; Lim, Siew Pheng; Rao, Ranga; Tania, Stefani; Wang, Gang; Lee, Gladys; Hunziker, Jürg; Karuna, Ratna; Manjunatha, Ujjini; Shi, Pei-Yong; Smith, Paul W

    2016-04-28

    The discovery and optimization of non-nucleoside dengue viral RNA-dependent-RNA polymerase (RdRp) inhibitors are described. An X-ray-based fragment screen of Novartis' fragment collection resulted in the identification of a biphenyl acetic acid fragment 3, which bound in the palm subdomain of RdRp. Subsequent optimization of the fragment hit 3, relying on structure-based design, resulted in a >1000-fold improvement in potency in vitro and acquired antidengue activity against all four serotypes with low micromolar EC50 in cell-based assays. The lead candidate 27 interacts with a novel binding pocket in the palm subdomain of the RdRp and exerts a promising activity against all clinically relevant dengue serotypes.

  13. Levels and patterns of HIV RNA viral load in untreated pregnant women

    DEFF Research Database (Denmark)

    NN, NN; Patel, Deven; Thorne, Claire;

    2008-01-01

    OBJECTIVE: To assess pregnancy levels and patterns of HIV RNA in the absence of antiretroviral therapy, while appropriately adjusting for potential confounders, including maternal immune status and race. METHODS: Data on > or = 1 antenatal HIV RNA measurements were available for 333 untreated HIV......-infected pregnant women enrolled in the European Collaborative Study. CD4 counts and HIV RNA measurements were routinely collected from 1992 and 1998, respectively. Linear mixed effects models based on 246 women for whom complete data were available examined changes in HIV RNA levels over pregnancy, with a nested...... random effects term accounting for measurement variability within women and period of sample collection. RESULTS: The change in HIV RNA over pregnancy varied significantly by race (p=0.005): from the second trimester until delivery, HIV RNA decreased significantly by an estimated 0.019 log(10) copies...

  14. Maternal Plasma DNA and RNA Sequencing for Prenatal Testing.

    Science.gov (United States)

    Tamminga, Saskia; van Maarle, Merel; Henneman, Lidewij; Oudejans, Cees B M; Cornel, Martina C; Sistermans, Erik A

    2016-01-01

    Cell-free DNA (cfDNA) testing has recently become indispensable in diagnostic testing and screening. In the prenatal setting, this type of testing is often called noninvasive prenatal testing (NIPT). With a number of techniques, using either next-generation sequencing or single nucleotide polymorphism-based approaches, fetal cfDNA in maternal plasma can be analyzed to screen for rhesus D genotype, common chromosomal aneuploidies, and increasingly for testing other conditions, including monogenic disorders. With regard to screening for common aneuploidies, challenges arise when implementing NIPT in current prenatal settings. Depending on the method used (targeted or nontargeted), chromosomal anomalies other than trisomy 21, 18, or 13 can be detected, either of fetal or maternal origin, also referred to as unsolicited or incidental findings. For various biological reasons, there is a small chance of having either a false-positive or false-negative NIPT result, or no result, also referred to as a "no-call." Both pre- and posttest counseling for NIPT should include discussing potential discrepancies. Since NIPT remains a screening test, a positive NIPT result should be confirmed by invasive diagnostic testing (either by chorionic villus biopsy or by amniocentesis). As the scope of NIPT is widening, professional guidelines need to discuss the ethics of what to offer and how to offer. In this review, we discuss the current biochemical, clinical, and ethical challenges of cfDNA testing in the prenatal setting and its future perspectives including novel applications that target RNA instead of DNA.

  15. Hairpin RNA derived from viral NIa gene confers immunity to wheat streak mosaic virus infection in transgenic wheat plants.

    Science.gov (United States)

    Fahim, Muhammad; Ayala-Navarrete, Ligia; Millar, Anthony A; Larkin, Philip J

    2010-09-01

    Wheat streak mosaic virus (WSMV), vectored by Wheat curl mite, has been of great economic importance in the Great Plains of the United States and Canada. Recently, the virus has been identified in Australia, where it has spread quickly to all major wheat growing areas. The difficulties in finding adequate natural resistance in wheat prompted us to develop transgenic resistance based on RNA interference (RNAi). An RNAi construct was designed to target the nuclear inclusion protein 'a' (NIa) gene of WSMV. Wheat was stably cotransformed with two plasmids: pStargate-NIa expressing hairpin RNA (hpRNA) including WSMV sequence and pCMneoSTLS2 with the nptII selectable marker. When T(1) progeny were assayed against WSMV, ten of sixteen families showed complete resistance in transgenic segregants. The resistance was classified as immunity by four criteria: no disease symptoms were produced; ELISA readings were as in uninoculated plants; viral sequences could not be detected by RT-PCR from leaf extracts; and leaf extracts failed to give infections in susceptible plants when used in test-inoculation experiments. Southern blot hybridization analysis indicated hpRNA transgene integrated into the wheat genome. Moreover, accumulation of small RNAs derived from the hpRNA transgene sequence positively correlated with immunity. We also showed that the selectable marker gene nptII segregated independently of the hpRNA transgene in some transgenics, and therefore demonstrated that it is possible using these techniques, to produce marker-free WSMV immune transgenic plants. This is the first report of immunity in wheat to WSMV using a spliceable intron hpRNA strategy.

  16. Human DDX3 interacts with the HIV-1 Tat protein to facilitate viral mRNA translation.

    Directory of Open Access Journals (Sweden)

    Ming-Chih Lai

    Full Text Available Nuclear export and translation of intron-containing viral mRNAs are required for HIV-1 gene expression and replication. In this report, we provide evidence to show that DDX3 regulates the translation of HIV-1 mRNAs. We found that knockdown of DDX3 expression effectively inhibited HIV-1 production. Translation of HIV-1 early regulatory proteins, Tat and rev, was impaired in DDX3-depleted cells. All HIV-1 transcripts share a highly structured 5' untranslated region (UTR with inhibitory elements on translation of viral mRNAs, yet DDX3 promoted translation of reporter mRNAs containing the HIV-1 5' UTR, especially with the transactivation response (TAR hairpin. Interestingly, DDX3 directly interacts with HIV-1 Tat, a well-characterized transcriptional activator bound to the TAR hairpin. HIV-1 Tat is partially targeted to cytoplasmic stress granules upon DDX3 overexpression or cell stress conditions, suggesting a potential role of Tat/DDX3 complex in translation. We further demonstrated that HIV-1 Tat remains associated with translating mRNAs and facilitates translation of mRNAs containing the HIV-1 5' UTR. Taken together, these findings indicate that DDX3 is recruited to the TAR hairpin by interaction with viral Tat to facilitate HIV-1 mRNA translation.

  17. Human DDX3 interacts with the HIV-1 Tat protein to facilitate viral mRNA translation.

    Science.gov (United States)

    Lai, Ming-Chih; Wang, Shainn-Wei; Cheng, Lie; Tarn, Woan-Yuh; Tsai, Shaw-Jenq; Sun, H Sunny

    2013-01-01

    Nuclear export and translation of intron-containing viral mRNAs are required for HIV-1 gene expression and replication. In this report, we provide evidence to show that DDX3 regulates the translation of HIV-1 mRNAs. We found that knockdown of DDX3 expression effectively inhibited HIV-1 production. Translation of HIV-1 early regulatory proteins, Tat and rev, was impaired in DDX3-depleted cells. All HIV-1 transcripts share a highly structured 5' untranslated region (UTR) with inhibitory elements on translation of viral mRNAs, yet DDX3 promoted translation of reporter mRNAs containing the HIV-1 5' UTR, especially with the transactivation response (TAR) hairpin. Interestingly, DDX3 directly interacts with HIV-1 Tat, a well-characterized transcriptional activator bound to the TAR hairpin. HIV-1 Tat is partially targeted to cytoplasmic stress granules upon DDX3 overexpression or cell stress conditions, suggesting a potential role of Tat/DDX3 complex in translation. We further demonstrated that HIV-1 Tat remains associated with translating mRNAs and facilitates translation of mRNAs containing the HIV-1 5' UTR. Taken together, these findings indicate that DDX3 is recruited to the TAR hairpin by interaction with viral Tat to facilitate HIV-1 mRNA translation.

  18. Poly(A) Tail Recognition by a Viral RNA Element Through Assembly of a Triple Helix

    Energy Technology Data Exchange (ETDEWEB)

    M Mitton-Fry; S DeGregorio; J Wang; T Steitz; J Steitz

    2011-12-31

    Kaposi's sarcoma-associated herpesvirus produces a highly abundant, nuclear noncoding RNA, polyadenylated nuclear (PAN) RNA, which contains an element that prevents its decay. The 79-nucleotide expression and nuclear retention element (ENE) was proposed to adopt a secondary structure like that of a box H/ACA small nucleolar RNA (snoRNA), with a U-rich internal loop that hybridizes to and protects the PAN RNA poly(A) tail. The crystal structure of a complex between the 40-nucleotide ENE core and oligo(A){sub 9} RNA at 2.5 angstrom resolution reveals that unlike snoRNAs, the U-rich loop of the ENE engages its target through formation of a major-groove triple helix. A-minor interactions extend the binding interface. Deadenylation assays confirm the functional importance of the triple helix. Thus, the ENE acts as an intramolecular RNA clamp, sequestering the PAN poly(A) tail and preventing the initiation of RNA decay.

  19. A functional selection of viral genetic elements in cultured cells to identify hepatitis C virus RNA translation inhibitors.

    Science.gov (United States)

    Jaffrelo, Loic; Chabas, Sandrine; Reigadas, Sandrine; Pflieger, Aude; Wychowski, Czeslaw; Rumi, Julie; Ventura, Michel; Toulmé, Jean-Jacques; Staedel, Cathy

    2008-09-01

    We developed a functional selection system based on randomized genetic elements (GE) to identify potential regulators of hepatitis C virus (HCV) RNA translation, a process initiated by an internal ribosomal entry site (IRES). A retroviral HCV GE library was introduced into HepG2 cells, stably expressing the Herpes simplex virus thymidine kinase (HSV-TK) under the control of the HCV IRES. Cells that expressed transduced GEs inhibiting HSV-TK were selected via their resistance to ganciclovir. Six major GEs were rescued by PCR on the selected cell DNA and identified as HCV elements. We validated our strategy by further studying the activity of one of them, GE4, encoding the 5' end of the viral NS5A gene. GE4 inhibited HCV IRES-, but not cap-dependent, reporter translation in human hepatic cell lines and inhibited HCV infection at a post-entry step, decreasing by 85% the number of viral RNA copies. This method can be applied to the identification of gene expression regulators.

  20. Role of viral RNA and lipid in the adverse events associated with the 2010 Southern Hemisphere trivalent influenza vaccine.

    Science.gov (United States)

    Rockman, Steve; Becher, Dorit; Dyson, Allison; Koernig, Sandra; Morelli, Adriana Baz; Barnden, Megan; Camuglia, Sarina; Soupourmas, Peter; Pearse, Martin; Maraskovsky, Eugene

    2014-06-24

    In Australia, during the 2010 Southern Hemisphere (SH) influenza season, there was an unexpected increase in post-marketing adverse event reports of febrile seizures (FS) in children under 5 years of age shortly after vaccination with the CSL 2010 SH trivalent influenza vaccine (CSL 2010 SH TIV) compared to previous CSL TIVs and other licensed 2010 SH TIVs. In an accompanying study, we described the contribution to these adverse events of the 2010 SH influenza strains as expressed in the CSL 2010 SH TIV using in vitro cytokine/chemokine secretion from whole blood cells and induction of NF-κB activation in HEK293 reporter cells. The aim of the present study was to identify the root cause components that elicited the elevated cytokine/chemokine and NF-κB signature. Our studies demonstrated that the pyrogenic signal was associated with a heat-labile, viral-derived component(s) in the CSL 2010 SH TIV. Further, it was found that viral lipid-mediated delivery of short, fragmented viral RNA was the key trigger for the increased cytokine/chemokine secretion and NF-κB activation. It is likely that the FS reported in children viral components of the new influenza strains (particularly B/Brisbane/60/2008 and to a lesser extent H1N1 A/California/07/2009). These combined to heighten immune activation of innate immune cells, which in a small proportion of children <5 years of age is associated with the occurrence of FS. The data also demonstrates that CSL TIVs formulated with increased levels of splitting agent (TDOC) for the B/Brisbane/60/2008 strain can attenuate the pro-inflammatory signals in vitro, identifying a potential path forward for generating a CSL TIV indicated for use in children <5 years.

  1. Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections

    Science.gov (United States)

    He, Hui; Li, Rongqun; Chen, Yi; Pan, Ping; Tong, Wenjuan; Dong, Xueyan; Chen, Yueming; Yu, Daojun

    2017-01-01

    Current extraction methods often extract DNA and RNA separately, and few methods are capable of co-extracting DNA and RNA from sputum. We established a nucleic acid co-extraction method from sputum based on magnetic beads and optimized the method by evaluating influencing factors, such as the guanidinium thiocyanate (GTC) and dithiothreitol (DTT) concentrations, magnetic bead amount, incubation temperature, lysis buffer pH and RNA carrier type. The feasibility of the simultaneous nucleic acid co-extraction method was evaluated by amplifying DNA and RNA viruses from a single clinical specimen with a multiplex RT-qPCR method. Both DNA and RNA were most efficiently extracted when the GTC and DTT concentrations were 2.0 M and 80 mM, respectively, 20 μl magnetic beads were added, the incubation temperature was 80 °C, the pH was 8 or 9, and RNA carrier A was used. Therefore, we established a simple method to extract nucleic acids from two important respiratory viruses compared with other commercial kits. This magnetic beads-based co-extraction method for sputum followed by a multiplex RT-qPCR can rapidly and precisely detect DNA and RNA viruses from a single clinical specimen and has many advantages, such as decreased time, low cost, and a lack of harmful chemicals. PMID:28332631

  2. [Synthesis of messenger-like RNA in plasma cels producing different clases of immunoglobulins].

    Science.gov (United States)

    Nikitin, A V; Babichev, V A

    1976-01-01

    Electrophoretic analysis of the nuclear poly A RNA cells of the MOPC 21, MOPC 406 and MOPC 41 plasmocytomas demonstrated a similarity in their distribution by the mol wt. Kinetics of the label accumulation in the nuclear poly A RNA of different plasma cells was unitypical. Individual peculiarities of the distribution of the cytoplasmic poly A RNA were expressed for each individual line of the myeloma cells. The majority of the molecules of the heterogenous RNA in the plasma cells were subjected to posttranscription polyadenylation.

  3. Viral delivery of shRNA to amygdala neurons leads to neurotoxicity and deficits in Pavlovian fear conditioning.

    Science.gov (United States)

    de Solis, Christopher A; Holehonnur, Roopashri; Banerjee, Anwesha; Luong, Jonathan A; Lella, Srihari K; Ho, Anthony; Pahlavan, Bahram; Ploski, Jonathan E

    2015-10-01

    The use of viral vector technology to deliver short hairpin RNAs (shRNAs) to cells of the nervous system of many model organisms has been widely utilized by neuroscientists to study the influence of genes on behavior. However, there have been numerous reports that delivering shRNAs to the nervous system can lead to neurotoxicity. Here we report the results of a series of experiments where adeno-associated viruses (AAV), that were engineered to express shRNAs designed to target known plasticity associated genes (i.e. Arc, Egr1 and GluN2A) or control shRNAs that were designed not to target any rat gene product for depletion, were delivered to the rat basal and lateral nuclei of the amygdala (BLA), and auditory Pavlovian fear conditioning was examined. In our first set of experiments we found that animals that received AAV (3.16E13-1E13 GC/mL; 1 μl/side), designed to knockdown Arc (shArc), or control shRNAs targeting either luciferase (shLuc), or nothing (shCntrl), exhibited impaired fear conditioning compared to animals that received viruses that did not express shRNAs. Notably, animals that received shArc did not exhibit differences in fear conditioning compared to animals that received control shRNAs despite gene knockdown of Arc. Viruses designed to harbor shRNAs did not induce obvious morphological changes to the cells/tissue of the BLA at any dose of virus tested, but at the highest dose of shRNA virus examined (3.16E13 GC/mL; 1 μl/side), a significant increase in microglia activation occurred as measured by an increase in IBA1 immunoreactivity. In our final set of experiments we infused viruses into the BLA at a titer of (1.60E+12 GC/mL; 1 μl/side), designed to express shArc, shLuc, shCntrl or shRNAs designed to target Egr1 (shEgr1), or GluN2A (shGluN2A), or no shRNA, and found that all groups exhibited impaired fear conditioning compared to the group which received a virus that did not express an shRNA. The shEgr1 and shGluN2A groups exhibited gene

  4. Adipose tissue interleukin-18 mRNA and plasma interleukin-18: effect of obesity and exercise

    DEFF Research Database (Denmark)

    Leick, Lotte; Lindegaard, Birgitte; Stensvold, Dorthe

    2007-01-01

    OBJECTIVES: Obesity and a physically inactive lifestyle are associated with increased risk of developing insulin resistance. The hypothesis that obesity is associated with increased adipose tissue (AT) interleukin (IL)-18 mRNA expression and that AT IL-18 mRNA expression is related to insulin......: AT IL-18 mRNA content and plasma IL-18 concentration were higher (p insulin resistance. While acute exercise did not affect IL-18 mRNA expression...... at the studied time-points, exercise training reduced AT IL-18 mRNA content by 20% in both sexes. DISCUSSION: Because obesity and insulin resistance were associated with elevated AT IL-18 mRNA and plasma IL-18 levels, the training-induced lowering of AT IL-18 mRNA content may contribute to the beneficial effects...

  5. Platelets confound the measurement of extracellular miRNA in archived plasma.

    Science.gov (United States)

    Mitchell, Adam J; Gray, Warren D; Hayek, Salim S; Ko, Yi-An; Thomas, Sheena; Rooney, Kim; Awad, Mosaab; Roback, John D; Quyyumi, Arshed; Searles, Charles D

    2016-09-13

    Extracellular miRNAs are detectable in biofluids and represent a novel class of disease biomarker. Although many studies have utilized archived plasma for miRNA biomarker discovery, the effects of processing and storage have not been rigorously studied. Previous reports have suggested plasma samples are commonly contaminated by platelets, significantly confounding the measurement of extracellular miRNA, which was thought to be easily addressed by additional post-thaw plasma processing. In a case-control study of archived plasma, we noted a significant correlation between miRNA levels and platelet counts despite post-thaw processing. We thus examined the effects of a single freeze/thaw cycle on microparticles (MPs) and miRNA levels, and show that a single freeze/thaw cycle of plasma dramatically increases the number of platelet-derived MPs, contaminates the extracellular miRNA pool, and profoundly affects the levels of miRNAs detected. The measurement of extracellular miRNAs in archived samples is critically dependent on the removal of residual platelets prior to freezing plasma samples. Many previous clinical studies of extracellular miRNA in archived plasma should be interpreted with caution and future studies should avoid the effects of platelet contamination.

  6. In vitro neutralization of viral hemorrhagic septicemia virus by plasma from immunized zebrafish

    NARCIS (Netherlands)

    Chinchilla, B.; Gomez-Casado, E.; Encinas, P.; Falco Gracia, J.A.; Estepa, A.; Coll, J.

    2013-01-01

    We studied humoral long-term adaptive viral neutralization responses in zebrafish (Danio rerio), an increasingly useful vertebrate model for viral diseases actually limited by the absence of standardized anti-zebrafish immunoglobulin M (IgM) antibodies. We established an alternative method, similar

  7. A Viral Protein Suppresses siRNA-directed Interference in Tobacco Mosaic Virus Infection

    Institute of Scientific and Technical Information of China (English)

    Ming-Min ZHAO; De-Rong AN; Guang-Hua HUANG; Zu-Hua HE; Jiang-Ye CHEN

    2005-01-01

    Plant viruses encode suppressors of post-transcriptional gene silencing (PTGS), an adaptive defense response that limits virus replication and its spread in plants. The helper component proteinase (HCPro) of the potato virus A (PVA, genus Potyvirus) suppresses PTGS of silenced transgenes. Here, the effect of HC-Pro on siRNA-directed interference in the tobacco mosaic virus (TMV) was examined by using a transient Agrobacterium tumefaciens-based delivery system in intact tissues. It was shown that the interference effect was completely blocked by co-infiltration with HC-Pro plus siRNA constructs in both systemic and hypersensitive hosts. In the system host, all plants agro-infiltrated with HC-Pro plus siRNA constructs displayed the same symptoms as the negative control. Meanwhile, TMV RNA accumulation was found to be abundant in the upper leaves using reverse transcriptase-PCR (RT-PCR) and Northern blot assays. On the contrary, plants agro-infiltrated with the siRNA construct alone were free of symptoms. Therefore, our study suggests that the transient expression of HC-Pro inhibited the siRNA-directed host defenses against TMV infection.

  8. A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis

    Science.gov (United States)

    Rey, Félix A.

    2017-01-01

    The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation. PMID:28151973

  9. An isothermal, label-free, and rapid one-step RNA amplification/detection assay for diagnosis of respiratory viral infections.

    Science.gov (United States)

    Koo, Bonhan; Jin, Choong Eun; Lee, Tae Yoon; Lee, Jeong Hoon; Park, Mi Kyoung; Sung, Heungsup; Park, Se Yoon; Lee, Hyun Jung; Kim, Sun Mi; Kim, Ji Yeun; Kim, Sung-Han; Shin, Yong

    2017-04-15

    Recently, RNA viral infections caused by respiratory viruses, such as influenza, parainfluenza, respiratory syncytial virus, coronavirus, and Middle East respiratory syndrome-coronavirus (MERS-CoV), and Zika virus, are a major public health threats in the world. Although myriads of diagnostic methods based on RNA amplification have been developed in the last decades, they continue to lack speed, sensitivity, and specificity for clinical use. A rapid and accurate diagnostic method is needed for appropriate control, including isolation and treatment of the patients. Here, we report an isothermal, label-free, one-step RNA amplification and detection system, termed as iROAD, for the diagnosis of respiratory diseases. It couples a one-step isothermal RNA amplification method and a bio-optical sensor for simultaneous viral RNA amplification/detection in a label-free and real-time manner. The iROAD assay offers a one-step viral RNA amplification/detection example to rapid analysis (<20min). The detection limit of iROAD assay was found to be 10-times more sensitive than that of real-time reverse transcription-PCR method. We confirmed the clinical utility of the iROAD assay by detecting viral RNAs obtained from 63 human respiratory samples. We envision that the iROAD assay will be useful and potentially adaptable for better diagnosis of emerging infectious diseases including respiratory diseases.

  10. Plasma HIV-2 RNA According to CD4 Count Strata among HIV-2-Infected Adults in the IeDEA West Africa Collaboration.

    Directory of Open Access Journals (Sweden)

    Didier K Ekouévi

    Full Text Available Plasma HIV-1 RNA monitoring is one of the standard tests for the management of HIV-1 infection. While HIV-1 RNA can be quantified using several commercial tests, no test has been commercialized for HIV-2 RNA quantification. We studied the relationship between plasma HIV-2 viral load (VL and CD4 count in West African patients who were either receiving antiretroviral therapy (ART or treatment-naïve.A cross sectional survey was conducted among HIV-2-infected individuals followed in three countries in West Africa from March to December 2012. All HIV-2 infected-patients who attended one of the participating clinics were proposed a plasma HIV-2 viral load measurement. HIV-2 RNA was quantified using the new ultrasensitive in-house real-time PCR assay with a detection threshold of 10 copies/ mL (cps/mL.A total of 351 HIV-2-infected individuals participated in this study, of whom 131 (37.3% were treatment naïve and 220 (62.7% had initiated ART. Among treatment-naïve patients, 60 (46.5% had undetectable plasma HIV-2 viral load (1000 cps/mL in 6.0% of the patients. Most of the treatment-naïve patients (70.2% had CD4-T cell count ≥500 cells/mm3 and 43 (46.7% of these patients had a detectable VL (≥10 cps/mL. Among the 220 patients receiving ART, the median CD4-T cell count rose from 231 to 393 cells/mm3 (IQR [259-561] after a median follow-up duration of 38 months and 145 (66.0% patients had CD4-T cell count ≤ 500 cells/mm3 with a median viral load of 10 cps/mL (IQR [10-33]. Seventy five (34.0% patients had CD4-T cell count ≥ 500 cells/mm3, among them 14 (18.7% had a VL between 10-100 cps/mL and 2 (2.6% had VL >100 cps/mL.This study suggests that the combination of CD4-T cell count and ultrasensitive HIV-2 viral load quantification with a threshold of 10 cps/mL, could improve ART initiation among treatment naïve HIV-2-infected patients and the monitoring of ART response among patients receiving treatment.

  11. A role for the H4 subunit of vaccinia RNA polymerase in transcription initiation at a viral early promoter.

    Science.gov (United States)

    Deng, L; Shuman, S

    1994-05-13

    The vaccinia virus H4 gene encodes an essential subunit of the DNA-dependent RNA polymerase holoenzyme encapsidated within virus particles (Ahn, B., and Moss, B. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3536-3540; Kane, E. M., and Shuman, S. (1992) J. Virol. 66, 5752-5762). The role of this protein in transcription of viral early genes was revealed by the effects of affinity-purified anti-H4 antibody on discrete phases of the early transcription reaction in vitro. Anti-H4 specifically prevented the synthesis of a 21-nucleotide nascent RNA chain but had no impact on elongation of the 21-mer RNA by preassembled ternary complexes. Inhibition of initiation but not elongation was also observed with affinity-purified anti-D6 antibody directed against the 70-kDa subunit of the vaccinia early transcription initiation factor (ETF). Native gel mobility-shift assays showed that anti-H4 prevented the NTP-dependent recruitment of RNA polymerase to the preinitiation complex of ETF bound at the early promoter. Two species of ternary complexes could be resolved by native gel electrophoresis. Addition of anti-H4 to preformed complexes elicited a supershift of both ternary species but not of the preinitiation complex. Supeshift by anti-D6 revealed that the more rapidly migrating species of ternary complex did not contain immunoreactive ETF. Loss of ETF from the ternary complex was time-dependent. Thus, whereas the H4 protein was a stable constituent of the elongation complex, ETF was dissociable. We suggest that H4 functions as a molecular bridge to ETF and thereby allows specific recognition of early promoters by the core RNA polymerase. H4 is unlike bacterial sigma factor in that it remains bound to polymerase after the elongation complex is established.

  12. Caenorhabditis elegans RIG-I Homolog Mediates Antiviral RNA Interference Downstream of Dicer-Dependent Biogenesis of Viral Small Interfering RNAs

    Science.gov (United States)

    Coffman, Stephanie R.; Lu, Jinfeng; Guo, Xunyang; Zhong, Jing; Broitman-Maduro, Gina; Li, Wan-Xiang; Lu, Rui; Maduro, Morris

    2017-01-01

    ABSTRACT Dicer enzymes process virus-specific double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) to initiate specific antiviral defense by related RNA interference (RNAi) pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans. Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans. Using a mapping-by-sequencing strategy, we uncovered four loss-of-function alleles of drh-1, three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsiRNAs) in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral dsRNA replicative intermediates. Interestingly, vsiRNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsiRNA hot spots when DRH-1 is functional. As RIG-I translocates on long dsRNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsiRNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long dsRNA precursors. PMID:28325765

  13. Structural basis for the recognition of cellular mRNA export factor REF by herpes viral proteins HSV-1 ICP27 and HVS ORF57.

    Directory of Open Access Journals (Sweden)

    Richard B Tunnicliffe

    2011-01-01

    Full Text Available The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104-112 and 103-120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway.

  14. Structural basis for the recognition of cellular mRNA export factor REF by herpes viral proteins HSV-1 ICP27 and HVS ORF57.

    Science.gov (United States)

    Tunnicliffe, Richard B; Hautbergue, Guillaume M; Kalra, Priti; Jackson, Brian R; Whitehouse, Adrian; Wilson, Stuart A; Golovanov, Alexander P

    2011-01-06

    The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104-112 and 103-120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway.

  15. Cytoplasmic translocation of polypyrimidine tract-binding protein and its binding to viral RNA during Japanese encephalitis virus infection inhibits virus replication.

    Directory of Open Access Journals (Sweden)

    Deepika Bhullar

    Full Text Available Japanese encephalitis virus (JEV has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5'- and 3'-non-coding regions (NCRs. The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB interacts in vitro with both the 5'-NCR of the positive-sense genomic RNA--5NCR(+, and its complementary sequence in the negative-sense replication intermediate RNA--3NCR(-. The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(- RNA with viral RNA-dependent RNA polymerase (NS5 protein, an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.

  16. Mutations in matrix and SP1 repair the packaging specificity of a Human Immunodeficiency Virus Type 1 mutant by reducing the association of Gag with spliced viral RNA

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    Ristic Natalia

    2010-09-01

    Full Text Available Abstract Background The viral genome of HIV-1 contains several secondary structures that are important for regulating viral replication. The stem-loop 1 (SL1 sequence in the 5' untranslated region directs HIV-1 genomic RNA dimerization and packaging into the virion. Without SL1, HIV-1 cannot replicate in human T cell lines. The replication restriction phenotype in the SL1 deletion mutant appears to be multifactorial, with defects in viral RNA dimerization and packaging in producer cells as well as in reverse transcription of the viral RNA in infected cells. In this study, we sought to characterize SL1 mutant replication restrictions and provide insights into the underlying mechanisms of compensation in revertants. Results HIV-1 lacking SL1 (NLΔSL1 did not replicate in PM-1 cells until two independent non-synonymous mutations emerged: G913A in the matrix domain (E42K on day 18 postinfection and C1907T in the SP1 domain (P10L on day 11 postinfection. NLΔSL1 revertants carrying either compensatory mutation showed enhanced infectivity in PM-1 cells. The SL1 revertants produced significantly more infectious particles per nanogram of p24 than did NLΔSL1. The SL1 deletion mutant packaged less HIV-1 genomic RNA and more cellular RNA, particularly signal recognition particle RNA, in the virion than the wild-type. NLΔSL1 also packaged 3- to 4-fold more spliced HIV mRNA into the virion, potentially interfering with infectious virus production. In contrast, both revertants encapsidated 2.5- to 5-fold less of these HIV-1 mRNA species. Quantitative RT-PCR analysis of RNA cross-linked with Gag in formaldehyde-fixed cells demonstrated that the compensatory mutations reduced the association between Gag and spliced HIV-1 RNA, thereby effectively preventing these RNAs from being packaged into the virion. The reduction of spliced viral RNA in the virion may have a major role in facilitating infectious virus production, thus restoring the infectivity of NLΔSL1

  17. Validation of an ultrasensitive digital droplet PCR assay for HIV-2 plasma RNA quantification.

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    Ruelle, Jean; Yfantis, Vasilieios; Duquenne, Armelle; Goubau, Patrick

    2014-01-01

    Low or undetectable plasma viral load (VL) using current qPCR assays is common for HIV-2 patients. Digital PCR is an emerging technology enabling more precision and reproducibility than qPCR at low DNA/RNA copy numbers. Available data related to digital droplet PCR (ddPCR, Bio-Rad) underscore issues linked to the threshold definition of positivity, coupled to the specificity of low copy results (1). A RT-PCR protocol was set up using the One-Step RT-ddPCR Kit for Probes on the QX200 platform (Bio-Rad, Hercules, CA) in an accredited environment (ISO15189:2012 norm). Parameters tested were in line with the digital MIQE guidelines (2). Inter-run coefficient of variation (CV) was established using synthetic RNA controls diluted in HIV-negative plasma. The ddPCR assay was compared to a qRT-PCR previously used in routine (LOQ 50 cop/mL (3)) using 46 clinical samples and the NIBSC international HIV-2 RNA standard. The optimal PCR efficiency and the best separation between positive and negative droplets were obtained with a mixture containing 0.5 mM manganese acetate, 700 nM primers and 250 nM of the 5'FAM-probe. Using a manual threshold to define positivity, 7.74% of negative controls (n=168) were scored as positive due to one positive droplet. The presence of two positive droplets or more was not observed for negative controls. Serial dilutions of a positive control showed excellent linearity (R2=0.999) and enabled us to define a limit of quantification of two positives droplets, which corresponds to 0.14 copies/μL in the reaction mixture and to seven copies per mL of plasma. The inter-run coefficient of variation was 3.37% at a mean value of 4,468 cop/mL, 19.59% at 416 cop/mL and 32.28% at 8 cop/mL. The NIBSC standard of 1,000 IU was quantified 1,400 copies by ddPCR and close to 5,000 copies by qPCR (delta log superior to 0.5). Among 46 clinical samples, 22 were undetectable with both qPCR and ddPCR, 12 were detected with both methods (respective means of 10,612 and 2

  18. Validation of an ultrasensitive digital droplet PCR assay for HIV-2 plasma RNA quantification

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    Jean Ruelle

    2014-11-01

    Full Text Available Introduction: Low or undetectable plasma viral load (VL using current qPCR assays is common for HIV-2 patients. Digital PCR is an emerging technology enabling more precision and reproducibility than qPCR at low DNA/RNA copy numbers. Available data related to digital droplet PCR (ddPCR, Bio-Rad underscore issues linked to the threshold definition of positivity, coupled to the specificity of low copy results (1. Materials and Methods: A RT-PCR protocol was set up using the One-Step RT-ddPCR Kit for Probes on the QX200 platform (Bio-Rad, Hercules, CA in an accredited environment (ISO15189:2012 norm. Parameters tested were in line with the digital MIQE guidelines (2. Inter-run coefficient of variation (CV was established using synthetic RNA controls diluted in HIV-negative plasma. The ddPCR assay was compared to a qRT-PCR previously used in routine (LOQ 50 cop/mL (3 using 46 clinical samples and the NIBSC international HIV-2 RNA standard. Results: The optimal PCR efficiency and the best separation between positive and negative droplets were obtained with a mixture containing 0.5 mM manganese acetate, 700 nM primers and 250 nM of the 5’FAM-probe. Using a manual threshold to define positivity, 7.74% of negative controls (n=168 were scored as positive due to one positive droplet. The presence of two positive droplets or more was not observed for negative controls. Serial dilutions of a positive control showed excellent linearity (R2=0.999 and enabled us to define a limit of quantification of two positives droplets, which corresponds to 0.14 copies/μL in the reaction mixture and to seven copies per mL of plasma. The inter-run coefficient of variation was 3.37% at a mean value of 4,468 cop/mL, 19.59% at 416 cop/mL and 32.28% at 8 cop/mL. The NIBSC standard of 1,000 IU was quantified 1,400 copies by ddPCR and close to 5,000 copies by qPCR (delta log superior to 0.5. Among 46 clinical samples, 22 were undetectable with both qPCR and ddPCR, 12 were

  19. A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication

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    Stoltzfus C Martin

    2006-02-01

    Full Text Available Abstract Background Inefficient alternative splicing of the human immunodeficiency virus type 1(HIV-1 primary RNA transcript results in greater than half of all viral mRNA remaining unspliced. Regulation of HIV-1 alternative splicing occurs through the presence of suboptimal viral 5' and 3' splice sites (5' and 3'ss, which are positively regulated by exonic splicing enhancers (ESE and negatively regulated by exonic splicing silencers (ESS and intronic splicing silencers (ISS. We previously showed that splicing at HIV-1 3'ss A2 is repressed by ESSV and enhanced by the downstream 5'ss D3 signal. Disruption of ESSV results in increased vpr mRNA accumulation and exon 3 inclusion, decreased accumulation of unspliced viral mRNA, and decreased virus production. Results Here we show that optimization of the 5'ss D2 signal results in increased splicing at the upstream 3'ss A1, increased inclusion of exon 2 into viral mRNA, decreased accumulation of unspliced viral mRNA, and decreased virus production. Virus production from the 5'ss D2 and ESSV mutants was rescued by transient expression of HIV-1 Gag and Pol. We further show that the increased inclusion of either exon 2 or 3 does not significantly affect the stability of viral mRNA but does result in an increase and decrease, respectively, in HIV-1 mRNA levels. The changes in viral mRNA levels directly correlate with changes in tat mRNA levels observed upon increased inclusion of exon 2 or 3. Conclusion These results demonstrate that splicing at HIV-1 3'ss A1 is regulated by the strength of the downstream 5'ss signal and that suboptimal splicing at 3'ss A1 is necessary for virus replication. Furthermore, the replication defective phenotype resulting from increased splicing at 3'ss A1 is similar to the phenotype observed upon increased splicing at 3'ss A2. Further examination of the role of 5'ss D2 and D3 in the alternative splicing of 3'ss A1 and A2, respectively, is necessary to delineate a role for non

  20. Structure of Hepatitis E Virion-Sized Particle Reveals an RNA-Dependent Viral Assembly Pathway

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    Xing, L.; Wall, J.; Li, T.-C.; Mayazaki, N.; Simon, M. N.; Moore, M.; Wang, C.-Y.; Takeda, N.; Wakita, T.; Miyamura, T.; Cheng, R. H.

    2010-10-22

    Hepatitis E virus (HEV) induces acute hepatitis in humans with a high fatality rate in pregnant women. There is a need for anti-HEV research to understand the assembly process of HEV native capsid. Here, we produced a large virion-sized and a small T=1 capsid by expressing the HEV capsid protein in insect cells with and without the N-terminal 111 residues, respectively, for comparative structural analysis. The virion-sized capsid demonstrates a T=3 icosahedral lattice and contains RNA fragment in contrast to the RNA-free T=1 capsid. However, both capsids shared common decameric organization. The in vitro assembly further demonstrated that HEV capsid protein had the intrinsic ability to form decameric intermediate. Our data suggest that RNA binding is the extrinsic factor essential for the assembly of HEV native capsids.

  1. The viral transmembrane superfamily: possible divergence of Arenavirus and Filovirus glycoproteins from a common RNA virus ancestor

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    Buchmeier Michael J

    2001-02-01

    Full Text Available Abstract Background Recent studies of viral entry proteins from influenza, measles, human immunodeficiency virus, type 1 (HIV-1, and Ebola virus have shown, first with molecular modeling, and then X-ray crystallographic or other biophysical studies, that these disparate viruses share a coiled-coil type of entry protein. Results Structural models of the transmembrane glycoproteins (GP-2 of the Arenaviruses, lymphochoriomeningitis virus (LCMV and Lassa fever virus, are presented, based on consistent structural propensities despite variation in the amino acid sequence. The principal features of the model, a hydrophobic amino terminus, and two antiparallel helices separated by a glycosylated, antigenic apex, are common to a number of otherwise disparate families of enveloped RNA viruses. Within the first amphipathic helix, demonstrable by circular dichroism of a peptide fragment, there is a highly conserved heptad repeat pattern proposed to mediate multimerization by coiled-coil interactions. The amino terminal 18 amino acids are 28% identical and 50% highly similar to the corresponding region of Ebola, a member of the Filovirus family. Within the second, charged helix just prior to membrane insertion there is also high similarity over the central 18 amino acids in corresponding regions of Lassa and Ebola, which may be further related to the similar region of HIV-1 defining a potent antiviral peptide analogue. Conclusions These findings indicate a common pattern of structure and function among viral transmembrane fusion proteins from a number of virus families. Such a pattern may define a viral transmembrane superfamily that evolved from a common precursor eons ago.

  2. Viral hemorrhagic fevers of animals caused by positive-stranded RNA viruses

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    Here we outline serious diseases of wildlife, food and fiber animals, and non-human primates that cause damaging economic effects on producers all over the world. While some zoonotic viruses that occasionally cause serious disease and death in humans are mentioned, the positive sense RNA viruses ge...

  3. The Ebola Virus VP30-NP Interaction Is a Regulator of Viral RNA Synthesis

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    Kirchdoerfer, Robert N.; Moyer, Crystal L.; Abelson, Dafna M.; Saphire, Erica Ollmann (Scripps)

    2016-10-18

    Filoviruses are capable of causing deadly hemorrhagic fevers. All nonsegmented negative-sense RNA-virus nucleocapsids are composed of a nucleoprotein (NP), a phosphoprotein (VP35) and a polymerase (L). However, the VP30 RNA-synthesis co-factor is unique to the filoviruses. The assembly, structure, and function of the filovirus RNA replication complex remain unclear. Here, we have characterized the interactions of Ebola, Sudan and Marburg virus VP30 with NP using in vitro biochemistry, structural biology and cell-based mini-replicon assays. We have found that the VP30 C-terminal domain interacts with a short peptide in the C-terminal region of NP. Further, we have solved crystal structures of the VP30-NP complex for both Ebola and Marburg viruses. These structures reveal that a conserved, proline-rich NP peptide binds a shallow hydrophobic cleft on the VP30 C-terminal domain. Structure-guided Ebola virus VP30 mutants have altered affinities for the NP peptide. Correlation of these VP30-NP affinities with the activity for each of these mutants in a cell-based mini-replicon assay suggests that the VP30-NP interaction plays both essential and inhibitory roles in Ebola virus RNA synthesis.

  4. Rational design of avian metapneumovirus live attenuated vaccines by inhibiting viral messenger RNA cap methyltransferase

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    Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis, is a non-segmented negative-sense RNA virus belonging to the family of Paramyxoviridae, the subfamily Pneumovirinae, and the genus Metapneumovirus. aMPV is the causative agent of respiratory tract infection and ...

  5. The viral RNA-based transfection of enhanced green fluorescent protein (EGFP) in the parasitic protozoan Trichomonas vaginalis.

    Science.gov (United States)

    Li, Wei; Ding, He; Zhang, Xinxin; Cao, Lili; Li, Jianhua; Gong, Pengtao; Li, He; Zhang, Guocai; Li, Shuhong; Zhang, Xichen

    2012-03-01

    Here we have developed methods to transiently and stably transfect the human pathogenic protist Trichomonas vaginalis. The viral RNA-based transfection vector pTVV-EGFP/NEO was constructed by using enhanced green fluorescent protein gene (EGFP) and neomycin resistance gene (NEO) in tandem to replace the whole gene encoding region of T. vaginalis virus (TVV). The in vitro transcripts of linearized pTVV-EGFP/NEO were electroporated into trophozoites and the transfectants transiently expressed EGFP after 16 h postincubation. Stable expression of EGFP was persistently detected by fluorescence microscopy and by RT-PCR in transfected trophozoites under G418 selection. Our study provides a novel and valuable approach for genetic study of T. vaginalis.

  6. Estimating the impact of plasma HIV-1 RNA reductions on heterosexual HIV-1 transmission risk.

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    Jairam R Lingappa

    Full Text Available BACKGROUND: The risk of sexual transmission of HIV-1 is strongly associated with the level of HIV-1 RNA in plasma making reduction in HIV-1 plasma levels an important target for HIV-1 prevention interventions. A quantitative understanding of the relationship of plasma HIV-1 RNA and HIV-1 transmission risk could help predict the impact of candidate HIV-1 prevention interventions that operate by reducing plasma HIV-1 levels, such as antiretroviral therapy (ART, therapeutic vaccines, and other non-ART interventions. METHODOLOGY/PRINCIPAL FINDINGS: We use prospective data collected from 2004 to 2008 in East and Southern African HIV-1 serodiscordant couples to model the relationship of plasma HIV-1 RNA levels and heterosexual transmission risk with confirmation of HIV-1 transmission events by HIV-1 sequencing. The model is based on follow-up of 3381 HIV-1 serodiscordant couples over 5017 person-years encompassing 108 genetically-linked HIV-1 transmission events. HIV-1 transmission risk was 2.27 per 100 person-years with a log-linear relationship to log(10 plasma HIV-1 RNA. The model predicts that a decrease in average plasma HIV-1 RNA of 0.74 log(10 copies/mL (95% CI 0.60 to 0.97 reduces heterosexual transmission risk by 50%, regardless of the average starting plasma HIV-1 level in the population and independent of other HIV-1-related population characteristics. In a simulated population with a similar plasma HIV-1 RNA distribution the model estimates that 90% of overall HIV-1 infections averted by a 0.74 copies/mL reduction in plasma HIV-1 RNA could be achieved by targeting this reduction to the 58% of the cohort with plasma HIV-1 levels ≥4 log(10 copies/mL. CONCLUSIONS/SIGNIFICANCE: This log-linear model of plasma HIV-1 levels and risk of sexual HIV-1 transmission may help estimate the impact on HIV-1 transmission and infections averted from candidate interventions that reduce plasma HIV-1 RNA levels.

  7. Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

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    Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

    2015-04-01

    Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm.

  8. RNA-seq Analysis of Host and Viral Gene Expression Highlights Interaction between Varicella Zoster Virus and Keratinocyte Differentiation

    Science.gov (United States)

    Singh, Manuraj; Kanda, Ravinder K.; Yee, Michael B.; Kellam, Paul; Hollinshead, Michael; Kinchington, Paul R.; O'Toole, Edel A.; Breuer, Judith

    2014-01-01

    Varicella zoster virus (VZV) is the etiological agent of chickenpox and shingles, diseases characterized by epidermal skin blistering. Using a calcium-induced keratinocyte differentiation model we investigated the interaction between epidermal differentiation and VZV infection. RNA-seq analysis showed that VZV infection has a profound effect on differentiating keratinocytes, altering the normal process of epidermal gene expression to generate a signature that resembles patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Further investigation by real-time PCR, protein analysis and electron microscopy revealed that VZV specifically reduced expression of specific suprabasal cytokeratins and desmosomal proteins, leading to disruption of epidermal structure and function. These changes were accompanied by an upregulation of kallikreins and serine proteases. Taken together VZV infection promotes blistering and desquamation of the epidermis, both of which are necessary to the viral spread and pathogenesis. At the same time, analysis of the viral transcriptome provided evidence that VZV gene expression was significantly increased following calcium treatment of keratinocytes. Using reporter viruses and immunohistochemistry we confirmed that VZV gene and protein expression in skin is linked with cellular differentiation. These studies highlight the intimate host-pathogen interaction following VZV infection of skin and provide insight into the mechanisms by which VZV remodels the epidermal environment to promote its own replication and spread. PMID:24497829

  9. RNA-seq analysis of host and viral gene expression highlights interaction between varicella zoster virus and keratinocyte differentiation.

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    Meleri Jones

    2014-01-01

    Full Text Available Varicella zoster virus (VZV is the etiological agent of chickenpox and shingles, diseases characterized by epidermal skin blistering. Using a calcium-induced keratinocyte differentiation model we investigated the interaction between epidermal differentiation and VZV infection. RNA-seq analysis showed that VZV infection has a profound effect on differentiating keratinocytes, altering the normal process of epidermal gene expression to generate a signature that resembles patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Further investigation by real-time PCR, protein analysis and electron microscopy revealed that VZV specifically reduced expression of specific suprabasal cytokeratins and desmosomal proteins, leading to disruption of epidermal structure and function. These changes were accompanied by an upregulation of kallikreins and serine proteases. Taken together VZV infection promotes blistering and desquamation of the epidermis, both of which are necessary to the viral spread and pathogenesis. At the same time, analysis of the viral transcriptome provided evidence that VZV gene expression was significantly increased following calcium treatment of keratinocytes. Using reporter viruses and immunohistochemistry we confirmed that VZV gene and protein expression in skin is linked with cellular differentiation. These studies highlight the intimate host-pathogen interaction following VZV infection of skin and provide insight into the mechanisms by which VZV remodels the epidermal environment to promote its own replication and spread.

  10. The HEX1 gene of Fusarium graminearum is required for fungal asexual reproduction and pathogenesis and for efficient viral RNA accumulation of Fusarium graminearum virus 1.

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    Son, Moonil; Lee, Kyung-Mi; Yu, Jisuk; Kang, Minji; Park, Jin Man; Kwon, Sun-Jung; Kim, Kook-Hyung

    2013-09-01

    The accumulation of viral RNA depends on many host cellular factors. The hexagonal peroxisome (Hex1) protein is a fungal protein that is highly expressed when the DK21 strain of Fusarium graminearum virus 1 (FgV1) infects its host, and Hex1 affects the accumulation of FgV1 RNA. The Hex1 protein is the major constituent of the Woronin body (WB), which is a peroxisome-derived electron-dense core organelle that seals the septal pore in response to hyphal wounding. To clarify the role of Hex1 and the WB in the relationship between FgV1 and Fusarium graminearum, we generated targeted gene deletion and overexpression mutants. Although neither HEX1 gene deletion nor overexpression substantially affected vegetative growth, both changes reduced the production of asexual spores and reduced virulence on wheat spikelets in the absence of FgV1 infection. However, the vegetative growth of deletion and overexpression mutants was increased and decreased, respectively, upon FgV1 infection compared to that of an FgV1-infected wild-type isolate. Viral RNA accumulation was significantly decreased in deletion mutants but was significantly increased in overexpression mutants compared to the viral RNA accumulation in the virus-infected wild-type control. Overall, these data indicate that the HEX1 gene plays a direct role in the asexual reproduction and virulence of F. graminearum and facilitates viral RNA accumulation in the FgV1-infected host fungus.

  11. The 3′ Untranslated Region of the Andes Hantavirus Small mRNA Functionally Replaces the Poly(A) Tail and Stimulates Cap-Dependent Translation Initiation from the Viral mRNA

    Science.gov (United States)

    Vera-Otarola, Jorge; Soto-Rifo, Ricardo; Ricci, Emiliano P.; Ohlmann, Théophile; Darlix, Jean-Luc; López-Lastra, Marcelo

    2010-01-01

    In the process of translation of eukaryotic mRNAs, the 5′ cap and the 3′ poly(A) tail interact synergistically to stimulate protein synthesis. Unlike its cellular counterparts, the small mRNA (SmRNA) of Andes hantavirus (ANDV), a member of the Bunyaviridae, lacks a 3′ poly(A) tail. Here we report that the 3′ untranslated region (3′UTR) of the ANDV SmRNA functionally replaces a poly(A) tail and synergistically stimulates cap-dependent translation initiation from the viral mRNA. Stimulation of translation by the 3′UTR of the ANDV SmRNA was found to be independent of viral proteins and of host poly(A)-binding protein. PMID:20660206

  12. Minor groove RNA triplex in the crystal structure of a ribosomal frameshifting viral pseudoknot

    Science.gov (United States)

    Su, L.; Chen, L.; Egli, M.; Berger, J. M.; Rich, A.

    1999-01-01

    Many viruses regulate translation of polycistronic mRNA using a -1 ribosomal frameshift induced by an RNA pseudoknot. A pseudoknot has two stems that form a quasi-continuous helix and two connecting loops. A 1.6 A crystal structure of the beet western yellow virus (BWYV) pseudoknot reveals rotation and a bend at the junction of the two stems. A loop base is inserted in the major groove of one stem with quadruple-base interactions. The second loop forms a new minor-groove triplex motif with the other stem, involving 2'-OH and triple-base interactions, as well as sodium ion coordination. Overall, the number of hydrogen bonds stabilizing the tertiary interactions exceeds the number involved in Watson-Crick base pairs. This structure will aid mechanistic analyses of ribosomal frameshifting.

  13. Frequent Zika Virus Sexual Transmission and Prolonged Viral RNA Shedding in an Immunodeficient Mouse Model

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    Nisha K. Duggal

    2017-02-01

    Full Text Available Circulation of Zika virus (ZIKV was first identified in the Western hemisphere in late 2014. Primarily transmitted through mosquito bite, ZIKV can also be transmitted through sex and from mother to fetus, and maternal ZIKV infection has been associated with fetal malformations. We assessed immunodeficient AG129 mice for their capacity to shed ZIKV in semen and to infect female mice via sexual transmission. Infectious virus was detected in semen between 7 and 21 days post-inoculation, and ZIKV RNA was detected in semen through 58 days post-inoculation. During mating, 73% of infected males transmitted ZIKV to uninfected females, and 50% of females became infected, with evidence of fetal infection in resulting pregnancies. Semen from vasectomized mice contained significantly lower levels of infectious virus, though sexual transmission still occurred. This model provides a platform for studying the kinetics of ZIKV sexual transmission and prolonged RNA shedding also observed in human semen.

  14. Kaposi's sarcoma-associated herpesvirus ORF57 functions as a viral splicing factor and promotes expression of intron-containing viral lytic genes in spliceosome-mediated RNA splicing.

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    Majerciak, Vladimir; Yamanegi, Koji; Allemand, Eric; Kruhlak, Michael; Krainer, Adrian R; Zheng, Zhi-Ming

    2008-03-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 facilitates the expression of both intronless viral ORF59 genes and intron-containing viral K8 and K8.1 genes (V. Majerciak, N. Pripuzova, J. P. McCoy, S. J. Gao, and Z. M. Zheng, J. Virol. 81:1062-1071, 2007). In this study, we showed that disruption of ORF57 in a KSHV genome led to increased accumulation of ORF50 and K8 pre-mRNAs and reduced expression of ORF50 and K-bZIP proteins but had no effect on latency-associated nuclear antigen (LANA). Cotransfection of ORF57 and K8beta cDNA, which retains a suboptimal intron of K8 pre-mRNA due to alternative splicing, promoted RNA splicing of K8beta and production of K8alpha (K-bZIP). Although Epstein-Barr virus EB2, a closely related homolog of ORF57, had a similar activity in the cotransfection assays, herpes simplex virus type 1 ICP27 was inactive. This enhancement of RNA splicing by ORF57 correlates with the intact N-terminal nuclear localization signal motifs of ORF57 and takes place in the absence of other viral proteins. In activated KSHV-infected B cells, KSHV ORF57 partially colocalizes with splicing factors in nuclear speckles and assembles into spliceosomal complexes in association with low-abundance viral ORF50 and K8 pre-mRNAs and essential splicing components. The association of ORF57 with snRNAs occurs by ORF57-Sm protein interaction. We also found that ORF57 binds K8beta pre-mRNAs in vitro in the presence of nuclear extracts. Collectively our data indicate that KSHV ORF57 functions as a novel splicing factor in the spliceosome-mediated splicing of viral RNA transcripts.

  15. Prevalence of hepatitis A viral RNA and antibodies among Chinese blood donors.

    Science.gov (United States)

    Sun, P; Su, N; Lin, F Z; Ma, L; Wang, H J; Rong, X; Dai, Y D; Li, J; Jian, Z W; Tang, L H; Xiao, W; Li, C Q

    2015-12-09

    Like other developing countries, China was reported to have a relatively high seroprevalence of anti-hepatitis A antibodies (anti-HAV). However, no studies have evaluated the prevalence of anti-HAV and HAV RNA among voluntary blood donors with or without elevated serum alanine transaminase (ALT) levels. Anti-HAV antibodies were detected using an enzyme-linked immunosorbent assay, and reverse transcription quantitative polymerase chain reaction was carried out for detection of HAV RNA. In the current study, we analyzed a total of 450 serum samples with elevated ALT levels (≥40 U/L) and 278 serum samples with non-elevated ALT levels. Seroprevalence rates of anti-HAV were 51.6% in donors with elevated ALT and 41.4% in donors with non-elevated ALT; however, none of the samples was positive for HAV RNA. The results of our study showed lower seroprevalence rates of anti-HAV in blood donors (irrespective of ALT levels) than those in published data on Chinese populations. Although donors with elevated ALT had statistically higher prevalence rates of anti- HAV than did those with non-elevated ALT, none of the serum samples had detectable levels of the active virus. In conclusion, our results demonstrate that the transmission of hepatitis A by blood transfusion will occur rarely.

  16. A novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis C viral RNA with armored RNA as internal control

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    Meng Shuang

    2010-06-01

    Full Text Available Abstract Background The hepatitis C virus (HCV genome is extremely heterogeneous. Several HCV infections can not be detected using currently available commercial assays, probably because of mismatches between the template and primers/probes. By aligning the HCV sequences, we developed a duplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR assay using 2 sets of primers/probes and a specific armored RNA as internal control. The 2 detection probes were labelled with the same fluorophore, namely, 6-carboxyfluorescein (FAM, at the 5' end; these probes could mutually combine, improving the power of the test. Results The limit of detection of the duplex primer/probe assay was 38.99 IU/ml. The sensitivity of the assay improved significantly, while the specificity was not affected. All HCV genotypes in the HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques could be detected. In the testing of 109 serum samples, the performance of the duplex real-time RT-PCR assay was identical to that of the COBAS AmpliPrep (CAP/COBAS TaqMan (CTM assay and superior to 2 commercial HCV assay kits. Conclusions The duplex real-time RT-PCR assay is an efficient and effective viral assay. It is comparable with the CAP/CTM assay with regard to the power of the test and is appropriate for blood-donor screening and laboratory diagnosis of HCV infection.

  17. The C-Terminal Domain of Chikungunya Virus nsP2 Independently Governs Viral RNA Replication, Cytopathicity, and Inhibition of Interferon Signaling

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    Fros, J. J.; van der Maten, E.; Vlak, J. M.; Pijlman, G.P.

    2013-01-01

    Alphavirus nonstructural protein 2 (nsP2) has pivotal roles in viral RNA replication, host cell shutoff, and inhibition of antiviral responses. Mutations that individually rendered other alphaviruses noncytopathic were introduced into chikungunya virus nsP2. Results show that (i) nsP2 mutation P718S only in combination with KR649AA or adaptive mutation D711G allowed noncytopathic replicon RNA replication, (ii) prohibiting nsP2 nuclear localization abrogates inhibition of antiviral interferon-...

  18. The internal initiation of translation in bovine viral diarrhea virus RNA depends on the presence of an RNA pseudoknot upstream of the initiation codon

    Directory of Open Access Journals (Sweden)

    Moes Lorin

    2007-11-01

    Full Text Available Abstract Background Bovine viral diarrhea virus (BVDV is the prototype representative of the pestivirus genus in the Flaviviridae family. It has been shown that the initiation of translation of BVDV RNA occurs by an internal ribosome entry mechanism mediated by the 5' untranslated region of the viral RNA 1. The 5' and 3' boundaries of the IRES of the cytopathic BVDV NADL have been mapped and it has been suggested that the IRES extends into the coding of the BVDV polyprotein 2. A putative pseudoknot structure has been recognized in the BVDV 5'UTR in close proximity to the AUG start codon. A pseudoknot structure is characteristic for flavivirus IRESes and in the case of the closely related classical swine fever virus (CSFV and the more distantly related Hepatitis C virus (HCV pseudoknot function in translation has been demonstrated. Results To characterize the BVDV IRESes in detail, we studied the BVDV translational initiation by transfection of dicistronic expression plasmids into mammalian cells. A region coding for the amino terminus of the BVDV SD-1 polyprotein contributes considerably to efficient initiation of translation. The translation efficiency mediated by the IRES of BVDV strains NADL and SD-1 approximates the poliovirus type I IRES directed translation in BHK cells. Compared to the poliovirus IRES increased expression levels are mediated by the BVDV IRES of strain SD-1 in murine cell lines, while lower levels are observed in human cell lines. Site directed mutagenesis revealed that a RNA pseudoknot upstream of the initiator AUG is an important structural element for IRES function. Mutants with impaired ability to base pair in stem I or II lost their translational activity. In mutants with repaired base pairing either in stem 1 or in stem 2 full translational activity was restored. Thus, the BVDV IRES translation is dependent on the pseudoknot integrity. These features of the pestivirus IRES are reminiscent of those of the classical

  19. Cap Snatching of Yeast L-A Double-stranded RNA Virus Can Operate in Trans and Requires Viral Polymerase Actively Engaging in Transcription*

    Science.gov (United States)

    Fujimura, Tsutomu; Esteban, Rosa

    2012-01-01

    Eukaryotic mRNA bears a cap structure (m7GpppX-) at the 5′ terminus crucial for efficient translation and stability. The yeast L-A double-stranded RNA virus furnishes its mRNA with this structure by a novel cap-snatching mechanism in which the virus transfers an m7Gp moiety from host mRNA to the diphosphorylated 5′ terminus of the viral transcript, thus forming on it an authentic cap structure (referred to as cap0) in the budding yeast. This capping reaction is essential for efficient viral expression. His-154 of the capsid protein Gag is involved in the cap transfer. Here we show that the virus can utilize an externally added viral transcript as acceptor in the capping reaction. The acceptor needs to be 5′ diphosphorylated, consistent with the fact that the viral transcript bears diphosphate at the 5′ terminus. A 5′ triphosphorylated or monophosphorylated transcript does not function as acceptor. N7 methylation at the 5′ cap guanine of mRNA is essential for cap donor activity. We also demonstrate that the capping reaction requires the viral polymerase actively engaging in transcription. Because the cap-snatching site of Gag is located at the cytoplasmic surface of the virion, whereas Pol is confined inside the virion, the result indicates coordination between the cap-snatching and polymerization sites. This will allow L-A virus to efficiently produce capsid proteins to form new virions when Pol is actively engaging in transcription. The coordination may also minimize the risk of accidental capping of nonviral RNA when Pol is dormant. PMID:22367202

  20. Systematic identification of spontaneous preterm birth-associated RNA transcripts in maternal plasma.

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    Stephen S C Chim

    Full Text Available BACKGROUND: Spontaneous preterm birth (SPB, before 37 gestational weeks is a major cause of perinatal mortality and morbidity, but its pathogenesis remains unclear. Studies on SPB have been hampered by the limited availability of markers for SPB in predelivery clinical samples that can be easily compared with gestational age-matched normal controls. We hypothesize that SPB involves aberrant placental RNA expression, and that such RNA transcripts can be detected in predelivery maternal plasma samples, which can be compared with gestational age-matched controls. PRINCIPAL FINDINGS: Using gene expression microarray to profile essentially all human genes, we observed that 426 probe signals were changed by >2.9-fold in the SPB placentas, compared with the spontaneous term birth (STB placentas. Among the genes represented by those probes, we observed an over-representation of functions in RNA stabilization, extracellular matrix binding, and acute inflammatory response. Using RT-quantitative PCR, we observed differences in the RNA concentrations of certain genes only between the SPB and STB placentas, but not between the STB and term elective cesarean delivery placentas. Notably, 36 RNA transcripts were observed at placental microarray signals higher than a threshold, which indicated the possibility of their detection in maternal plasma. Among them, the IL1RL1 mRNA was tested in plasma samples taken from 37 women. It was detected in 6 of 10 (60% plasma samples collected during the presentation of preterm labor (≤32.9 weeks in women eventually giving SPB, but was detected in only 1 of 27 (4% samples collected during matched gestational weeks from women with no preterm labor (Fisher exact test, p = 0.00056. CONCLUSION: We have identified 36 SPB-associated RNA transcripts, which are possibly detectable in maternal plasma. We have illustrated that the IL1RL1 mRNA was more frequently detected in predelivery maternal plasma samples collected from women

  1. Cigarette smoking substantially alters plasma microRNA profiles in healthy subjects

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Kei; Yokota, Shin-ichi; Tatsumi, Naoyuki; Fukami, Tatsuki; Yokoi, Tsuyoshi; Nakajima, Miki, E-mail: nmiki@p.kanazawa-u.ac.jp

    2013-10-01

    Circulating microRNAs (miRNAs) are receiving attention as potential biomarkers of various diseases, including cancers, chronic obstructive pulmonary disease, and cardiovascular disease. However, it is unknown whether the levels of circulating miRNAs in a healthy subject might vary with external factors in daily life. In this study, we investigated whether cigarette smoking, a habit that has spread throughout the world and is a risk factor for various diseases, affects plasma miRNA profiles. We determined the profiles of 11 smokers and 7 non-smokers by TaqMan MicroRNA array analysis. A larger number of miRNAs were detected in smokers than in non-smokers, and the plasma levels of two-thirds of the detected miRNAs (43 miRNAs) were significantly higher in smokers than in non-smokers. A principal component analysis of the plasma miRNA profiles clearly separated smokers and non-smokers. Twenty-four of the miRNAs were previously reported to be potential biomarkers of disease, suggesting the possibility that smoking status might interfere with the diagnosis of disease. Interestingly, we found that quitting smoking altered the plasma miRNA profiles to resemble those of non-smokers. These results suggested that the differences in the plasma miRNA profiles between smokers and non-smokers could be attributed to cigarette smoking. In addition, we found that an acute exposure of ex-smokers to cigarette smoke (smoking one cigarette) did not cause a dramatic change in the plasma miRNA profile. In conclusion, we found that repeated cigarette smoking substantially alters the plasma miRNA profile, interfering with the diagnosis of disease or signaling potential smoking-related diseases. - Highlights: • Plasma miRNA profiles were unambiguously different between smokers and non-smokers. • Smoking status might interfere with the diagnosis of disease using plasma miRNAs. • Changes of plasma miRNA profiles may be a signal of smoking-related diseases.

  2. Long non-coding RNA GAS5 inhibited hepatitis C virus replication by binding viral NS3 protein.

    Science.gov (United States)

    Qian, Xijing; Xu, Chen; Zhao, Ping; Qi, Zhongtian

    2016-05-01

    HCV infection has a complex and dynamic process which involves a large number of viral and host factors. Long non-coding RNA GAS5 inhibits liver fibrosis and liver tumor migration and invasion. However, the contribution of GAS5 on HCV infection remains unknown. In this study, GAS5 was gradually upregulated during HCV infection in Huh7 cells. In addition, GAS5 attenuated virus replication with its 5' end sequences, as confirmed by different GAS5 truncations. Moreover, this 5' end sequences showed RNA-protein interaction with HCV NS3 protein that could act as a decoy to inhibit its functions, which contributed to the suppression of HCV replication. Finally, the innate immune responses remained low in HCV infected Huh7 cells, ruling out the possibility of GAS5 to modulate innate immunity. Thus, HCV stimulated endogenous GAS5 can suppress HCV infection by acting as HCV NS3 protein decoy, providing a potential role of GAS5 as a diagnostic or therapeutic target.

  3. Use of Dried Plasma Spots for HIV-1 Viral Load Determination and Drug Resistance Genotyping in Mexican Patients

    Science.gov (United States)

    Rodriguez-Auad, Juan Pablo; Rojas-Montes, Othon; Maldonado-Rodriguez, Angelica; Alvarez-Muñoz, Ma. Teresa; Muñoz, Onofre; Torres-Ibarra, Rocio; Vazquez-Rosales, Guillermo

    2015-01-01

    Monitoring antiretroviral therapy using measurements of viral load (VL) and the genotyping of resistance mutations is not routinely performed in low- to middle-income countries because of the high costs of the commercial assays that are used. The analysis of dried plasma spot (DPS) samples on filter paper may represent an alternative for resource-limited settings. Therefore, we evaluated the usefulness of analyzing DPS samples to determine VL and identify drug resistance mutations (DRM) in a group of HIV-1 patients. The VL was measured from 22 paired plasma and DPS samples. In these samples, the average VL was 4.7 log10 copies/mL in liquid plasma and 4.1 log10 copies/mL in DPS, with a correlation coefficient of R = 0.83. A 1.1 kb fragment of HIV pol could be amplified in 14/22 (63.6%) of the DPS samples and the same value was amplified in plasma samples. A collection of ten paired DPS and liquid plasma samples was evaluated for the presence of DRM; an excellent correlation was found in the identification of DRM between the paired samples. All HIV-1 pol sequences that were obtained corresponded to HIV subtype B. The analysis of DPS samples offers an attractive alternative for monitoring ARV therapy in resource-limited settings. PMID:26779533

  4. Use of Dried Plasma Spots for HIV-1 Viral Load Determination and Drug Resistance Genotyping in Mexican Patients

    Directory of Open Access Journals (Sweden)

    Juan Pablo Rodriguez-Auad

    2015-01-01

    Full Text Available Monitoring antiretroviral therapy using measurements of viral load (VL and the genotyping of resistance mutations is not routinely performed in low- to middle-income countries because of the high costs of the commercial assays that are used. The analysis of dried plasma spot (DPS samples on filter paper may represent an alternative for resource-limited settings. Therefore, we evaluated the usefulness of analyzing DPS samples to determine VL and identify drug resistance mutations (DRM in a group of HIV-1 patients. The VL was measured from 22 paired plasma and DPS samples. In these samples, the average VL was 4.7 log10 copies/mL in liquid plasma and 4.1 log10 copies/mL in DPS, with a correlation coefficient of R = 0.83. A 1.1 kb fragment of HIV pol could be amplified in 14/22 (63.6% of the DPS samples and the same value was amplified in plasma samples. A collection of ten paired DPS and liquid plasma samples was evaluated for the presence of DRM; an excellent correlation was found in the identification of DRM between the paired samples. All HIV-1 pol sequences that were obtained corresponded to HIV subtype B. The analysis of DPS samples offers an attractive alternative for monitoring ARV therapy in resource-limited settings.

  5. The C-terminal domain of chikungunya virus nsP2 independently governs viral RNA replication, cytopathicity, and inhibition of interferon signaling

    NARCIS (Netherlands)

    Fros, J.J.; Maten, van der E.; Vlak, J.M.; Pijlman, G.P.

    2013-01-01

    Alphavirus nonstructural protein 2 (nsP2) has pivotal roles in viral RNA replication, host cell shutoff, and inhibition of antiviral responses. Mutations that individually rendered other alphaviruses noncytopathic were introduced into chikungunya virus nsP2. Results show that (i) nsP2 mutation P718S

  6. The C-terminal domain of chikungunya virus nsP2 independently governs viral RNA replication, cytopathicity, and inhibition of interferon signaling

    NARCIS (Netherlands)

    Fros, J.J.; Maten, van der E.; Vlak, J.M.; Pijlman, G.P.

    2013-01-01

    Alphavirus nonstructural protein 2 (nsP2) has pivotal roles in viral RNA replication, host cell shutoff, and inhibition of antiviral responses. Mutations that individually rendered other alphaviruses noncytopathic were introduced into chikungunya virus nsP2. Results show that (i) nsP2 mutation P718S

  7. Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus

    Science.gov (United States)

    Xu, Kai; Nagy, Peter D.

    2016-01-01

    Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs). The VRCs built by Tomato bushy stunt virus (TBSV) are enriched with phosphatidylethanolamine (PE) through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP)-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5–positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment. PMID:27760128

  8. Visualising a viral RNA genome poised for release from its receptor complex.

    Science.gov (United States)

    Toropova, Katerina; Stockley, Peter G; Ranson, Neil A

    2011-05-06

    We describe the cryo-electron microscopy structure of bacteriophage MS2 bound to its receptor, the bacterial F-pilus. The virus contacts the pilus at a capsid 5-fold vertex, thus locating the surface-accessible portion of the single copy of the pilin-binding maturation protein present in virions. This arrangement allows a 5-fold averaged map to be calculated, showing for the first time in any virus-receptor complex the nonuniform distribution of RNA within the capsid. Strikingly, at the vertex that contacts the pilus, a rod of density that may include contributions from both genome and maturation protein sits above a channel that goes through the capsid to the outside. This density is reminiscent of the DNA density observed in the exit channel of double-stranded DNA phages, suggesting that the RNA-maturation protein complex is poised to leave the capsid as the first step of the infection process. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Patterns of oligonucleotide sequences in viral and host cell RNA identify mediators of the host innate immune system.

    Directory of Open Access Journals (Sweden)

    Benjamin D Greenbaum

    Full Text Available The innate immune response provides a first line of defense against pathogens by targeting generic differential features that are present in foreign organisms but not in the host. These innate responses generate selection forces acting both in pathogens and hosts that further determine their co-evolution. Here we analyze the nucleic acid sequence fingerprints of these selection forces acting in parallel on both host innate immune genes and ssRNA viral genomes. We do this by identifying dinucleotide biases in the coding regions of innate immune response genes in plasmacytoid dendritic cells, and then use this signal to identify other significant host innate immune genes. The persistence of these biases in the orthologous groups of genes in humans and chickens is also examined. We then compare the significant motifs in highly expressed genes of the innate immune system to those in ssRNA viruses and study the evolution of these motifs in the H1N1 influenza genome. We argue that the significant under-represented motif pattern of CpG in an AU context--which is found in both the ssRNA viruses and innate genes, and has decreased throughout the history of H1N1 influenza replication in humans--is immunostimulatory and has been selected against during the co-evolution of viruses and host innate immune genes. This shows how differences in host immune biology can drive the evolution of viruses that jump into species with different immune priorities than the original host.

  10. Correlation of mRNA Profiles, miRNA Profiles, and Functional Immune Response in Rainbow Trout (Oncorrhynkus Mykiss) Infected With Viral Hemorrhagic Septicemia Virus (VHSV) and in Fish Vaccinated With a DNA Vaccine Against VHSV

    DEFF Research Database (Denmark)

    Bela-Ong, Dennis; Schyth, Brian Dall; Jørgensen, Hanne

    2011-01-01

    and are incorporated into the RNA-Induced Silencing Complex (RISC), which target specific mRNA sequences, causing either mRNA degradation or translation repression. This results in altered mRNA and protein profiles characteristic of a particular cellular phenotype or physiological state. By targeting immune relevant m...... with the highly pathogenic fish rhabdovirus Viral hemorrhagic septicemia virus (VHSV). This talk will discuss our overall strategy and present preliminary data on the expression of miRNAs and the type I interferon-inducible Mx gene in the liver and the skeletal muscle tissue of fish injected with a DNA vaccine...

  11. Lipid droplet-binding protein TIP47 regulates hepatitis C Virus RNA replication through interaction with the viral NS5A protein.

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    Dorothee A Vogt

    Full Text Available The nonstructural protein NS5A has emerged as a new drug target in antiviral therapies for Hepatitis C Virus (HCV infection. NS5A is critically involved in viral RNA replication that takes place at newly formed membranes within the endoplasmic reticulum (membranous web and assists viral assembly in the close vicinity of lipid droplets (LDs. To identify host proteins that interact with NS5A, we performed a yeast two-hybrid screen with the N-terminus of NS5A (amino acids 1-31, a well-studied α-helical domain important for the membrane tethering of NS5A. Our studies identified the LD-associated host protein, Tail-Interacting Protein 47 (TIP47 as a novel NS5A interaction partner. Coimmunoprecipitation experiments in Huh7 hepatoma cells confirmed the interaction of TIP47 with full-length NS5A. shRNA-mediated knockdown of TIP47 caused a more than 10-fold decrease in the propagation of full-length infectious HCV in Huh7.5 hepatoma cells. A similar reduction was observed when TIP47 was knocked down in cells harboring an autonomously replicating HCV RNA (subgenomic replicon, indicating that TIP47 is required for efficient HCV RNA replication. A single point mutation (W9A in NS5A that disrupts the interaction with TIP47 but preserves proper subcellular localization severely decreased HCV RNA replication. In biochemical membrane flotation assays, TIP47 cofractionated with HCV NS3, NS5A, NS5B proteins, and viral RNA, and together with nonstructural viral proteins was uniquely distributed to lower-density LD-rich membrane fractions in cells actively replicating HCV RNA. Collectively, our data support a model where TIP47--via its interaction with NS5A--serves as a novel cofactor for HCV infection possibly by integrating LD membranes into the membranous web.

  12. Extracellular tumor-related mRNA in plasma of lymphoma patients and survival implications.

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    Vanesa Garcia

    Full Text Available BACKGROUND: We studied anomalous extracellular mRNAs in plasma from patients with diffuse large B-cell lymphoma (DLBCL and their survival implications. mRNAs studied have been reported in the literature as markers of poor (BCL2, CCND2, MYC and favorable outcome (LMO2, BCL6, FN1 in tumors. These markers were also analyzed in lymphoma tissues to test possible associations with their presence in plasma. METHODOLOGY/PRINCIPAL FINDINGS: mRNA from 42 plasma samples and 12 tumors from patients with DLBCL was analyzed by real-time PCR. Samples post-treatment were studied. The immunohistochemistry of BCL2 and BCL6 was defined. Presence of circulating tumor cells was determined by analyzing the clonality of the immunoglobulin heavy-chain genes by PCR. In DLBCL, MYC mRNA was associated with short overall survival. mRNA targets with unfavorable outcome in tumors were associated with characteristics indicative of poor prognosis, with partial treatment response and with short progression-free survival in patients with complete response. In patients with low IPI score, unfavorable mRNA targets were related to shorter overall survival, partial response, high LDH levels and death. mRNA disappeared in post-treatment samples of patients with complete response, and persisted in those with partial response or death. No associations were found between circulating tumor cells and plasma mRNA. Absence of BCL6 protein in tumors was associated with presence of unfavorable plasma mRNA. CONCLUSIONS/SIGNIFICANCE: Through a non-invasive procedure, tumor-derived mRNAs can be obtained in plasma. mRNA detected in plasma did not proceed from circulating tumor cells. In our study, unfavorable targets in plasma were associated with poor prognosis in B-cell lymphomas, mainly MYC mRNA. Moreover, the unfavorable targets in plasma could help us to classify patients with poor outcome within the good prognosis group according to IPI.

  13. Total Extracellular Small RNA Profiles from Plasma, Saliva, and Urine of Healthy Subjects

    Science.gov (United States)

    Yeri, Ashish; Courtright, Amanda; Reiman, Rebecca; Carlson, Elizabeth; Beecroft, Taylor; Janss, Alex; Siniard, Ashley; Richholt, Ryan; Balak, Chris; Rozowsky, Joel; Kitchen, Robert; Hutchins, Elizabeth; Winarta, Joseph; McCoy, Roger; Anastasi, Matthew; Kim, Seungchan; Huentelman, Matthew; Van Keuren-Jensen, Kendall

    2017-01-01

    Interest in circulating RNAs for monitoring and diagnosing human health has grown significantly. There are few datasets describing baseline expression levels for total cell-free circulating RNA from healthy control subjects. In this study, total extracellular RNA (exRNA) was isolated and sequenced from 183 plasma samples, 204 urine samples and 46 saliva samples from 55 male college athletes ages 18–25 years. Many participants provided more than one sample, allowing us to investigate variability in an individual’s exRNA expression levels over time. Here we provide a systematic analysis of small exRNAs present in each biofluid, as well as an analysis of exogenous RNAs. The small RNA profile of each biofluid is distinct. We find that a large number of RNA fragments in plasma (63%) and urine (54%) have sequences that are assigned to YRNA and tRNA fragments respectively. Surprisingly, while many miRNAs can be detected, there are few miRNAs that are consistently detected in all samples from a single biofluid, and profiles of miRNA are different for each biofluid. Not unexpectedly, saliva samples have high levels of exogenous sequence that can be traced to bacteria. These data significantly contribute to the current number of sequenced exRNA samples from normal healthy individuals. PMID:28303895

  14. Persistence of Hepatitis C RNA in Liver Allografts Is Associated with Histologic Progression Independent of Serologic Viral Clearance

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    M. Ghabril

    2009-01-01

    Full Text Available Background. Hepatitis C virus (HCV nondetectability in the liver may predict a sustained viral response (SVR in patients with an end of treatment response. HCV RNA can be detected in liver tissue by in situ hybridization (ISH. Aim. To determine if HCV nondetectability in liver allografts by ISH can predict SVR in patients who cleared virus serologically on treatment. Methods. Twenty five patients with undetectable serum HCV on Interferon/Ribavirin therapy for HCV recurrence post liver transplant (LT were studied. All had biopsies at 4 months post LT (baseline and follow up with HCV ISH analysis performed. Results. 10 were ISH positive (group 1; 15 were ISH negative (group 2. Groups 1 and 2 had similar patient, donor, and viral characteristics at LT, as well as treatment duration at the time of the ISH assayed liver biopsy (13±16 versus 10±4 months P = .24. However, group 1 had longer total treatment duration (24±10 versus 14±5 months, P = .001. Eight (80% group 1 and 9 (60% group 2 patients achieved SVR. Mean grade and stage (modified Ishak score were similar at 4 months, however, group 1 had higher grade (3±1.7 versus 1.6±1.3, P = .039 and stage (1.4±1.4 versus 0.5±0.6, P = .084 on the ISH assayed biopsy, after similar post LT intervals (23±10 versus 24±12 months, P = .91. Conclusion. Allograft HCV ISH nondetectability does not predict SVR in treatment responsive HCV recurrence, but is associated with less severe histologic disease.

  15. A human herpesvirus miRNA attenuates interferon signaling and contributes to maintenance of viral latency by targeting IKKε

    Institute of Scientific and Technical Information of China (English)

    Deguang Liang; Yuan Gao; Xianzhi Lin; Zhiheng He; Qinglan Zhao; Qiang Deng; Ke Lan

    2011-01-01

    Type I interferon(IFN)signaling is the principal response mediating antiviral innate immunity. IFN transcription is dependent upon the activation of transcription factors IRF3/IRF7 and NF-KB. Many viral proteins have been shown as being capable of interfering with IFN signaling to facilitate evasion from the host innate immune response.Here, we report that a viral miRNA, miR-K12-11, encoded by Kaposi's sarcoma-associated herpesvirus(KSHV)is critical for the modulation of IFN signaling and acts through targeting 1-kappa-B kinase epsilon(IKKε). Ectopic expression of miR-K12-11 resulted in decreased IKKε expression, while inhibition of miR-K12-11 was found to restore IKKE expression in KSHV-infected cells. Importantly, expression of milk-K12-I1 attenuated IFN signaling by decreasing IKKε-mediated IRF3/IRF7 phosphorylation and by inhibiting the activation of IKKE-dependent IFN stimulating genes(ISGs), allowing miR-K12-11 suppression of antiviral immunity. Our data suggest that IKKE targeting by miR-K12-11 is an important strategy utilized by KSHV to modulate IFN signaling during the KSHV lifecycle, especially in latency. We also demonstrated that IKKE was able to enhance KSHV reactivation synergistically with the treatment of 12-O-tetradecanoylphorbol 13-acetate. Moreover, inhibition of miR-K12-11enhanced KSHV reactivation induced by vesicular stomatitis virus infection. Taken together, our findings also suggest that miR-K12-11 can contribute to maintenance of KSHV latency by targeting IKKE.

  16. The C-terminal domain of chikungunya virus nsP2 independently governs viral RNA replication, cytopathicity, and inhibition of interferon signaling.

    Science.gov (United States)

    Fros, Jelke J; van der Maten, Erika; Vlak, Just M; Pijlman, Gorben P

    2013-09-01

    Alphavirus nonstructural protein 2 (nsP2) has pivotal roles in viral RNA replication, host cell shutoff, and inhibition of antiviral responses. Mutations that individually rendered other alphaviruses noncytopathic were introduced into chikungunya virus nsP2. Results show that (i) nsP2 mutation P718S only in combination with KR649AA or adaptive mutation D711G allowed noncytopathic replicon RNA replication, (ii) prohibiting nsP2 nuclear localization abrogates inhibition of antiviral interferon-induced JAK-STAT signaling, and (iii) nsP2 independently affects RNA replication, cytopathicity, and JAK-STAT signaling.

  17. Biodegradable Nanoparticles of mPEG-PLGA-PLL Triblock Copolymers as Novel Non-Viral Vectors for Improving siRNA Delivery and Gene Silencing

    Directory of Open Access Journals (Sweden)

    Qiu-Sheng Shi

    2012-01-01

    Full Text Available Degradation of mRNA by RNA interference is one of the most powerful and specific mechanisms for gene silencing. However, insufficient cellular uptake and poor stability have limited its usefulness. Here, we report efficient delivery of siRNA via the use of biodegradable nanoparticles (NPs made from monomethoxypoly(ethylene glycol-poly(lactic-co-glycolic acid-poly-l-lysine (mPEG-PLGA-PLL triblock copolymers. Various physicochemical properties of mPEG-PLGA-PLL NPs, including morphology, size, surface charge, siRNA encapsulation efficiency, and in vitro release profile of siRNA from NPs, were characterized by scanning electron microscope, particle size and zeta potential analyzer, and high performance liquid chromatography. The levels of siRNA uptake and targeted gene inhibition were detected in human lung cancer SPC-A1-GFP cells stably expressing green fluorescent protein. Examination of the cultured SPC-A1-GFP cells with fluorescent microscope and flow cytometry showed NPs loading Cy3-labeled siRNA had much higher intracellular siRNA delivery efficiencies than siRNA alone and Lipofectamine-siRNA complexes. The gene silencing efficiency of mPEG-PLGA-PLL NPs was higher than that of commercially available transfecting agent Lipofectamine while showing no cytotoxicity. Thus, the current study demonstrates that biodegradable NPs of mPEG-PLGA-PLL triblock copolymers can be potentially applied as novel non-viral vectors for improving siRNA delivery and gene silencing.

  18. Biodegradable nanoparticles of mPEG-PLGA-PLL triblock copolymers as novel non-viral vectors for improving siRNA delivery and gene silencing.

    Science.gov (United States)

    Du, Jing; Sun, Ying; Shi, Qiu-Sheng; Liu, Pei-Feng; Zhu, Ming-Jie; Wang, Chun-Hui; Du, Lian-Fang; Duan, You-Rong

    2012-01-01

    Degradation of mRNA by RNA interference is one of the most powerful and specific mechanisms for gene silencing. However, insufficient cellular uptake and poor stability have limited its usefulness. Here, we report efficient delivery of siRNA via the use of biodegradable nanoparticles (NPs) made from monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly-l-lysine (mPEG-PLGA-PLL) triblock copolymers. Various physicochemical properties of mPEG-PLGA-PLL NPs, including morphology, size, surface charge, siRNA encapsulation efficiency, and in vitro release profile of siRNA from NPs, were characterized by scanning electron microscope, particle size and zeta potential analyzer, and high performance liquid chromatography. The levels of siRNA uptake and targeted gene inhibition were detected in human lung cancer SPC-A1-GFP cells stably expressing green fluorescent protein. Examination of the cultured SPC-A1-GFP cells with fluorescent microscope and flow cytometry showed NPs loading Cy3-labeled siRNA had much higher intracellular siRNA delivery efficiencies than siRNA alone and Lipofectamine-siRNA complexes. The gene silencing efficiency of mPEG-PLGA-PLL NPs was higher than that of commercially available transfecting agent Lipofectamine while showing no cytotoxicity. Thus, the current study demonstrates that biodegradable NPs of mPEG-PLGA-PLL triblock copolymers can be potentially applied as novel non-viral vectors for improving siRNA delivery and gene silencing.

  19. Association of human mitochondrial lysyl-tRNA synthetase with HIV-1 GagPol does not require other viral proteins

    Directory of Open Access Journals (Sweden)

    Lydia Kobbi

    2016-06-01

    Full Text Available In human, the cytoplasmic (cLysRS and mitochondrial (mLysRS species of lysyl-tRNA synthetase are encoded by a single gene. Following HIV-1 infection, mLysRS is selectively taken up into viral particles along with the three tRNALys isoacceptors. The GagPol polyprotein precursor is involved in this process. With the aim to reconstitute in vitro the HIV-1 tRNA3Lys packaging complex, we first searched for the putative involvement of another viral protein in the selective viral hijacking of mLysRS only. After screening all the viral proteins, we observed that Vpr and Rev have the potential to interact with mLysRS, but that this association does not take place at the level of the assembly of mLysRS into the packaging complex. We also show that tRNA3Lys can form a ternary complex with the two purified proteins mLysRS and the Pol domain of GagPol, which mimicks its packaging complex.

  20. Genetics and Molecular Biology of Epstein-Barr Virus-Encoded BART MicroRNA: A Paradigm for Viral Modulation of Host Immune Response Genes and Genome Stability

    Directory of Open Access Journals (Sweden)

    David H. Dreyfus

    2017-01-01

    Full Text Available Epstein-Barr virus, a ubiquitous human herpesvirus, is associated through epidemiologic evidence with common autoimmune syndromes and cancers. However, specific genetic mechanisms of pathogenesis have been difficult to identify. In this review, the author summarizes evidence that recently discovered noncoding RNAs termed microRNA encoded by Epstein-Barr virus BARF (BamHI A right frame termed BART (BamHI A right transcripts are modulators of human immune response genes and genome stability in infected and bystander cells. BART expression is apparently regulated by complex feedback loops with the host immune response regulatory NF-κB transcription factors. EBV-encoded BZLF-1 (ZEBRA protein could also regulate BART since ZEBRA contains a terminal region similar to ankyrin proteins such as IκBα that regulate host NF-κB. BALF-2 (BamHI A left frame transcript, a viral homologue of the immunoglobulin and T cell receptor gene recombinase RAG-1 (recombination-activating gene-1, may also be coregulated with BART since BALF-2 regulatory sequences are located near the BART locus. Viral-encoded microRNA and viral mRNA transferred to bystander cells through vesicles, defective viral particles, or other mechanisms suggest a new paradigm in which bystander or hit-and-run mechanisms enable the virus to transiently or chronically alter human immune response genes as well as the stability of the human genome.

  1. Evolution of viral RNA in a Chinese patient to interferon/ribavirin therapy for hepatitis C

    Institute of Scientific and Technical Information of China (English)

    Xian-Zi Wen; Zhi-Hai Chen; Ya-Zhi Wei; Jia-Fu Ji

    2012-01-01

    Objective:The combination of interferon (IFN) and ribavirin (RBV) is the standard therapy for hepatitis C virus (HCV) infection.HCV genotype 2a has proved more amenable to the therapy,but its efficacy is yet limited.This study aimed to investigate the mechanism of the poor response in a case of HCV genotype 2a infection.Methods:We analyzed dynamic change of HCV RNA from a patient,infected with HCV genotype 2a,showing a poor virological response to IFN/RBV as judged 12 weeks after initiation of the therapy by HCV clone sequencing.Then we constructed subgenomic Japanese fulminant hepatitis-1 (JFH1) replicon and different chimeric replicons with humanized Gaussia luciferase gene.The chimeric replicons were derived from subgenomic JFH1 replicon,in which the NS5A region was replaced by the patient's sequence from the pre/post-treatment,and the chimeric replicons' susceptibility to IFN were evaluated by relative Gausia Luciferase activity.Results:The pretreatment HCV sequences appeared almost uniform,and the quasispecies variation was further more simplified after 12 weeks of therapy.Besides,the quasispecies variation seemed to be more diversified in the NS5A,relatively,a region crucial for IFN response,and each of chimeric replicons exhibited distinct response to IFN.Conclusions:During the course of the chronic infection,HCV population seems to be adapted to the patient's immunological system,and further to be selected by combination of IFN/RBV therapy,indicating quasispecies may completely eliminated by addition of other drugs with targets different from those of IFN.In addition,each different response of chimeric replicon to IFN is most likely related to amino acid changes in or near the IFN-sensitivity determining region (ISDR) of NS5A during chronic infection and IFN/RBV therapy.

  2. Non-Viral, Lipid-Mediated DNA and mRNA Gene Therapy of the Central Nervous System (CNS): Chemical-Based Transfection.

    Science.gov (United States)

    Hecker, James G

    2016-01-01

    Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Cationic lipid-mediated delivery is an alternative to viral vector-mediated gene delivery. Lipid-mediated delivery of DNA or mRNA is usually more rapid than viral-mediated delivery, offers a larger payload, and has a nearly zero risk of incorporation. Lipid-mediated delivery of DNA or RNA is therefore preferable to viral DNA delivery in those clinical applications that do not require long-term expression for chronic conditions. Delivery of RNA may be preferable to non-viral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary and onset of expression with RNA is therefore even faster than with DNA, although both are faster than most viral vectors. Here, we describe techniques for cationic lipid-mediated delivery of nucleic acids encoding reporter genes in a variety of cell lines. We describe optimized formulations and transfection procedures that we previously assessed by bioluminescence and flow cytometry. RNA transfection demonstrates increased efficiency relative to DNA transfection in non-dividing cells. Delivery of mRNA results in onset of expression within 1 h after transfection and a peak in expression 5-7 h after transfection. Duration of expression in eukaryotic cells after mRNA transcript delivery depends on multiple factors, including transcript stability, protein turnover, and cell type. Delivery of DNA results in onset of expression within 5 h after transfection, a peak in expression 24-48 h after transfection, and a return to baseline that can be as long as several weeks after transfection. In vitro results are consistent with our in vivo delivery results, techniques for which are described as well. RNA delivery is suitable for short-term transient gene expression due to its rapid onset, short duration of expression and greater efficiency, particularly in non-dividing cells, while the longer duration and

  3. Small interfering RNAs targeting peste des petits ruminants virus M mRNA increase virus-mediated fusogenicity and inhibit viral replication in vitro.

    Science.gov (United States)

    Liu, Fuxiao; Wu, Xiaodong; Zou, Yanli; Li, Lin; Liu, Shan; Chi, Tianying; Wang, Zhiliang

    2015-11-01

    Peste des petits ruminants (PPR), caused by peste des petits ruminants virus (PPRV), is an acute or subacute, highly contagious and economically important disease of small ruminants. The PPRV is classified into the genus Morbillivirus in the family Paramyxoviridae. The PPRV matrix (M) protein possesses an intrinsic ability to bind to lipid membranes, and plays a crucial role in viral assembly and further budding. In this study, three different small interfering RNAs (siRNA) were designed on the basis of translated region for PPRV Nigeria 75/1M mRNA, and were subsequently synthesized for their transfection into Vero-SLAM cells, followed by infection with PPRVs. The results showed that two out of three siRNAs robustly induced cell-to-cell fusion as early as 36h post-infection with PPRVs, effectively suppressed expression of the M protein by interference for the M mRNA, and eventually inhibited viral replication in vitro. These findings led us to speculate that siRNA-mediated knockdown of the M protein would alter its interaction with viral glycoproteins, thus exacerbating intercellular fusion but hampering virus release. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Comparison of EBV DNA viral load in whole blood, plasma, B-cells and B-cell culture supernatant.

    Science.gov (United States)

    Ouedraogo, David Eric; Bollore, Karine; Viljoen, Johannes; Foulongne, Vincent; Reynes, Jacques; Cartron, Guillaume; Vendrell, Jean-Pierre; Van de Perre, Philippe; Tuaillon, Edouard

    2014-05-01

    Epstein-Barr virus (EBV) genome quantitation in whole blood is used widely for therapeutic monitoring of EBV-associated disorders in immunosuppressed individuals and in patients with EBV-associated lymphoma. However, the most appropriate biological material to be used for EBV DNA quantitation remains a subject of debate. This study compare the detection rate and levels of EBV DNA from whole blood, plasma, enriched B-cells, and B-cell short-term culture supernatant using quantitative real-time PCR. Samples were collected from 33 subjects with either HIV infection or B-cell lymphoma. Overall, EBV DNA was detected in 100% of enriched B-cell samples, in 82% of B-cell culture supernatants, in 57% of plasma, and 42% of whole blood samples. A significant correlation for EBV viral load was found between enriched B-cell and B-cell culture supernatant material (ρ = 0.92; P cells (ρ = -0.02; P = 0.89), whole blood and plasma (ρ = 0.24; P = 0.24), or enriched B-cells and plasma (ρ = 0.08; P = 0.77). Testing of enriched B-cells appeared to be the most sensitive method for detection of EBV DNA as well as for exploration of the cellular reservoir. Quantitation of EBV DNA in plasma and B-cell culture supernatant may be of interest to assess EBV reactivation dynamics and response to treatment as well as to decipher EBV host-pathogen interactions in various clinical scenarios.

  5. Suppression of injuries caused by a lytic RNA virus (mengovirus) and their uncoupling from viral reproduction by mutual cell/virus disarmament.

    Science.gov (United States)

    Mikitas, Olga V; Ivin, Yuri Y; Golyshev, Sergey A; Povarova, Natalia V; Galkina, Svetlana I; Pletjushkina, Olga Y; Nadezhdina, Elena S; Gmyl, Anatoly P; Agol, Vadim I

    2012-05-01

    Viruses often elicit cell injury (cytopathic effect [CPE]), a major cause of viral diseases. CPE is usually considered to be a prerequisite for and/or consequence of efficient viral growth. Recently, we proposed that viral CPE may largely be due to host defensive and viral antidefensive activities. This study aimed to check the validity of this proposal by using as a model HeLa cells infected with mengovirus (MV). As we showed previously, infection of these cells with wild-type MV resulted in necrosis, whereas a mutant with incapacitated antidefensive ("security") viral leader (L) protein induced apoptosis. Here, we showed that several major morphological and biochemical signs of CPE (e.g., alterations in cellular and nuclear shape, plasma membrane, cytoskeleton, chromatin, and metabolic activity) in cells infected with L(-) mutants in the presence of an apoptosis inhibitor were strongly suppressed or delayed for long after completion of viral reproduction. These facts demonstrate that the efficient reproduction of a lytic virus may not directly require development of at least some pathological alterations normally accompanying infection. They also imply that L protein is involved in the control of many apparently unrelated functions. The results also suggest that the virus-activated program with competing necrotic and apoptotic branches is host encoded, with the choice between apoptosis and necrosis depending on a variety of intrinsic and extrinsic conditions. Implementation of this defensive suicidal program could be uncoupled from the viral reproduction. The possibility of such uncoupling has significant implications for the pathogenesis and treatment of viral diseases.

  6. Specific anti-viral effects of RNA interference on replication and expression of hepatitis B virus in mice

    Institute of Scientific and Technical Information of China (English)

    WU Ying; HUANG Ai-long; TANG Ni; ZHANG Bing-qiang; LU Nian-fang

    2005-01-01

    Background RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally. Our previous study has demonstrated that small interfering RNAs (siRNAs) have sufficiently inhibited hepatitis B virus (HBV) replication and expression in vitro. In this study we observed the RNAi-mediated inhibitory effects on HBV replication in mice models and accessed the specificity of these effects.Methods A mutant RNAi vector (pSI-C mut) with two base pairs different from the original target gene sequence at the RNAi vector (pSI-C) was constructed according to the method described in this study. A mouse model of acute hepatitis B virus infection was established by injecting naked plasmid pHBV1.3 via the tail vein with acute circulatory overload. pSI-C, pSI-C mut and the irrelevant RNAi control plasmid for green fluorescent protein (GFP) gene, pSIGFP were respectively delivered with pHBV1.3 by tail vein injection method. Six days post injection, enzyme-linked immunosorbent assay (ELISA) assay was used to measure the concentration of HBV surface antigen (HBsAg) in mouse serum, immunohistochemical straining method was used to visualize the expressin of HBV core protein (HBcAg) in liver tissues, and the transcriptional level of HBV C mRNA in liver tissues was detectedd by reverse transcriptase PCR (RT-PCR) analysis.Results Injection of pSI-C exerted magnificent and specific inhibitory effects on the replication and expression of HBV in the murine model. After 6-day post-injection (p.i.), the OD values were shown to be 5.07±1.07 in infecting group and 0.62±0.59 in pSI-C group. The concentration of HBsAg in pSI-C group was significantly lower than that in infecting group (P<0.01). Liver intracellular synthesis of viral core protein was sharply reduced to 0.9%±0.1%, compared with 5.4%±1.2% of positive hepatocytes in infecting group (P<0.01), and the transcriptional level of HBV C mRNA was greatly reduced by 84.7%. However, the irrelevant RNAi control plasmid

  7. Use of plasma human herpesvirus-8 viral load measurement: evaluation of practice in three UK HIV treatment centres.

    Science.gov (United States)

    Nugent, D B; Webster, D; Mabayoje, D; Chung, E; El Bouzidi, K; O'Sullivan, A; Ainsworth, J; Miller, R F

    2017-02-01

    A retrospective audit of plasma human herpesvirus-8 (HHV-8) viral load testing was performed in three HIV treatment centres over 24 months. Reasons for testing (360 tests) were: symptoms of systemic inflammatory response syndrome (SIRS) (fever, lymphadenopathy and raised inflammatory markers); monitoring in known HHV-8 pathology other than Kaposi sarcoma (KS); investigation of known/suspected KS, and other/no reason. Of patients with multicentric Castleman disease (MCD), 14/16 (88%) had detectable plasma HHV-8, as did 27/45 (60%) with biopsy proven or clinically confirmed KS, and 6/19 (32%) with lymphoma. Neither of the two patients with MCD and no detectable HHV-8 had SIRS symptoms at the time of the test. There was wide variation between centres in the indications prompting HHV-8 testing, with a more conservative approach resulting in a higher proportion of positive results. Measuring plasma HHV-8 in the absence of SIRS symptoms, established HHV-8 disease monitoring, or confirmed/suspected KS is unlikely to yield detectable HHV-8 thus allowing potential cost savings.

  8. HIV-1 Viral Escape in Cerebrospinal Fluid of Subjects on Suppressive Antiretroviral Treatment

    OpenAIRE

    Edén, Arvid; Fuchs, Dietmar; Hagberg, Lars; Nilsson, Staffan; Spudich, Serena; SVENNERHOLM, BO; Price, Richard W.; Gisslén, Magnus

    2010-01-01

    Background. Occasional cases of viral escape in cerebrospinal fluid (CSF) despite suppression of plasma human immunodeficiency virus type 1 (HIV-1) RNA have been reported. We investigated CSF viral escape in subjects treated with commonly used antiretroviral therapy regimens in relation to intrathecal immune activation and central nervous system penetration effectiveness (CPE) rank.

  9. Ultra Structural Characterisation of Tetherin - a Protein Capable of Preventing Viral Release from the Plasma Membrane

    Directory of Open Access Journals (Sweden)

    Ravindra K. Gupta

    2010-04-01

    Full Text Available Tetherin is an antiviral restriction factor made by mammalian cells to protect them from viral infection. It prevents newly formed virus particles from leaving infected cells. Its antiviral mechanism appears to be remarkably uncomplicated. In 2 studies published in PLoS Pathogens electron microscopy is used to support the hypothesis that the tethers that link HIV-1 virions to tetherin expressing cells contain tetherin and are likely to contain tetherin alone. They also show that the HIV-1 encoded tetherin antagonist that is known to cause tetherin degradation, Vpu, serves to reduce the amount of tetherin in the particles thereby allowing their release.

  10. The expanding functions of cellular helicases: the tombusvirus RNA replication enhancer co-opts the plant eIF4AIII-like AtRH2 and the DDX5-like AtRH5 DEAD-box RNA helicases to promote viral asymmetric RNA replication.

    Directory of Open Access Journals (Sweden)

    Nikolay Kovalev

    2014-04-01

    Full Text Available Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. Several of the co-opted host factors bind to the viral RNA, which plays multiple roles, including mRNA function, as an assembly platform for the viral replicase (VRC, template for RNA synthesis, and encapsidation during infection. It is likely that remodeling of the viral RNAs and RNA-protein complexes during the switch from one step to another requires RNA helicases. In this paper, we have discovered a second group of cellular RNA helicases, including the eIF4AIII-like yeast Fal1p and the DDX5-like Dbp3p and the orthologous plant AtRH2 and AtRH5 DEAD box helicases, which are co-opted by tombusviruses. Unlike the previously characterized DDX3-like AtRH20/Ded1p helicases that bind to the 3' terminal promoter region in the viral minus-strand (-RNA, the other class of eIF4AIII-like RNA helicases bind to a different cis-acting element, namely the 5' proximal RIII(- replication enhancer (REN element in the TBSV (-RNA. We show that the binding of AtRH2 and AtRH5 helicases to the TBSV (-RNA could unwind the dsRNA structure within the RIII(- REN. This unique characteristic allows the eIF4AIII-like helicases to perform novel pro-viral functions involving the RIII(- REN in stimulation of plus-strand (+RNA synthesis. We also show that AtRH2 and AtRH5 helicases are components of the tombusvirus VRCs based on co-purification experiments. We propose that eIF4AIII-like helicases destabilize dsRNA replication intermediate within the RIII(- REN that promotes bringing the 5' and 3' terminal (-RNA sequences in close vicinity via long-range RNA-RNA base pairing. This newly formed RNA structure promoted by eIF4AIII helicase together with AtRH20 helicase might facilitate the recycling of the viral replicases for multiple rounds of (+-strand synthesis, thus resulting in asymmetrical viral replication.

  11. Viral escape from HIV-1 neutralizing antibodies drives increased plasma neutralization breadth through sequential recognition of multiple epitopes and immunotypes.

    Directory of Open Access Journals (Sweden)

    Constantinos Kurt Wibmer

    2013-10-01

    Full Text Available Identifying the targets of broadly neutralizing antibodies to HIV-1 and understanding how these antibodies develop remain important goals in the quest to rationally develop an HIV-1 vaccine. We previously identified a participant in the CAPRISA Acute Infection Cohort (CAP257 whose plasma neutralized 84% of heterologous viruses. In this study we showed that breadth in CAP257 was largely due to the sequential, transient appearance of three distinct broadly neutralizing antibody specificities spanning the first 4.5 years of infection. The first specificity targeted an epitope in the V2 region of gp120 that was also recognized by strain-specific antibodies 7 weeks earlier. Specificity for the autologous virus was determined largely by a rare N167 antigenic variant of V2, with viral escape to the more common D167 immunotype coinciding with the development of the first wave of broadly neutralizing antibodies. Escape from these broadly neutralizing V2 antibodies through deletion of the glycan at N160 was associated with exposure of an epitope in the CD4 binding site that became the target for a second wave of broadly neutralizing antibodies. Neutralization by these CD4 binding site antibodies was almost entirely dependent on the glycan at position N276. Early viral escape mutations in the CD4 binding site drove an increase in wave two neutralization breadth, as this second wave of heterologous neutralization matured to recognize multiple immunotypes within this site. The third wave targeted a quaternary epitope that did not overlap any of the four known sites of vulnerability on the HIV-1 envelope and remains undefined. Altogether this study showed that the human immune system is capable of generating multiple broadly neutralizing antibodies in response to a constantly evolving viral population that exposes new targets as a consequence of escape from earlier neutralizing antibodies.

  12. Specific binding of Fusarium graminearum Hex1 protein to untranslated regions of the genomic RNA of Fusarium graminearum virus 1 correlates with increased accumulation of both strands of viral RNA.

    Science.gov (United States)

    Son, Moonil; Choi, Hoseong; Kim, Kook-Hyung

    2016-02-01

    The HEX1 gene of Fusarium graminearum was previously reported to be required for the efficient accumulation of Fusarium graminearum virus 1 (FgV1) RNA in its host. To investigate the molecular mechanism underlying the production of FgHEX1 and the replication of FgV1 viral RNA, we conducted electrophoretic mobility shift assays (EMSA) with recombinant FgHex1 protein and RNA sequences derived from various regions of FgV1 genomic RNA. These analyses demonstrated that FgHex1 and both the 5'- and 3'-untranslated regions of plus-strand FgV1 RNA formed complexes. To determine whether FgHex1 protein affects FgV1 replication, we quantified accumulation viral RNAs in protoplasts and showed that both (+)- and (-)-strands of FgV1 RNAs were increased in the over-expression mutant and decreased in the deletion mutant. These results indicate that the FgHex1 functions in the synthesis of both strands of FgV1 RNA and therefore in FgV1 replication probably by specifically binding to the FgV1 genomic RNA. Copyright © 2016. Published by Elsevier Inc.

  13. Replicative homeostasis II: Influence of polymerase fidelity on RNA virus quasispecies biology: Implications for immune recognition, viral autoimmunity and other "virus receptor" diseases

    Directory of Open Access Journals (Sweden)

    Sallie Richard

    2005-08-01

    Full Text Available Abstract Much of the worlds' population is in active or imminent danger from established infectious pathogens, while sporadic and pandemic infections by these and emerging agents threaten everyone. RNA polymerases (RNApol generate enormous genetic and consequent antigenic heterogeneity permitting both viruses and cellular pathogens to evade host defences. Thus, RNApol causes more morbidity and premature mortality than any other molecule. The extraordinary genetic heterogeneity defining viral quasispecies results from RNApol infidelity causing rapid cumulative genomic RNA mutation a process that, if uncontrolled, would cause catastrophic loss of sequence integrity and inexorable quasispecies extinction. Selective replication and replicative homeostasis, an epicyclical regulatory mechanism dynamically linking RNApol fidelity and processivity with quasispecies phenotypic diversity, modulating polymerase fidelity and, hence, controlling quasispecies behaviour, prevents this happening and also mediates immune escape. Perhaps more importantly, ineluctable generation of broad phenotypic diversity after viral RNA is translated to protein quasispecies suggests a mechanism of disease that specifically targets, and functionally disrupts, the host cell surface molecules – including hormone, lipid, cell signalling or neurotransmitter receptors – that viruses co-opt for cell entry. This mechanism – "Viral Receptor Disease (VRD" – may explain so-called "viral autoimmunity", some classical autoimmune disorders and other diseases, including type II diabetes mellitus, and some forms of obesity. Viral receptor disease is a unifying hypothesis that may also explain some diseases with well-established, but multi-factorial and apparently unrelated aetiologies – like coronary artery and other vascular diseases – in addition to diseases like schizophrenia that are poorly understood and lack plausible, coherent, pathogenic explanations.

  14. Plasma microRNA profiles distinguish lethal injury in acetaminophen toxicity: A research study

    Institute of Scientific and Technical Information of China (English)

    Jeanine Ward; Shashi Bala; Jan Petrasek; Gyongyi Szabo

    2012-01-01

    AIM:To investigate plasma microRNA (miRNA) profiles indicative of hepatotoxicity in the setting of lethal acetaminophen (APAP) toxicity in mice.METHODS:Using plasma from APAP poisoned mice,either lethally (500 mg/kg) or sublethally (150 mg/kg) dosed,we screened commercially available murine microRNA libraries (SABiosciences,Qiagen Sciences,MD) to evaluate for unique miRNA profiles between these two dosing parameters.RESULTS:We distinguished numerous,unique plasma miRNAs both up- and downregulated in lethally compared to sublethally dosed mice.Of note,many of the greatest up- and downregulated miRNAs,namely 574-5p,466g,466f-3p,375,29c,and 148a,have been shown to be associated with asthma in prior studies.Interestingly,a relationship between APAP and asthma has been previously well described in the literature,with an as yet unknown mechanism of pathology.There was a statistically significant increase in alanine aminotransferase levels in the lethal compared to sublethal APAP dosing groups at the 12 h time point (P <0.001).There was 90% mortality in the lethally compared to sublethally dosed mice at the 48 h time point (P =0.011).CONCLUSION:We identified unique plasma miRNAs both up- and downregulated in APAP poisoning which are correlated to asthma development.

  15. Association between the miRNA Signatures in Plasma and Bronchoalveolar Fluid in Respiratory Pathologies

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    Sonia Molina-Pinelo

    2012-01-01

    Full Text Available The identification of new less invasive biomarkers is necessary to improve the detection and prognostic outcome of respiratory pathological processes. The measurement of miRNA expression through less invasive techniques such as plasma and serum have been suggested to analysis of several lung malignancies including lung cancer. These studies are assuming a common deregulated miRNA expression both in blood and lung tissue. The present study aimed to obtain miRNA representative signatures both in plasma and bronchoalveolar cell fraction that could serve as biomarker in respiratory diseases. Ten patients were evaluated to assess the expression levels of 381 miRNAs. We found that around 50% miRNAs were no detected in both plasma and bronchoalveolar cell fraction and only 20% of miRNAs showed similar expression in both samples. These results show a lack of association of miRNA signatures between plasma and bronchoalveolar cytology in the same patient. The profiles are not comparable; however, there is a similarity in the relative expression in a very small subset of miRNAs (miR-17, miR-19b, miR-195 and miR-20b between both biological samples in all patients. This finding supports that the miRNAs profiles obtained from different biological samples have to be carefully validated to link with respiratory diseases.

  16. A viral nuclear noncoding RNA binds re-localized poly(A binding protein and is required for late KSHV gene expression.

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    Sumit Borah

    2011-10-01

    Full Text Available During the lytic phase of infection, the gamma herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV expresses a highly abundant, 1.1 kb nuclear noncoding RNA of unknown function. We observe that this polyadenylated nuclear (PAN RNA avidly binds host poly(A-binding protein C1 (PABPC1, which normally functions in the cytoplasm to bind the poly(A tails of mRNAs, regulating mRNA stability and translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nucleus as a consequence of expression of the viral shutoff exonuclease (SOX protein; SOX also mediates the host shutoff effect in which host mRNAs are downregulated while viral mRNAs are selectively expressed. We show that whereas PAN RNA is not required for the host shutoff effect or for PABPC1 re-localization, SOX strongly upregulates the levels of PAN RNA in transient transfection experiments. This upregulation is destroyed by the same SOX mutation that ablates the host shutoff effect and PABPC1 nuclear re-localization or by removal of the poly(A tail of PAN. In cells induced into the KSHV lytic phase, depletion of PAN RNA using RNase H-targeting antisense oligonucleotides reveals that it is necessary for the production of late viral proteins from mRNAs that are themselves polyadenylated. Our results add to the repertoire of functions ascribed to long noncoding RNAs and suggest a mechanism of action for nuclear noncoding RNAs in gamma herpesvirus infection.

  17. Optimized Collection Protocol for Plasma MicroRNA Measurement in Patients with Cardiovascular Disease

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    Chi-Sheng Wu

    2016-01-01

    Full Text Available Background. Various microRNAs (miRNAs are used as markers of acute coronary syndrome, in which heparinization is considered mandatory therapy. Nevertheless, a standard method of handling plasma samples has not been proposed, and the effects of heparin treatment on miRNA detection are rarely discussed. Materials and Method. This study used quantitative polymerase chain reaction (qPCR analysis to investigate how storage temperature, standby time, hemolysis, and heparin treatment affect miRNA measurement in plasma samples from 25 patients undergoing cardiac catheterization. Results. For most miRNAs, the qPCR results remained consistent during the first 2 hours. The miRNA signals did not significantly differ between samples stored at 4°C before processing and samples stored at room temperature (RT before processing. miR-451a/miR-23a ratio < 60 indicated < 0.12% hemolysis with 100% sensitivity and 100% specificity. Pretreatment with 0.25 U heparinase I recovered qPCR signals that were reduced by in vivo heparinization. Conclusions. For miRNA measurement, blood samples stored at RT should be processed into plasma within 2 hours after withdrawal and should be pretreated with 0.25 U heparinase I to overcome heparin-attenuated miRNA signals. The miR-451a/miR-23a ratio is a reliable indicator of significant hemolysis.

  18. RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Liu, Chao, E-mail: liuchao9@mail.sysu.edu.cn [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Zhang, Hui [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China)

    2015-12-15

    Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. - Highlights: • MOV10 can function as a co-factor of HIV-1 Rev. • MOV10 facilitates Rev/RRE-dependent transport of viral mRNAs. • MOV10 interacts with Rev in an RNA-independent manner. • The DEAG-box of MOV10 is required for the enhancement of Rev/RRE-dependent export.

  19. Zika viral dynamics and shedding in rhesus and cynomolgus macaques

    Science.gov (United States)

    Osuna, Christa E; Lim, So-Yon; Deleage, Claire; Griffin, Bryan D; Stein, Derek; Schroeder, Lukas T; Omange, Robert Were; Best, Katharine; Luo, Ma; Hraber, Peter T; Andersen-Elyard, Hanne; Ojeda, Erwing Fabian Cardozo; Huang, Scott; Vanlandingham, Dana L; Higgs, Stephen; Perelson, Alan S; Estes, Jacob D; Safronetz, David; Lewis, Mark G; Whitney, James B

    2017-01-01

    Infection with Zika virus has been associated with serious neurological complications and fetal abnormalities. However, the dynamics of viral infection, replication and shedding are poorly understood. Here we show that both rhesus and cynomolgus macaques are highly susceptible to infection by lineages of Zika virus that are closely related to, or are currently circulating in, the Americas. After subcutaneous viral inoculation, viral RNA was detected in blood plasma as early as 1 d after infection. Viral RNA was also detected in saliva, urine, cerebrospinal fluid (CSF) and semen, but transiently in vaginal secretions. Although viral RNA during primary infection was cleared from blood plasma and urine within 10 d, viral RNA was detectable in saliva and seminal fluids until the end of the study, 3 weeks after the resolution of viremia in the blood. The control of primary Zika virus infection in the blood was correlated with rapid innate and adaptive immune responses. We also identified Zika RNA in tissues, including the brain and male and female reproductive tissues, during early and late stages of infection. Re-infection of six animals 45 d after primary infection with a heterologous strain resulted in complete protection, which suggests that primary Zika virus infection elicits protective immunity. Early invasion of Zika virus into the nervous system of healthy animals and the extent and duration of shedding in saliva and semen underscore possible concern for additional neurologic complications and nonarthropod-mediated transmission in humans. PMID:27694931

  20. Isolation of infectious Zika virus from saliva and prolonged viral RNA shedding in a traveller returning from the Dominican Republic to Italy, January 2016.

    Science.gov (United States)

    Barzon, Luisa; Pacenti, Monia; Berto, Alessandro; Sinigaglia, Alessandro; Franchin, Elisa; Lavezzo, Enrico; Brugnaro, Pierluigi; Palù, Giorgio

    2016-01-01

    We report the isolation of infectious Zika virus (ZIKV) in cell culture from the saliva of a patient who developed a febrile illness after returning from the Dominican Republic to Italy, in January 2016. The patient had prolonged shedding of viral RNA in saliva and urine, at higher load than in blood, for up to 29 days after symptom onset. Sequencing of ZIKV genome showed relatedness with strains from Latin America.

  1. Generation of a safe and effective live viral vaccine by virus self-attenuation using species-specific artificial microRNA.

    Science.gov (United States)

    Li, Junwei; Arévalo, Maria T; Diaz-Arévalo, Diana; Chen, Yanping; Choi, Jang-Gi; Zeng, Mingtao

    2015-06-10

    Vaccination with live attenuated vaccines (LAVs) is an effective way for prevention of infectious disease. While several methods are employed to create them, efficacy and safety are still a challenge. In this study, we evaluated the feasibility of creating a self-attenuated RNA virus expressing a functional species-specific artificial microRNA. Using influenza virus as a model, we produced an attenuated virus carrying a mammalian-specific miR-93 expression cassette that expresses a viral nucleoprotein (NP)-specific artificial microRNA from an insertion site within the non-structural (NS) gene segment. The resulting engineered live-attenuated influenza virus, PR8-amiR-93NP, produced mature and functional artificial microRNA against NP in mammalian cells, but not in avian cells. Furthermore, PR8-amiR-93NP was attenuated by 10(4) fold in mice compared with its wild-type counterpart. Importantly, intranasal immunization with PR8-amiR-93NP conferred cross-protective immunity against heterologous influenza virus strains. In short, this method provides a safe and effective platform for creation of live attenuated RNA viral vaccines. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Extraction of nuclei from sonchus yellow net rhabdovirus-infected plants yields a polymerase that synthesizes viral mRNAs and polyadenylated plus-strand leader RNA.

    Science.gov (United States)

    Wagner, J D; Choi, T J; Jackson, A O

    1996-01-01

    Although the primary sequence of the genome of the plant rhabdovirus sonchus yellow net virus (SYNV) has been determined, little is known about the composition of the viral polymerase or the mechanics of viral transcription and replication. In this paper, we report the partial isolation and characterization of an active SYNV polymerase from nuclei of SYNV-infected leaf tissue. A salt extraction procedure is shown to be an effective purification step for recovery of the polymerase from the nuclei. Full-length, polyadenylated SYNV N and M2 mRNAs and plus-strand leader RNA are among the products of the in vitro polymerase reactions. Polyadenylation of the plus-strand leader RNA in vitro is shown with RNase H and specific oligonucleotides. This is the first report of a polyadenylated plus-strand leader RNA for a minus-strand RNA virus, a feature that may reflect adaptation of SYNV to replication in the nucleus. Analysis of the SYNV proteins present in the polymerase extract suggests that the N, M2, and L proteins are components of the transcription complex. Overall, the system we developed promises to be useful for an in-depth characterization of the mechanics of SYNV RNA synthesis.

  3. Exposure to the viral by-product dsRNA or Coxsackievirus B5 triggers pancreatic beta cell apoptosis via a Bim / Mcl-1 imbalance.

    Directory of Open Access Journals (Sweden)

    Maikel L Colli

    2011-09-01

    Full Text Available The rise in type 1 diabetes (T1D incidence in recent decades is probably related to modifications in environmental factors. Viruses are among the putative environmental triggers of T1D. The mechanisms regulating beta cell responses to viruses, however, remain to be defined. We have presently clarified the signaling pathways leading to beta cell apoptosis following exposure to the viral mimetic double-stranded RNA (dsRNA and a diabetogenic enterovirus (Coxsackievirus B5. Internal dsRNA induces cell death via the intrinsic mitochondrial pathway. In this process, activation of the dsRNA-dependent protein kinase (PKR promotes eIF2α phosphorylation and protein synthesis inhibition, leading to downregulation of the antiapoptotic Bcl-2 protein myeloid cell leukemia sequence 1 (Mcl-1. Mcl-1 decrease results in the release of the BH3-only protein Bim, which activates the mitochondrial pathway of apoptosis. Indeed, Bim knockdown prevented both dsRNA- and Coxsackievirus B5-induced beta cell death, and counteracted the proapoptotic effects of Mcl-1 silencing. These observations indicate that the balance between Mcl-1 and Bim is a key factor regulating beta cell survival during diabetogenic viral infections.

  4. Exposure to the viral by-product dsRNA or Coxsackievirus B5 triggers pancreatic beta cell apoptosis via a Bim / Mcl-1 imbalance.

    Directory of Open Access Journals (Sweden)

    Maikel L Colli

    2011-09-01

    Full Text Available The rise in type 1 diabetes (T1D incidence in recent decades is probably related to modifications in environmental factors. Viruses are among the putative environmental triggers of T1D. The mechanisms regulating beta cell responses to viruses, however, remain to be defined. We have presently clarified the signaling pathways leading to beta cell apoptosis following exposure to the viral mimetic double-stranded RNA (dsRNA and a diabetogenic enterovirus (Coxsackievirus B5. Internal dsRNA induces cell death via the intrinsic mitochondrial pathway. In this process, activation of the dsRNA-dependent protein kinase (PKR promotes eIF2α phosphorylation and protein synthesis inhibition, leading to downregulation of the antiapoptotic Bcl-2 protein myeloid cell leukemia sequence 1 (Mcl-1. Mcl-1 decrease results in the release of the BH3-only protein Bim, which activates the mitochondrial pathway of apoptosis. Indeed, Bim knockdown prevented both dsRNA- and Coxsackievirus B5-induced beta cell death, and counteracted the proapoptotic effects of Mcl-1 silencing. These observations indicate that the balance between Mcl-1 and Bim is a key factor regulating beta cell survival during diabetogenic viral infections.

  5. Maternal And Neonatal Plasma MicroRNA Biomarkers For Fetal Alcohol Exposure In An Ovine Model

    Science.gov (United States)

    Balaraman, Sridevi; Lunde, E. Raine; Sawant, Onkar; Cudd, Timothy A.; Washburn, Shannon E.; Miranda, Rajesh C.

    2014-01-01

    Background Plasma or circulating miRNAs (cirmiRNAs) have potential diagnostic value as biomarkers for a range of diseases. Based on observations that ethanol altered intracellular miRNAs during development, we tested the hypothesis that plasma miRNAs were biomarkers for maternal alcohol exposure, and for past in utero exposure, in the neonate. Methods Pregnant sheep were exposed to a binge model of ethanol consumption resulting in an average peak blood alcohol content of 243 mg/dl, for a three-trimester equivalent period from gestational day (GD) 4 to GD 132. MiRNA profiles were assessed by quantitative PCR analysis in plasma, erythrocyte and leukocytes obtained from non-pregnant ewes, and plasma from pregnant ewes 24 hours following the last binge ethanol episode, and from newborn lambs, at birth on ~GD 147. Results Pregnant ewe and newborn lamb cirmiRNA profiles were similar to each other and different from non-pregnant female plasma, erythrocyte or leukocyte miRNAs. Significant changes in cirmiRNA profiles were observed in the ethanol-exposed ewe, and at birth, in the in utero, ethanol-exposed lamb. CirmiRNAs including miR-9, -15b, -19b and -20a were sensitive and specific measures of ethanol exposure in both pregnant ewe and newborn lamb. Additionally, ethanol exposure altered guide to passenger strand cirmiRNA ratios in the pregnant ewe, but not in the lamb. Conclusion Shared profiles between pregnant dam and neonate suggest possible maternal-fetal miRNA transfer. CirmiRNAs are biomarkers for alcohol exposure during pregnancy, in both mother and neonate, and may constitute an important shared endocrine biomarker that is vulnerable to the maternal environment. PMID:24588274

  6. Viral uncoating is directional: exit of the genomic RNA in a common cold virus starts with the poly-(A tail at the 3'-end.

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    Shushan Harutyunyan

    Full Text Available Upon infection, many RNA viruses reorganize their capsid for release of the genome into the host cell cytosol for replication. Often, this process is triggered by receptor binding and/or by the acidic environment in endosomes. In the genus Enterovirus, which includes more than 150 human rhinovirus (HRV serotypes causing the common cold, there is persuasive evidence that the viral RNA exits single-stranded through channels formed in the protein shell. We have determined the time-dependent emergence of the RNA ends from HRV2 on incubation of virions at 56°C using hybridization with specific oligonucleotides and detection by fluorescence correlation spectroscopy. We report that psoralen UV crosslinking prevents complete RNA release, allowing for identification of the sequences remaining inside the capsid. We also present the structure of uncoating intermediates in which parts of the RNA are condensed and take the form of a rod that is directed roughly towards a two-fold icosahedral axis, the presumed RNA exit point. Taken together, in contrast to schemes frequently depicted in textbooks and reviews, our findings demonstrate that exit of the RNA starts from the 3'-end. This suggests that packaging also occurs in an ordered manner resulting in the 3'-poly-(A tail becoming located close to a position of pore formation during conversion of the virion into a subviral particle. This directional genome release may be common to many icosahedral non-enveloped single-stranded RNA viruses.

  7. Viral uncoating is directional: exit of the genomic RNA in a common cold virus starts with the poly-(A) tail at the 3'-end.

    Science.gov (United States)

    Harutyunyan, Shushan; Kumar, Mohit; Sedivy, Arthur; Subirats, Xavier; Kowalski, Heinrich; Köhler, Gottfried; Blaas, Dieter

    2013-01-01

    Upon infection, many RNA viruses reorganize their capsid for release of the genome into the host cell cytosol for replication. Often, this process is triggered by receptor binding and/or by the acidic environment in endosomes. In the genus Enterovirus, which includes more than 150 human rhinovirus (HRV) serotypes causing the common cold, there is persuasive evidence that the viral RNA exits single-stranded through channels formed in the protein shell. We have determined the time-dependent emergence of the RNA ends from HRV2 on incubation of virions at 56°C using hybridization with specific oligonucleotides and detection by fluorescence correlation spectroscopy. We report that psoralen UV crosslinking prevents complete RNA release, allowing for identification of the sequences remaining inside the capsid. We also present the structure of uncoating intermediates in which parts of the RNA are condensed and take the form of a rod that is directed roughly towards a two-fold icosahedral axis, the presumed RNA exit point. Taken together, in contrast to schemes frequently depicted in textbooks and reviews, our findings demonstrate that exit of the RNA starts from the 3'-end. This suggests that packaging also occurs in an ordered manner resulting in the 3'-poly-(A) tail becoming located close to a position of pore formation during conversion of the virion into a subviral particle. This directional genome release may be common to many icosahedral non-enveloped single-stranded RNA viruses.

  8. Evaluation of the VACUTAINER PPT Plasma Preparation Tube for use with the Bayer VERSANT assay for quantification of human immunodeficiency virus type 1 RNA.

    Science.gov (United States)

    Elbeik, Tarek; Nassos, Patricia; Kipnis, Patricia; Haller, Barbara; Ng, Valerie L

    2005-08-01

    Separation and storage of plasma within 2 h of phlebotomy is required for the VACUTAINER PPT Plasma Preparation Tube (PPT) versus 4 h for the predecessor VACUTAINER EDTA tube for human immunodeficiency virus type 1 (HIV-1) viral load (HIVL) testing by the VERSANT HIV-1 RNA 3.0 assay (branched DNA). The 2-h limit for PPT imposes time constraints for handling and transporting to the testing laboratory. This study compares HIVL reproducibility from matched blood in EDTA tubes and PPTs and between PPT pairs following processing within 4 h of phlebotomy, stability of plasma HIV-1 RNA at 24- and 72-h room temperature storage in the tube, and comparative labor and supply requirements. Blood from 159 patients was collected in paired tubes (EDTA/PPT or PPT/PPT): 86 paired EDTA tubes and PPTs were processed 4 h following phlebotomy and their HIVLs were compared, 42 paired PPT/PPT pairs were analyzed for intertube HIVL reproducibility, and 31 PPT/PPT pairs were analyzed for HIV-1 RNA stability by HIVL. Labor and supply requirements were compared between PPT and EDTA tubes. PPTs produce results equivalent to standard EDTA tube results when processed 4 h after phlebotomy. PPT intertube analyte results are reproducible. An average decrease of 13% and 37% in HIVL was observed in PPT plasma after 24 and 72 h of room temperature storage, respectively; thus, plasma can be stored at room temperature up to 24 h in the original tube. PPTs offer labor and supply savings over EDTA tubes.

  9. Development of an RNA Assay to Assess HIV I Latency

    Science.gov (United States)

    1993-01-10

    current study is limited to examining cellular RNAs rather than free genomic RNA in the plasma. Ottmann and colleagues demonstrated HIV-1 genomic RNA in 95...aberrant pattern of viral RNA expression: a molecular model for latency. Cell 1990; 61:1271-1276. 25 Ottmann M, Innocenti P, Tenadey M, Micoud M

  10. Viral RNA silencing suppression

    NARCIS (Netherlands)

    Hedil, Marcio; Kormelink, Richard

    2016-01-01

    The Bunyaviridae is a family of arboviruses including both plant-and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmissio

  11. A novel functional site in the PB2 subunit of influenza A virus essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication.

    Science.gov (United States)

    Hatakeyama, Dai; Shoji, Masaki; Yamayoshi, Seiya; Hirota, Takenori; Nagae, Monami; Yanagisawa, Shin; Nakano, Masahiro; Ohmi, Naho; Noda, Takeshi; Kawaoka, Yoshihiro; Kuzuhara, Takashi

    2014-09-05

    The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, are essential for viral transcription and replication. The PB2 subunit binds to the host RNA cap (7-methylguanosine triphosphate (m(7)GTP)) and supports the endonuclease activity of PA to "snatch" the cap from host pre-mRNAs. However, the structure of PB2 is not fully understood, and the functional sites remain unknown. In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is involved in interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs). In vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m(7)GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m(7)GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino acid mutations in the VRG site. Substitutions of the valine and arginine residues or of all 3 residues of the VRG site to alanine significantly reduced the binding ability of PB2 to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. APOBEC3G induces a hypermutation gradient: purifying selection at multiple steps during HIV-1 replication results in levels of G-to-A mutations that are high in DNA, intermediate in cellular viral RNA, and low in virion RNA

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    Pathak Vinay K

    2009-02-01

    Full Text Available Abstract Background Naturally occurring Vif variants that are unable to inhibit the host restriction factor APOBEC3G (A3G have been isolated from infected individuals. A3G can potentially induce G-to-A hypermutation in these viruses, and hypermutation could contribute to genetic variation in HIV-1 populations through recombination between hypermutant and wild-type genomes. Thus, hypermutation could contribute to the generation of immune escape and drug resistant variants, but the genetic contribution of hypermutation to the viral evolutionary potential is poorly understood. In addition, the mechanisms by which these viruses persist in the host despite the presence of A3G remain unknown. Results To address these questions, we generated a replication-competent HIV-1 Vif mutant in which the A3G-binding residues of Vif, Y40RHHY44, were substituted with five alanines. As expected, the mutant was severely defective in an A3G-expressing T cell line and exhibited a significant delay in replication kinetics. Analysis of viral DNA showed the expected high level of G-to-A hypermutation; however, we found substantially reduced levels of G-to-A hypermutation in intracellular viral RNA (cRNA, and the levels of G-to-A mutations in virion RNA (vRNA were even further reduced. The frequencies of hypermutation in DNA, cRNA, and vRNA were 0.73%, 0.12%, and 0.05% of the nucleotides sequenced, indicating a gradient of hypermutation. Additionally, genomes containing start codon mutations and early termination codons within gag were isolated from the vRNA. Conclusion These results suggest that sublethal levels of hypermutation coupled with purifying selection at multiple steps during the early phase of viral replication lead to the packaging of largely unmutated genomes, providing a mechanism by which mutant Vif variants can persist in infected individuals. The persistence of genomes containing mutated gag genes despite this selection pressure indicates that dual

  13. hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yimeng; Zhou, Jianhong; Du, Yuchun, E-mail: ydu@uark.edu

    2014-01-20

    The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to the cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation. - Highlights: • Cellular protein hnRNP A2/B1 interacts with influenza viral protein NS1. • hnRNP A2/B1 suppresses the levels of NS1 protein, vRNA and mRNA in infected cells. • hnRNP A2/B1 protein is associated with NS1 and NS2 mRNAs. • hnRNP A2/B1 inhibits the nuclear export of NS1 mRNAs. • hnRNP A2/B1 inhibits influenza virus replication.

  14. Plasma microRNA profiles in rat models of hepatocellular injury, cholestasis, and steatosis.

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    Yu Yamaura

    Full Text Available MicroRNAs (miRNAs are small RNA molecules that function to modulate the expression of target genes, playing important roles in a wide range of physiological and pathological processes. The miRNAs in body fluids have received considerable attention as potential biomarkers of various diseases. In this study, we compared the changes of the plasma miRNA expressions by acute liver injury (hepatocellular injury or cholestasis and chronic liver injury (steatosis, steatohepatitis and fibrosis using rat models made by the administration of chemicals or special diets. Using miRNA array analysis, we found that the levels of a large number of miRNAs (121-317 miRNAs were increased over 2-fold and the levels of a small number of miRNAs (6-35 miRNAs were decreased below 0.5-fold in all models except in a model of cholestasis caused by bile duct ligation. Interestingly, the expression profiles were different between the models, and the hierarchical clustering analysis discriminated between the acute and chronic liver injuries. In addition, miRNAs whose expressions were typically changed in each type of liver injury could be specified. It is notable that, in acute liver injury models, the plasma level of miR-122, the most abundant miRNA in the liver, was more quickly and dramatically increased than the plasma aminotransferase level, reflecting the extent of hepatocellular injury. This study demonstrated that the plasma miRNA profiles could reflect the types of liver injury (e.g. acute/chronic liver injury or hepatocellular injury/cholestasis/steatosis/steatohepatitis/fibrosis and identified the miRNAs that could be specific and sensitive biomarkers of liver injury.

  15. MicroRNA-134 as a potential plasma biomarker for the diagnosis of acute pulmonary embolism

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    Liu Yi

    2011-09-01

    Full Text Available Abstract Background Acute pulmonary embolism (APE remains a diagnostic challenge due to a variable clinical presentation and the lack of a reliable screening tool. MicroRNAs (miRNAs regulate gene expression in a wide range of pathophysiologic processes. Circulating miRNAs are emerging biomarkers in heart failure, type 2 diabetes and other disease states; however, using plasma miRNAs as biomarkers for the diagnosis of APE is still unknown. Methods Thirty-two APE patients, 32 healthy controls, and 22 non-APE patients (reported dyspnea, chest pain, or cough were enrolled in this study. The TaqMan miRNA microarray was used to identify dysregulated miRNAs in the plasma of APE patients. The TaqMan-based miRNA quantitative real-time reverse transcription polymerase chain reactions were used to validate the dysregulated miRNAs. The receiver-operator characteristic (ROC curve analysis was conducted to evaluate the diagnostic accuracy of the miRNA identified as the candidate biomarker. Results Plasma miRNA-134 (miR-134 level was significantly higher in the APE patients than in the healthy controls or non-APE patients. The ROC curve showed that plasma miR-134 was a specific diagnostic predictor of APE with an area under the curve of 0.833 (95% confidence interval, 0.737 to 0.929; P Conclusions Our findings indicated that plasma miR-134 could be an important biomarker for the diagnosis of APE. Because of this finding, large-scale investigations are urgently needed to pave the way from basic research to clinical utilization.

  16. RNA interference-mediated targeting of human cytomegalovirus immediate-early or early gene products inhibits viral replication with differential effects on cellular functions.

    Science.gov (United States)

    Xiaofei, E; Stadler, Bradford M; Debatis, Michelle; Wang, Shixia; Lu, Shan; Kowalik, Timothy F

    2012-05-01

    Viral drug toxicity, resistance, and an increasing immunosuppressed population warrant continued research into new avenues for limiting diseases associated with human cytomegalovirus (HCMV). In this study, a small interfering RNA (siRNA), siX3, was designed to target coding sequences within shared exon 3 of UL123 and UL122 transcripts encoding IE1 and IE2 immediate-early proteins of HCMV. Pretreatment of cells with siX3 reduced the levels of viral protein expression, DNA replication, and progeny virus production compared to control siRNA. Two siRNAs against UL54 and overlapping transcripts (UL55-57) were compared to siX3 in HCMV infection and were also found to be effective at inhibiting HCMV replication. Further investigation into the effects of the siRNAs on viral replication showed that pretreatment with each of the siRNAs resulted in an inhibition in the formation of mature replication compartments. The ability of these siRNAs to prevent or reduce certain cytopathic effects associated with HCMV infection was also examined. Infected cells pretreated with siX3, but not siUL54, retained promyelocytic leukemia (PML) protein in cellular PML bodies, an essential component of this host intrinsic antiviral defense. DNA damage response proteins, which are localized in nuclear viral replication compartments, were reduced in the siX3- and siUL54-treated cells. siX3, but not siUL54, prevented DNA damage response signaling early after infection. Therapeutic efficacy was demonstrated by treating cells with siRNAs after HCMV replication had commenced. Together, these findings suggest that siRNAs targeting exon 3 of the major IE genes or the UL54-57 transcripts be further studied for their potential development into anti-HCMV therapeutics.

  17. Human Immunodeficiency Virus Type 1 Vif Protein Is an Integral Component of an mRNP Complex of Viral RNA and Could Be Involved in the Viral RNA Folding and Packaging Process

    OpenAIRE

    Zhang, Hui; Pomerantz, Roger J.; Dornadula, Geethanjali; Sun, Yong

    2000-01-01

    Virion infectivity factor (Vif) is a protein encoded by human immunodeficiency virus types 1 and 2 (HIV-1 and -2) and simian immunodeficiency virus, plus other lentiviruses, and is essential for viral replication either in vivo or in culture for nonpermissive cells such as peripheral blood lymphoid cells, macrophages, and H9 T cells. Defects in the vif gene affect virion morphology and reverse transcription but not the expression of viral components. It has been shown that Vif colocalizes wit...

  18. Extraction of HCV-RNA from Plasma Samples: Development towards Semiautomation

    Directory of Open Access Journals (Sweden)

    Imran Amin

    2015-01-01

    Full Text Available A semiautomated extraction protocol of HCV-RNA using Favorgen RNA extraction kit has been developed. The kit provided protocol was modified by replacing manual spin steps with vacuum filtration. The assay performance was evaluated by real-time qPCR based on Taqman technology. Assay linearity was confirmed with the serial dilutions of RTA (Turkey containing 1 × (106, 105, 104, and 103 IU mL−1. Comparison of test results obtained by two extraction methods showed a good correlation (r=0.95, n=30 with detection limit of 102 IU mL−1. The semiautomated vacuum filtration based protocol demonstrated high throughput: 35 minutes for the extraction of a batch of 30 samples (150 µL each with reduced labor, time, waste, and cost. Performance characteristics of semiautomated system make it suitable for use in diagnostic purpose and viral load determinations.

  19. Molecular characterization of a dual inhibitory and mutagenic activity of 5-fluorouridine triphosphate on viral RNA synthesis. Implications for lethal mutagenesis.

    Science.gov (United States)

    Agudo, Rubén; Arias, Armando; Pariente, Nonia; Perales, Celia; Escarmís, Cristina; Jorge, Alberto; Marina, Anabel; Domingo, Esteban

    2008-10-10

    The basis for a dual inhibitory and mutagenic activity of 5-fluorouracil (5-FU) on foot-and-mouth disease virus (FMDV) RNA replication has been investigated with purified viral RNA-dependent RNA polymerase (3D) in vitro. 5-Fluorouridine triphosphate acted as a potent competitive inhibitor of VPg uridylylation, the initial step of viral replication. Peptide analysis by mass spectrometry has identified a VPg fragment containing 5-fluorouridine monophosphate (FUMP) covalently attached to Tyr3, the amino acid target of the uridylylation reaction. During RNA elongation, FUMP was incorporated in the place of UMP or CMP by FMDV 3D, using homopolymeric and heteropolymeric templates. Incorporation of FUMP did not prevent chain elongation, and, in some sequence contexts, it favored misincorporations at downstream positions. When present in the template, FUMP directed the incorporation of AMP and GMP, with ATP being a more effective substrate than GTP. The misincorporation of GMP was 17-fold faster opposite FU than opposite U in the template. These results in vitro are consistent with the mutational bias observed in the mutant spectra of 5-FU-treated FMDV populations. The dual mutagenic and inhibitory activity of 5-fluorouridine triphosphate may contribute to the effective extinction of FMDV by 5-FU through virus entry into error catastrophe.

  20. The miRNA plasma signature in response to acute aerobic exercise and endurance training.

    Directory of Open Access Journals (Sweden)

    Søren Nielsen

    Full Text Available MiRNAs are potent intracellular posttranscriptional regulators and are also selectively secreted into the circulation in a cell-specific fashion. Global changes in miRNA expression in skeletal muscle in response to endurance exercise training have been reported. Therefore, our aim was to establish the miRNA signature in human plasma in response to acute exercise and chronic endurance training by utilizing a novel methodological approach. RNA was isolated from human plasma collected from young healthy men before and after an acute endurance exercise bout and following 12 weeks of endurance training. Global miRNA (742 miRNAs measurements were performed as a screening to identify detectable miRNAs in plasma. Using customized qPCR panels we quantified the expression levels of miRNAs detected in the screening procedure (188 miRNAs. We demonstrate a dynamic regulation of circulating miRNA (ci-miRNA levels following 0 hour (miR-106a, miR-221, miR-30b, miR-151-5p, let-7i, miR-146, miR-652 and miR-151-3p, 1 hour (miR-338-3p, miR-330-3p, miR-223, miR-139-5p and miR-143 and 3 hours (miR-1 after an acute exercise bout (P<0.00032. Where ci-miRNAs were all downregulated immediately after an acute exercise bout (0 hour the 1 and 3 hour post exercise timepoints were followed by upregulations. In response to chronic training, we identified seven ci-miRNAs with decreased levels in plasma (miR-342-3p, let-7d, miR-766, miR-25, miR-148a, miR-185 and miR-21 and two miRNAs that were present at higher levels after the training period (miR-103 and miR-107 (P<0.00032. In conclusion, acute exercise and chronic endurance training, likely through specific mechanisms unique to each stimulus, robustly modify the miRNA signature of human plasma.

  1. A Tiny RNA that Packs a Big Punch: The Critical Role of a Viral miR-155 Ortholog in Lymphomagenesis in Marek’s Disease

    Directory of Open Access Journals (Sweden)

    Guoqing Zhuang

    2017-06-01

    Full Text Available MicroRNAs (miRNAs are small non-coding RNAs that have been identified in animals, plants, and viruses. These small RNAs play important roles in post-transcriptional regulation of various cellular processes, including development, differentiation, and all aspects of cancer biology. Rapid-onset T-cell lymphoma of chickens, namely Marek’s disease (MD, induced by Gallid alphaherpesvirus 2 (GaHV2, could provide an ideal natural animal model for herpesvirus-related cancer research. GaHV2 encodes 26 mature miRNAs derived from 14 precursors assembled in three distinct gene clusters in the viral genome. One of the most highly expressed GaHV2 miRNAs, miR-M4-5p, shows high sequence similarity to the cellular miR-155 and the miR-K12-11 encoded by Kaposi’s sarcoma-associated herpesvirus, particularly in the miRNA “seed region.” As with miR-K12-11, miR-M4-5p shares a common set of host and viral target genes with miR-155, suggesting that they may target the same regulatory cellular networks; however, differences in regulatory function between miR-155 and miR-M4-5p may distinguish non-viral and viral mediated tumorigenesis. In this review, we focus on the functions of miR-M4-5p as the viral ortholog of miR-155 to explore how the virus mimics a host pathway to benefit the viral life cycle and trigger virus-induced tumorigenesis.

  2. Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot.

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    Jae-Su Moon

    Full Text Available The hepatitis C virus (HCV internal ribosome entry site (IRES that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts 277-343. Based on their antiviral activity, we mapped a druggable region (nts 313-343 where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 5' or 3' direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log10 in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens.

  3. Rational Design of Human Metapneumovirus Live Attenuated Vaccine Candidates by Inhibiting Viral mRNA Cap Methyltransferase

    Science.gov (United States)

    Zhang, Yu; Wei, Yongwei; Zhang, Xiaodong; Cai, Hui; Niewiesk, Stefan

    2014-01-01

    ABSTRACT The paramyxoviruses human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (hPIV3) are responsible for the majority of pediatric respiratory diseases and inflict significant economic loss, health care costs, and emotional burdens. Despite major efforts, there are no vaccines available for these viruses. The conserved region VI (CR VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at positions guanine N-7 (G-N-7) and ribose 2′-O. In this study, we generated a panel of recombinant hMPVs carrying mutations in the S-adenosylmethionine (SAM) binding site in CR VI of L protein. These recombinant viruses were specifically defective in ribose 2′-O methylation but not G-N-7 methylation and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of cotton rats. Importantly, vaccination of cotton rats with these recombinant hMPVs (rhMPVs) with defective MTases triggered a high level of neutralizing antibody, and the rats were completely protected from challenge with wild-type rhMPV. Collectively, our results indicate that (i) amino acid residues in the SAM binding site in the hMPV L protein are essential for 2′-O methylation and (ii) inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for hMPV and perhaps other paramyxoviruses, such as hRSV and hPIV3. IMPORTANCE Human paramyxoviruses, including hRSV, hMPV, and hPIV3, cause the majority of acute upper and lower respiratory tract infections in humans, particularly in infants, children, the elderly, and immunocompromised individuals. Currently, there is no licensed vaccine available. A formalin-inactivated vaccine is not suitable for these viruses because it causes enhanced lung damage upon reinfection with the same virus. A live attenuated vaccine

  4. Non-Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co-Delivery of Cas9 mRNA and sgRNA.

    Science.gov (United States)

    Miller, Jason B; Zhang, Shuyuan; Kos, Petra; Xiong, Hu; Zhou, Kejin; Perelman, Sofya S; Zhu, Hao; Siegwart, Daniel J

    2017-01-19

    CRISPR/Cas is a revolutionary gene editing technology with wide-ranging utility. The safe, non-viral delivery of CRISPR/Cas components would greatly improve future therapeutic utility. We report the synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to (co)deliver long RNAs including Cas9 mRNA and sgRNAs. ZAL nanoparticle (ZNP) delivery of low sgRNA doses (15 nm) reduces protein expression by >90 % in cells. In contrast to transient therapies (such as RNAi), we show that ZNP delivery of sgRNA enables permanent DNA editing with an indefinitely sustained 95 % decrease in protein expression. ZNP delivery of mRNA results in high protein expression at low doses in vitro (gene editing.

  5. [Study of the correlation between the plasma viral load and protective immunity induced by the equine infectious anemia attenuated vaccine and its parental virulent strain].

    Science.gov (United States)

    Cao, Xue-Zhi; Lin, Yue-Zhi; Li, Li; Jiang, Cheng-Gang; Zhao, Li-Ping; Lv, Xiao-Ling; Zhou, Jian-Hua

    2010-03-01

    The threshold hypothesis of attenuated lentiviral vaccine considers that the type of host response to infections of lentiviruses depends on the viral load. To evaluate the correlation between viral loads of the attenuated vaccine strain of equine infectious anemia virus (EIAV) and their effects to induce protective immunity, longitudinal plasma viral loads in groups of horses inoculated with either an attenuated EIAV vaccine strain (EIAV(DLV125)) or sub-lethal dose of an EIAV virulent strain (EIAV(LN40)) were compared. Similar levels of plasma viral loads ranging from 10(3)-10(5) copies/mL were detected from samples of these two groups of animals (P > 0.05) during 23 weeks post the inoculation. However, different responses to the challenge performed thereafter with lethal dose of the EIAV virulent strain were observed from the groups of horses inoculated with either EIAV(DLV125) or sub-lethal dose of EIAV(LN40). The protective efficiency was 67% (3 of 4 cases) and 0 (none of 2 cases), respectively. Our results implicate that the viral load of EIAV attenuated vaccine is not the primary factor, or at least not the solo primary factor, to determine the establishment of immune protection.

  6. Immunological and viral determinants of dengue severity in hospitalized adults in Ha Noi, Viet Nam.

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    Annette Fox

    Full Text Available BACKGROUND: The relationships between the infecting dengue serotype, primary and secondary infection, viremia and dengue severity remain unclear. This cross-sectional study examined these interactions in adult patients hospitalized with dengue in Ha Noi. METHODS AND FINDINGS: 158 patients were enrolled between September 16 and November 11, 2008. Quantitative RT-PCR, serology and NS1 detection were used to confirm dengue infection, determine the serotype and plasma viral RNA concentration, and categorize infections as primary or secondary. 130 (82% were laboratory confirmed. Serology was consistent with primary and secondary infection in 34% and 61%, respectively. The infecting serotype was DENV-1 in 42 (32%, DENV-2 in 39 (30% and unknown in 49 (38%. Secondary infection was more common in DENV-2 infections (79% compared to DENV-1 (36%, p<0.001. The proportion that developed dengue haemorrhagic fever (DHF was 32% for secondary infection compared to 18% for primary infection (p = 0.14, and 26% for DENV-1 compared to 28% for DENV-2. The time until NS1 and plasma viral RNA were undetectable was shorter for DENV-2 compared to DENV-1 (p≤0.001 and plasma viral RNA concentration on day 5 was higher for DENV-1 (p = 0.03. Plasma viral RNA concentration was higher in secondary infection on day 5 of illness (p = 0.046. We didn't find an association between plasma viral RNA concentration and clinical severity. CONCLUSION: Dengue is emerging as a major public health problem in Ha Noi. DENV-1 and DENV-2 were the prevalent serotypes with similar numbers and clinical presentation. Secondary infection may be more common amongst DENV-2 than DENV-1 infections because DENV-2 infections resulted in lower plasma viral RNA concentrations and viral RNA concentrations were higher in secondary infection. The drivers of dengue emergence in northern Viet Nam need to be elucidated and public health measures instituted.

  7. D471G Mutation in LCMV-NP Affects Its Ability to Self-associate and Results in a Dominant Negative Effect in Viral RNA Synthesis

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    Luis Martínez-Sobrido

    2012-10-01

    Full Text Available Arenaviruses merit significant interest because several family members are etiological agents of severe hemorrhagic fevers, representing a major burden to public health. Currently, there are no FDA-licensed vaccines against arenaviruses and the only available antiviral therapy is limited to the use of ribavirin that is partially effective. Arenavirus nucleoprotein (NP is found associated with the genomic RNA forming the viral ribonucleoproteins (vRNPs that together with the polymerase (L direct viral replication and transcription. Virion formation requires the recruitment of vRNPs into budding sites, a process in which the arenavirus matrix-like protein (Z plays a major role. Therefore, proper NP-NP and NP-Z interactions are required for the generation of infectious progeny. In this work we demonstrate the role of the amino acid residue D471 in the self-association of lymphocytic choriomeningitis virus nucleoprotein (LCMV-NP. Amino acid substitutions at this position abrogate NP oligomerization, affecting its ability to mediate replication and transcription of a minigenome reporter plasmid. However, its ability to interact with the Z protein, counteract the cellular interferon response and bind to dsRNA analogs was retained. Additionally, we also document the dominant negative effect of D471G mutation on viral infection, suggesting that NP self-association is an excellent target for the development of new antivirals against arenaviruses.

  8. RNA-binding protein hnRNPLL regulates mRNA splicing and stability during B-cell to plasma-cell differentiation.

    Science.gov (United States)

    Chang, Xing; Li, Bin; Rao, Anjana

    2015-04-14

    Posttranscriptional regulation is a major mechanism to rewire transcriptomes during differentiation. Heterogeneous nuclear RNA-binding protein LL (hnRNPLL) is specifically induced in terminally differentiated lymphocytes, including effector T cells and plasma cells. To study the molecular functions of hnRNPLL at a genome-wide level, we identified hnRNPLL RNA targets and binding sites in plasma cells through integrated Photoactivatable-Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP) and RNA sequencing. hnRNPLL preferentially recognizes CA dinucleotide-containing sequences in introns and 3' untranslated regions (UTRs), promotes exon inclusion or exclusion in a context-dependent manner, and stabilizes mRNA when associated with 3' UTRs. During differentiation of primary B cells to plasma cells, hnRNPLL mediates a genome-wide switch of RNA processing, resulting in loss of B-cell lymphoma 6 (Bcl6) expression and increased Ig production--both hallmarks of plasma-cell maturation. Our data identify previously unknown functions of hnRNPLL in B-cell to plasma-cell differentiation and demonstrate that the RNA-binding protein hnRNPLL has a critical role in tuning transcriptomes of terminally differentiating B lymphocytes.

  9. A central role for the primary microRNA stem in guiding the position and efficiency of Drosha processing of a viral pri-miRNA.

    Science.gov (United States)

    Burke, James M; Kelenis, Demetra P; Kincaid, Rodney P; Sullivan, Christopher S

    2014-07-01

    Processing of primary microRNA (pri-miRNA) stem-loops by the Drosha-DGCR8 complex is the initial step in miRNA maturation and crucial for miRNA function. Nonetheless, the underlying mechanism that determines the Drosha cleavage site of pri-miRNAs has remained unclear. Two prevalent but seemingly conflicting models propose that Drosha-DGCR8 anchors to and directs cleavage a fixed distance from either the basal single-stranded (ssRNA) or the terminal loop. However, recent studies suggest that the basal ssRNA and/or the terminal loop may influence the Drosha cleavage site dependent upon the sequence/structure of individual pri-miRNAs. Here, using a panel of closely related pri-miRNA variants, we further examine the role of pri-miRNA structures on Drosha cleavage site selection in cells. Our data reveal that both the basal ssRNA and terminal loop influence the Drosha cleavage site within three pri-miRNAs, the Simian Virus 40 (SV40) pri-miRNA, pri-miR-30a, and pri-miR-16. In addition to the flanking ssRNA regions, we show that an internal loop within the SV40 pri-miRNA stem strongly influences Drosha cleavage position and efficiency. We further demonstrate that the positions of the internal loop, basal ssRNA, and the terminal loop of the SV40 pri-miRNA cooperatively coordinate Drosha cleavage position and efficiency. Based on these observations, we propose that the pri-miRNA stem, defined by internal and flanking structural elements, guides the binding position of Drosha-DGCR8, which consequently determines the cleavage site. This study provides mechanistic insight into pri-miRNA processing in cells that has numerous biological implications and will assist in refining Drosha-dependent shRNA design.

  10. Sequence requirements for viral RNA replication and VPg uridylylation directed by the internal cis-acting replication element (cre) of human rhinovirus type 14.

    Science.gov (United States)

    Yang, Yan; Rijnbrand, Rene; McKnight, Kevin L; Wimmer, Eckard; Paul, Aniko; Martin, Annette; Lemon, Stanley M

    2002-08-01

    Until recently, the cis-acting signals required for replication of picornaviral RNAs were believed to be restricted to the 5' and 3' noncoding regions of the genome. However, an RNA stem-loop in the VP1-coding sequence of human rhinovirus type 14 (HRV-14) is essential for viral minus-strand RNA synthesis (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998). The nucleotide sequence of the apical loop of this internal cis-acting replication element (cre) was critical for RNA synthesis, while secondary RNA structure, but not primary sequence, was shown to be important within the duplex stem. Similar cres have since been identified in other picornaviral genomes. These RNA segments appear to serve as template for the uridylylation of the genome-linked protein, VPg, providing the VPg-pUpU primer required for viral RNA transcription (A. V. Paul et al., J. Virol. 74:10359-10370, 2000). Here, we show that the minimal functional HRV-14 cre resides within a 33-nucleotide (nt) RNA segment that is predicted to form a simple stem-loop with a 14-nt loop sequence. An extensive mutational analysis involving every possible base substitution at each position within the loop segment defined the sequence that is required within this loop for efficient replication of subgenomic HRV-14 replicon RNAs. These results indicate that three consecutive adenosine residues (nt 2367 to 2369) within the 5' half of this loop are critically important for cre function and suggest that a common RNNNAARNNNNNNR loop motif exists among the cre sequences of enteroviruses and rhinoviruses. We found a direct, positive correlation between the capacity of mutated cres to support RNA replication and their ability to function as template in an in vitro VPg uridylylation reaction, suggesting that these functions are intimately linked. These data thus define more precisely the sequence and structural requirements of the HRV-14 cre and provide additional support for a model in which the role of the cre in RNA

  11. Mal de Río Cuarto Virus Infection Triggers the Production of Distinctive Viral-Derived siRNA Profiles in Wheat and Its Planthopper Vector

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    Luis A. de Haro

    2017-05-01

    Full Text Available Plant reoviruses are able to multiply in gramineae plants and delphacid vectors encountering different defense strategies with unique features. This study aims to comparatively assess alterations of small RNA (sRNA populations in both hosts upon virus infection. For this purpose, we characterized the sRNA profiles of wheat and planthopper vectors infected by Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae and quantified virus genome segments by quantitative reverse transcription PCR We provide evidence that plant and insect silencing machineries differentially recognize the viral genome, thus giving rise to distinct profiles of virus-derived small interfering RNAs (vsiRNAs. In plants, most of the virus genome segments were targeted preferentially within their upstream sequences and vsiRNAs mapped with higher density to the smaller genome segments than to the medium or larger ones. This tendency, however, was not observed in insects. In both hosts, vsiRNAs were equally derived from sense and antisense RNA strands and the differences in vsiRNAs accumulation did not correlate with mRNAs accumulation. We also established that the piwi-interacting RNA (piRNA pathway was active in the delphacid vector but, contrary to what is observed in virus-infected mosquitoes, virus-specific piRNAs were not detected. This work contributes to the understanding of the silencing response in insect and plant hosts.

  12. Evaluation of microRNA stability in plasma and serum from healthy dogs

    DEFF Research Database (Denmark)

    Enelund, Lars; Nielsen, Lise Nikolic; Cirera, Susanna

    2017-01-01

    . METHODS: The levels of four microRNAs (cfa-let-7a, cfa-miR-16, cfa-miR-23a and cfa-miR-26a) known to be stably expressed from other canine studies, have been measured by real-time quantitative PCR. RESULTS: MicroRNA levels were found sufficiently stable for gene profiling in serum- and plasma stored...

  13. HoBi-Like Virus RNA Detected in Foetuses Following Challenge of Pregnant Cows that had Previously Given Birth to Calves Persistently Infected with Bovine Viral Diarrhoea Virus.

    Science.gov (United States)

    Bauermann, F V; Falkenberg, S M; Ridpath, J F

    2016-09-11

    The ability of ruminant pestivirus including bovine viral diarrhoea virus (BVDV) and the related emerging pestivirus, HoBi-like virus, to establish persistent infection (PI) following foetal infection is central to keeping these viruses in circulation. Non-PI dams carrying BVDV PI calves develop high levels of immunity due constantly viral exposure. A study to determine whether the immunity developed following the generation of a BVDV PI is enough to prevent HoBi-like virus infection of a subsequent foetus was performed. This study consisted of nine pregnant cows, four had birthed BVDV-1 PI calves in a previous pregnancy, three cows had birthed BVDV-2 PIs and two had birthed pestivirus negative calves. From this, six pregnant cows were challenged with HoBi-like virus about day 85 of gestation (four BVDV-1 and two BVDV-2 cows) and three non-challenged cows (negative control). At the day of challenge, the serum neutralizing titres against the homologous BVDV strains of the first inoculation ranged from 1148 to 5793. At day 6 post-challenge, HoBi-like RNA was detected in the serum of all four BVDV-1 cows but not in the two BVDV-2 cows. The foetuses harvested from five of the exposed dams (three BVDV-1 and two BVDV-2 cows) at day 30 post-challenge were positive for HoBi-like virus RNA. The sixth cow, BVDV-1 cow #541, while pregnant at the time of exposure, had no foetus 30 days after exposure. Foetuses from HoBi-like virus exposed dams were significantly smaller and lighter than control foetuses. HoBi-like RNA was detected in samples of all challenged foetuses. The identification of viral RNA in the serum of 4 cows at day 6 post-challenge, as well viral RNA detection in all foetuses 30 days post-inoculation, indicates that the foetuses of dams with high antibodies titres against BVDV-1 or BVDV-2 would not be protected from challenge with a HoBi-like virus.

  14. Virological and immunological profiles among patients with undetectable viral load followed prospectively for 24 months

    DEFF Research Database (Denmark)

    Katzenstein, T L; Ullum, H; Røge, Birgit T

    2003-01-01

    OBJECTIVE: To quantify HIV-RNA in plasma, in lymphoid tissue and proviral DNA in peripheral blood mononuclear cells and to relate these to immunological markers among patients with plasma viral load counts of RNA copies/mL. METHODS: A prospective study of one hundred and three patient......-DNA and IgA levels were higher among patients with less complete virological suppression relative to patients with persistently undetectable plasma HIV-RNA. Hence, a high cellular level of HIV-DNA and high plasma IgA may predict subsequent development of low-grade viraemia....

  15. The Life-Cycle of the HIV-1 Gag–RNA Complex

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    Elodie Mailler

    2016-09-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 replication is a highly regulated process requiring the recruitment of viral and cellular components to the plasma membrane for assembly into infectious particles. This review highlights the recent process of understanding the selection of the genomic RNA (gRNA by the viral Pr55Gag precursor polyprotein, and the processes leading to its incorporation into viral particles.

  16. Development of a walleye spleen stromal cell line sensitive to viral hemorrhagic septicemia virus (VHSV IVb) and to protection by synthetic dsRNA.

    Science.gov (United States)

    Vo, Nguyen T K; Bender, Aaron W; Ammendolia, Dustin A; Lumsden, John S; Dixon, Brian; Bols, Niels C

    2015-07-01

    A cell line, WE-spleen6, has been developed from the stromal layer of primary spleen cell cultures. On conventional plastic, WE-spleen6 cells had a spindle-shaped morphology at low cell density but grew to become epithelial-like at confluency. On the commercial extracellular matrix (ECM), Matrigel, the cells remained spindle-shaped and formed lumen-like structures. WE-spleen6 cells had intermediate filament protein, vimentin and the ECM protein, collagen I, but not smooth muscle α-actin (SMA) and von Willebrand factor (vWF) and lacked alkaline phosphatase and phagocytic activities. WE-spleen6 was more susceptible to infection with VHSV IVb than a fibroblast and epithelial cell lines from the walleye caudal fin, WE-cfin11f and WE-cfin11e, respectively. Viral transcripts and proteins appeared earlier in WE-spleen6 cultures as did cytopathic effect (CPE) and significant virus production. The synthetic double-stranded RNA (dsRNA), polyinosinic: polycytidylic acid (pIC), induced the antiviral protein Mx in both cell lines. Treating WE-spleen6 cultures with pIC prior to infection with VHSV IVb inhibited the early accumulation of viral transcripts and proteins and delayed the appearance of CPE and significant viral production. Of particular note, pIC caused the disappearance of viral P protein 2 days post infection. WE-spleen6 should be useful for investigating the impact of VHSV IVb on hematopoietic organs and the actions of pIC on the rhabdovirus life cycle.

  17. hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export.

    Science.gov (United States)

    Wang, Yimeng; Zhou, Jianhong; Du, Yuchun

    2014-01-20

    The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to the cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation.

  18. New tools to study RNA interference to fish viruses: Fish cell lines permanently expressing siRNAs targeting the viral polymerase of viral hemorrhagic septicemia virus

    DEFF Research Database (Denmark)

    Ruiz, S.; Schyth, Brian Dall; Encinas, P.

    2009-01-01

    Previous studies have indicated that low transfection efficiency can be a major problem when gene inhibition by the use of small interfering RNAs (siRNAs) is attempted in fish cells. This may especially be true when targeting genes of viruses which are fast replicating and which can still infect ...... stably expressing rhabdoviral specific siRNA sequences could be a strategy to further investigate the use of RNA interference for targeting costly fish pathogenic viruses.......Previous studies have indicated that low transfection efficiency can be a major problem when gene inhibition by the use of small interfering RNAs (siRNAs) is attempted in fish cells. This may especially be true when targeting genes of viruses which are fast replicating and which can still infect...... cells that have not been transfected with the antiviral siRNAs. To increase the amount of antiviral siRNAs per cell a different strategy than transfection was taken here. Thus, we describe carp epithelioma papulosum cyprinid (EPC) cell clones expressing siRNAs designed to target the L polymerase gene...

  19. Phosphorylation of the human respiratory syncytial virus P protein mediates M2-2 regulation of viral RNA synthesis, a process that involves two P proteins.

    Science.gov (United States)

    Asenjo, Ana; Villanueva, Nieves

    2016-01-04

    The M2-2 protein regulates the balance between human respiratory syncytial virus (HRSV) transcription and replication. Here it is shown that M2-2 mediated transcriptional inhibition is managed through P protein phosphorylation. Transcription inhibition by M2-2 of the HRSV based minigenome pRSVluc, required P protein phosphorylation at serines (S) in positions 116, 117, 119 and increased inhibition is observed if S232 or S237 is also phosphorylated. Phosphorylation of these residues is required for viral particle egression from infected cells. Viral RNA synthesis complementation assays between P protein variants, suggest that two types of P proteins participate in the process as components of RNA dependent RNA polymerase (RdRp). Type I is only functional when, as a homotetramer, it is bound to N and L proteins through residues 203-241. Type II is functionally independent of these interactions and binds to N protein at a region outside residues 232-241. P protein type I phosphorylation at S116, S117 and S119, did not affect the activity of RdRp but this phosphorylation in type II avoids its interaction with N protein and impairs RdRp functionality for transcription and replication. Structural changes in the RdRp, mediated by phosphorylation turnover at the indicated residues, in the two types of P proteins, may result in a fine adjustment, late in the infectious cycle, of transcription, replication and progression in the morphogenetic process that ends in egression of the viral particles from infected cells.

  20. Rice grassy stunt virus nonstructural protein p5 serves as a viral suppressor of RNA silencing and interacts with nonstructural protein p3.

    Science.gov (United States)

    Zhang, Chao; Liu, Xiao-juan; Wu, Kang-cheng; Zheng, Lu-Ping; Ding, Zuo-mei; Li, Fei; Zou, Peng; Yang, Liang; Wu, Jian-guo; Wu, Zu-jian

    2015-11-01

    Rice grassy stunt virus (RGSV), a member of the genus Tenuivirus, causes serious rice disease in Southeast Asian countries. In this study, a green fluorescent protein (GFP)-based transient expression assay was conducted to show that p5, encoded on RNA5 in the viral sense, is a viral suppressor of RNA silencing (VSR). Protein-protein interactions (PPIs) between p5 and all RGSV proteins except pC1 and pC2 were investigated using Gal4-based yeast two-hybrid (Y2H) experiments. The results demonstrated that p5 interacts with itself and with p3 encoded on RNA3 in the viral sense. p5-p5 and p5-p3 interactions were detected by bimolecular fluorescence complementation (BiFC) assay, and the p5-p3 interaction was confirmed by subcellular co-localization and co-immunoprecipitation (Co-IP) assays. Using the Y2H system, we demonstrated that the p5-p3 interaction requires both the N-terminal (amino acid residues 1 to 99) and C-terminal (amino acid residues 94 to 191) domains of p5. In addition, either p5 or p3 could enhance the pathogenicity of potato virus X (PVX) in Nicotiana benthamiana plants. A much more significant enhancement of PVX pathogenicity and accumulation was observed when p5 and p3 were expressed together. Our data also showed that RGSV p3 does not function as a VSR, and it had no effect on the VSR activity of p5 or the subcellular localization pattern of p5 in plant cells from Nicotiana benthamiana.

  1. Dynamics of viral replication in blood and lymphoid tissues during SIVmac251 infection of macaques

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    Mannioui Abdelkrim

    2009-01-01

    Full Text Available Abstract Background Extensive studies of primary infection are crucial to our understanding of the course of HIV disease. In SIV-infected macaques, a model closely mimicking HIV pathogenesis, we used a combination of three markers -- viral RNA, 2LTR circles and viral DNA -- to evaluate viral replication and dissemination simultaneously in blood, secondary lymphoid tissues, and the gut during primary and chronic infections. Subsequent viral compartmentalization in the main target cells of the virus in peripheral blood during the chronic phase of infection was evaluated by cell sorting and viral quantification with the three markers studied. Results The evolutions of viral RNA, 2LTR circles and DNA levels were correlated in a given tissue during primary and early chronic infection. The decrease in plasma viral load principally reflects a large decrease in viral replication in gut-associated lymphoid tissue (GALT, with viral RNA and DNA levels remaining stable in the spleen and peripheral lymph nodes. Later, during chronic infection, a progressive depletion of central memory CD4+ T cells from the peripheral blood was observed, accompanied by high levels of viral replication in the cells of this subtype. The virus was also found to replicate at this point in the infection in naive CD4+ T cells. Viral RNA was frequently detected in monocytes, but no SIV replication appeared to occur in these cells, as no viral DNA or 2LTR circles were detected. Conclusion We demonstrated the persistence of viral replication and dissemination, mostly in secondary lymphoid tissues, during primary and early chronic infection. During chronic infection, the central memory CD4+ T cells were the major site of viral replication in peripheral blood, but viral replication also occurred in naive CD4+ T cells. The role of monocytes seemed to be limited to carrying the virus as a cargo because there was an observed lack of replication in these cells. These data may have important

  2. TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus

    Science.gov (United States)

    Senís, Elena; Mockenhaupt, Stefan; Rupp, Daniel; Bauer, Tobias; Paramasivam, Nagarajan; Knapp, Bettina; Gronych, Jan; Grosse, Stefanie; Windisch, Marc P.; Schmidt, Florian; Theis, Fabian J.; Eils, Roland; Lichter, Peter; Schlesner, Matthias; Bartenschlager, Ralf; Grimm, Dirk

    2017-01-01

    Successful RNAi applications depend on strategies allowing robust and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. Here, we propose a novel avenue which is integration of a promoterless shmiRNA, i.e. a shRNA embedded in a micro-RNA (miRNA) scaffold, into an engineered genomic miRNA locus. For proof-of-concept, we used TALE or CRISPR/Cas9 nucleases to site-specifically integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr locus in hepatoma cells, with the aim to obtain cellular clones that are genetically protected against HCV infection. Using reporter assays, Northern blotting and qRT-PCR, we confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality in selected cellular progeny. Moreover, we employed a comprehensive battery of PCR, cDNA/miRNA profiling and whole genome sequencing analyses to validate targeted integration of a single shmiRNA molecule at the expected position, and to rule out deleterious effects on the genomes or transcriptomes of the engineered cells. Importantly, a subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNAi expression technologies benefits numerous applications, from miRNA, genome and transgenesis research, to human gene therapy. PMID:27614072

  3. Strong resistance against Rice grassy stunt virus is induced in transgenic rice plants expressing double-stranded RNA of the viral genes for nucleocapsid or movement proteins as targets for RNA interference.

    Science.gov (United States)

    Shimizu, Takumi; Ogamino, Takumi; Hiraguri, Akihiro; Nakazono-Nagaoka, Eiko; Uehara-Ichiki, Tamaki; Nakajima, Masami; Akutsu, Katsumi; Omura, Toshihiro; Sasaya, Takahide

    2013-05-01

    Rice grassy stunt virus (RGSV), a member of the genus Tenuivirus, causes significant economic losses in rice production in South, Southeast, and East Asian countries. Growing resistant varieties is the most efficient method to control RGSV; however, suitable resistance genes have not yet been found in natural rice resources. One of the most promising methods to confer resistance against RGSV is the use of RNA interference (RNAi). It is important to target viral genes that play important roles in viral infection and proliferation at an early stage of viral replication. Our recent findings obtained from an RNAi experiment with Rice stripe virus (RSV), a tenuivirus, revealed that the genes for nucleocapsid and movement proteins were appropriate targets for RNAi to confer resistance against RSV. In this study, we transformed rice plants by introducing an RNAi construct of the RGSV genes for the nucelocapsid protein pC5 or movement protein pC6. All progenies from self-fertilized transgenic plants had strong resistance against RGSV infection and did not allow the proliferation of RGSV. Thus, our strategy to target genes for nucleocapsid and movement proteins for conferring viral resistance might be applicable to the plant viruses in the genus Tenuivirus.

  4. Modification of picornavirus genomic RNA using 'click' chemistry shows that unlinking of the VPg peptide is dispensable for translation and replication of the incoming viral RNA

    NARCIS (Netherlands)

    Langereis, Martijn A; Feng, Qian; Nelissen, Frank H T; Virgen-Slane, Richard; van der Heden van Noort, Gerbrand J; Maciejewski, Sonia; Filippov, Dmitri V; Semler, Bert L; van Delft, Floris L; van Kuppeveld, Frank J M

    2014-01-01

    Picornaviruses constitute a large group of viruses comprising medically and economically important pathogens such as poliovirus, coxsackievirus, rhinovirus, enterovirus 71 and foot-and-mouth disease virus. A unique characteristic of these viruses is the use of a viral peptide (VPg) as primer for vir

  5. Plasma miRNA levels correlate with sensitivity to bone mineral density in postmenopausal osteoporosis patients.

    Science.gov (United States)

    Li, Hongqiu; Wang, Zhe; Fu, Qin; Zhang, Jing

    2014-11-01

    In our study, we detect the levels of three micro-RNAs (miRNAs; miR-21, miR-133a and miR-146a) in the plasma of 120 Chinese postmenopausal women who were divided into three groups (normal, osteopenia and osteoporosis) according to the T-scores. Downregulation of miR-21, as well as upregulation of miR-133a, was validated in the plasma of osteoporosis and osteopenia patients versus the normal group. The difference in expression regarding the miR-146a level in plasma among the three groups was not significant (p > 0.01). The circulating miRNA expression levels and bone mineral density (BMD) were examined during a multiple correlation analysis as a dependent variable after adjusting for age, weight and height. We have demonstrated that specific miRNAs species are significantly changed in the plasma of osteoporosis and osteopenia patients and correlated with the BMD. Our study suggested a potential use of miR-21 and miR-133a as sensitive and plasma biomarkers for postmenopausal osteoporosis.

  6. EVALUATION OF THE PLASMA MICRO RNA EXPRESSION LEVELS IN SECONDARY HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS

    Directory of Open Access Journals (Sweden)

    Ali Bay

    2013-11-01

    Full Text Available Background: Hemophagocytic lymphohistiocytosis (HLH is a life threatening hyper inflammatory disease. Micro RNAs (miRNA are about 22 nucleotide-long, small RNAs encoded with genes, and they have regulatory functions in immune response. Objective: To determine the miRNA expression levels of 11 secondary HLH patients, we evaluated the associations of miRNA levels with pathogenesis, clinical presentation, and prognosis of the disease. Patients and Methods: Patients who were diagnosed with secondary HLH from January 2011 to December 2012 were included in this study. We profiled the expressions of 379 miRNAs in plasma of both HLH patients and healthy controls. Patients were evaluated regarding with age, clinical findings, miRNA expresions, laboratory data, treatment, and prognosis, by using descriptive statistics. Results: A total of 11 secondary HLH patients and 11 healthy children were included in this study. miR-205-5p was expressed in all case and controls and expression level of miR-205-5p was found 6.21 fold higher than control group (p=0.01. We detected the second highest expression percent in miR-194-5p with 81% of cases and controls. Expression level of miR-194-5p was found to have 163 fold higher than controls (p= 0.009. miR-30c-5p showed 77% expression percent in cases and controls together. The expression level of this miRNA was detected 9 fold decreased in HLH patients compared to healthy children (p= 0.031. Conclusion: We showed that miR-205-5p, miR-194-5p and miR-30c-5p could be useful plasma biomarkers for HLH. Further research is needed in larger and homogenous study groups, especially for these miRNAs as biomarkers for HLH.

  7. A single-center prospective study on the safety of plasma exchange procedures using a double-viral-inactivated and prion-reduced solvent/detergent fresh-frozen plasma as the replacement fluid in the treatment of thrombotic microangiopathy.

    Science.gov (United States)

    Vendramin, Chiara; McGuckin, Siobhan; Alwan, Ferras; Westwood, John-Paul; Thomas, Mari; Scully, Marie

    2017-01-01

    Patients presenting with acute episodes of thrombotic microangiopathies (TMAs) require urgent access to plasma exchange (PEX). OctaplasLG, a solvent/detergent fresh-frozen plasma product that has undergone viral inactivation and prion reduction step, has been used in our institution since 2013, replacing Octaplas. We prospectively reviewed 981 PEX procedures where OctaplasLG was the replacement fluid in 90 patients admitted acutely with a TMA presentation within our institution from January 1, 2013, to December 31, 2015. We recorded citrate toxicities, plasma reactions, viral transfer, complications related to central venous catheter, and venous thrombotic events (VTEs). Citrate toxicities were 5.4%, plasma reactions were 2%, and all were classified as Grade 1 or 2. VTE had an incidence of 12.2%, although 50% of the episodes occurred in early remission when patients were not receiving PEX. No line insertions complications were recorded. Line-associated infections were 2.2%. Hepatitis B and C serology and human immunodeficiency virus (HIV) were checked on admission. There were four patients who may have had passive transient transfer of hepatitis B antibodies from pooled plasma. No hepatitis C or HIV viral transfer was documented after treatment and no seroconversion was detected after treatment. Our data have demonstrated that the incidence of complications during PEX is low and using OctaplasLG is comparable to the low incidence of reactions. No cases of anaphylaxis, transfusion-related acute lung injury, or fatal plasma reactions were seen. There was no evidence of viral transmission or seroconversion after treatment. © 2016 AABB.

  8. The microRNA-221/-222 cluster balances the antiviral and inflammatory response in viral myocarditis

    NARCIS (Netherlands)

    Corsten, Maarten; Heggermont, Ward; Papageorgiou, Anna-Pia; Deckx, Sophie; Tijsma, Aloys; Verhesen, Wouter; van Leeuwen, Rick; Carai, Paolo; Thibaut, Hendrik-Jan; Custers, Kevin; Summer, Georg; Hazebroek, Mark; Verheyen, Fons; Neyts, Johan; Schroen, Blanche; Heymans, Stephane

    2015-01-01

    AIMS: Viral myocarditis (VM) is an important cause of heart failure and sudden cardiac death in young healthy adults; it is also an aetiological precursor of dilated cardiomyopathy. We explored the role of the miR-221/-222 family that is up-regulated in VM. METHODS AND RESULTS: Here, we show that mi

  9. Targeting the human DEAD-box polypeptide 3 (DDX3) RNA helicase as a novel strategy to inhibit viral replication.

    Science.gov (United States)

    Garbelli, A; Radi, M; Falchi, F; Beermann, S; Zanoli, S; Manetti, F; Dietrich, U; Botta, M; Maga, G

    2011-01-01

    Compounds currently used for the treatment of HIV-1 Infections are targeted to viral proteins. However, the high intrinsic mutation and replication rates of HIV-1 often lead to the emergence of drug resistant strains and consequent therapeutic failure. On this basis, cellular cofactors represent attractive new targets for HIV-1 chemotherapy, since targeting a cellular factor that is required for viral replication should help to overcome the problem of viral resistance. We and others have recently reported the identification of compounds suppressing HIV-1 replication by targeting the cellular DEAD-box helicase DDX3. These results provide a proof-of-principle for the feasibility of blocking HIV-1 infection by rendering the host cell environment less favorable for the virus. The rationale for such an approach and its implications in potentially overcoming the problem of drug resistance related to drugs targeting viral proteins will be discussed in the context of the known cellular functions of the DEAD-box helicase DDX3.

  10. The Distribution of HIV DNA and RNA in Cell Subsets Differs in Gut and Blood of HIV-Positive Patients on ART: Implications for Viral Persistence

    Science.gov (United States)

    Yukl, Steven A.; Shergill, Amandeep K.; Ho, Terence; Killian, Maudi; Girling, Valerie; Epling, Lorrie; Li, Peilin; Wong, Lisa K.; Crouch, Pierre; Deeks, Steven G.; Havlir, Diane V.; McQuaid, Kenneth; Sinclair, Elizabeth; Wong, Joseph K.

    2013-01-01

    Even with optimal antiretroviral therapy, human immunodeficiency virus (HIV) persists in plasma, blood cells, and tissues. To develop new therapies, it is essential to know what cell types harbor residual HIV. We measured levels of HIV DNA, RNA, and RNA/DNA ratios in sorted subsets of CD4+ T cells (CCR7+, transitional memory, and effector memory) and non-CD4+ T leukocytes from blood, ileum, and rectum of 8 ART-suppressed HIV-positive subjects. Levels of HIV DNA/million cells in CCR7+ and effector memory cells were higher in the ileum than blood. When normalized by cell frequencies, most HIV DNA and RNA in the blood were found in CCR7+ cells, whereas in both gut sites, most HIV DNA and RNA were found in effector memory cells. HIV DNA and RNA were observed in non-CD4+ T leukocytes at low levels, particularly in gut tissues. Compared to the blood, the ileum had higher levels of HIV DNA and RNA in both CD4+ T cells and non-CD4+ T leukocytes, whereas the rectum had higher HIV DNA levels in both cell types but lower RNA levels in CD4+ T cells. Future studies should determine whether different mechanisms allow HIV to persist in these distinct reservoirs, and the degree to which different therapies can affect each reservoir. PMID:23852128

  11. Non-invasive aneuploidy detection using free fetal DNA and RNA in maternal plasma: recent progress and future possibilities.

    NARCIS (Netherlands)

    Go, A.T.; Vugt, J.M.G. van; Oudejans, C.B.

    2011-01-01

    BACKGROUND: Cell-free fetal DNA (cff DNA) and RNA can be detected in maternal plasma and used for non-invasive prenatal diagnostics. Recent technical advances have led to a drastic change in the clinical applicability and potential uses of free fetal DNA and RNA. This review summarizes the latest cl

  12. High-intensity endurance training improves adiponectin mRNA and plasma concentrations.

    Science.gov (United States)

    Moghadasi, Mehrzad; Mohebbi, Hamid; Rahmani-Nia, Farhad; Hassan-Nia, Sadegh; Noroozi, Hamid; Pirooznia, Nazanin

    2012-04-01

    Adiponectin is an anti-inflammatory protein that reduced in obesity. Exercise training may reduce the adipose tissue (AT), although it is not well known whether exercise-induced change in AT, increases the adiponectin mRNA expression and plasma concentrations or not; therefore, the purpose of this study was to investigate the adiponectin mRNA and plasma concentrations in middle-aged men after 12 weeks high-intensity exercise training and after a week detraining. Sixteen sedentary overweight and obese middle-aged men (age 41.18 ± 6.1 years; ± SD) volunteered to participate in this study. The subjects were randomly assigned to training group (n = 8) or control group (n = 8). The training group performed endurance training 4 days a week for 12 weeks at an intensity corresponding to 75-80% individual maximum oxygen consumption for 45 min. After 12 weeks of training, subjects underwent a week of detraining. The results showed that the BMI as well as central and peripheral AT volume were decreased in the training group compared to the control group (P training group resulted in a significant increase (P training compared to the control group (P training group. In conclusion, high-intensity endurance training caused an increase adiponectin mRNA in obese middle-aged men.

  13. Persistence of viral RNA in fish infected with VHSV-IVb at 15°C and then moved to warmer temperatures after the onset of disease.

    Science.gov (United States)

    Goodwin, A E; Merry, G E; Noyes, A D

    2012-07-01

    Smallmouth bass, Micropterus dolomieu Lacepède, bluegill, Lepomis macrochirus Rafinesque (coppernose strain), koi carp, Cyprinus carpio L., and channel catfish Ictalurus punctatus (Rafinesque), were infected by intraperitoneal injection with viral haemorrhagic septicaemia virus genotype IVb (VHSV-IVb) at 15 °C. When clinical signs of disease developed, one-third of the fish was moved to 20°C and one-third to 25°C. Mortality in challenged fish at all three temperatures ranged from 25 to 45% in smallmouth bass and from 70 to 90% in bluegill. No koi carp or channel catfish died during the study. Viral copy numbers detected by quantitative real-time reverse transcriptase PCR (qrt-RTPCR) in fish dying at 20 and 25°C decreased over time. In survivors of the challenge, viral copy numbers were higher in the more susceptible species (smallmouth bass and bluegill) than in the more VHSV-IVb disease-resistant species (koi carp and channel catfish). In fish surviving 28days post-infection, prevalence of infection was 66-100% depending on species and temperature, and VHSV-IVb was detected at 10(3) -10(5) copies μg(-1) host RNA. Our results show that qrt-RTPCR is a useful tool to investigate fish kills even 28days after temperatures are elevated above those known to be permissive for VHSV replication.

  14. High-throughput sequencing of plasma microRNA in chronic fatigue syndrome/myalgic encephalomyelitis.

    Directory of Open Access Journals (Sweden)

    Ekua W Brenu

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are known to regulate many biological processes and their dysregulation has been associated with a variety of diseases including Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME. The recent discovery of stable and reproducible miRNA in plasma has raised the possibility that circulating miRNAs may serve as novel diagnostic markers. The objective of this study was to determine the role of plasma miRNA in CFS/ME. RESULTS: Using Illumina high-throughput sequencing we identified 19 miRNAs that were differentially expressed in the plasma of CFS/ME patients in comparison to non-fatigued controls. Following RT-qPCR analysis, we were able to confirm the significant up-regulation of three miRNAs (hsa-miR-127-3p, hsa-miR-142-5p and hsa-miR-143-3p in the CFS/ME patients. CONCLUSION: Our study is the first to identify circulating miRNAs from CFS/ME patients and also to confirm three differentially expressed circulating miRNAs in CFS/ME patients, providing a basis for further study to find useful CFS/ME biomarkers.

  15. The relative prognostic value of plasma HIV RNA levels and CD4 lymphocyte counts in advanced HIV infection

    DEFF Research Database (Denmark)

    Cozzi-Lepri, A; Katzenstein, T L; Ullum, H

    1998-01-01

    OBJECTIVE: It has been suggested that the plasma HIV RNA level is a better predictor of AIDS and death than the CD4 lymphocyte count. We assessed whether the prognostic value of plasma virus levels was different according to the CD4 count. DESIGN: Prospective cohort study of HIV-infected patients...

  16. Fugu double U6 promoter-driven long double-stranded RNA inhibits proliferation of viral hemorrhagic septicemia virus (VHSV) in fish cell lines.

    Science.gov (United States)

    Kim, Min Sun; Jee, Bo Young; Cho, Mi Young; Kim, Jin Woo; Jeong, Hyun Do; Kim, Ki Hong

    2012-06-01

    A long double-stranded RNA (dsRNA)-producing vector driven by fugu double U6 promotors, in which the two promoters were arranged in a head-to-head fashion, was newly constructed. To determine whether the DNA-vector-based long dsRNAs can induce sequence-specific RNA interference (RNAi), Epithelioma papulosum cyprini (EPC) cells and chinook salmon embryonic (CHSE-214) cells were transfected with the long dsRNA vector targeting the G gene of VHSV, and its effect on expression of the G gene and viral proliferation was investigated. The sequence-specific inhibitory effect was further confirmed by analysis of interferon (IFN)-triggered Mx1 gene expression and cross-protection against infectious hematopoietic necrosis virus (IHNV). The fugu double U6 promoter-driven vector successfully produced long dsRNAs in EPC cells, a system that allows continuous production of long dsRNAs in transfected cells. The plasmid-based long dsRNAs targeting the VHSV G gene effectively suppressed G gene expression, but control dsRNAs targeting the EGFP gene did not. Furthermore, there was no significant difference in Mx gene expression between cells transfected with the long dsRNA-producing vector and those transfected with the control empty vector. These results suggest that G gene expression was suppressed not by type-I-IFN-mediated nonspecific inhibition but in a sequence-specific manner. Both EPC and CHSE-214 cells transfected with plasmids producing long dsRNAs targeting the VHSV G gene were protected against VHSV infection but were not protected against IHNV infection, suggesting sequence-specific RNAi-mediated inhibition of viral proliferation. In conclusion, we show, for the first time, long-dsRNA-mediated RNAi in fish cells. The DNA-vector-based long dsRNAs may provide an efficient tool for analysis of gene function in fish cells without preliminary burdensome work for selection of effective siRNA clones, and it may be applied as an antiviral measure in cultured fish.

  17. Quantification of BCR-ABL mRNA in Plasma/Serum of Patients with Chronic Myelogenous Leukemia

    Directory of Open Access Journals (Sweden)

    Miwako Narita, Anri Saito, Aya Kojima, Minami Iwabuchi, Naoya Satoh, Takayoshi Uchiyama, Akie Yamahira, Tatsuo Furukawa, Hirohito Sone, Masuhiro Takahashi

    2012-01-01

    Full Text Available Quantification of tumor-associated mRNA extracted from blood cells/tissues containing tumor cells is used for evaluation of treatment efficacy or residual tumor cell burden in tumors including leukemia. However, this method using tumor cell-containing blood/tissue is difficult to evaluate the whole tumor cell burden in the body. In order to establish an efficient method to evaluate the whole tumor cell burden in the body, we tried to quantify tumor-associated mRNA existing in plasma/serum instead of leukemia cell-containing blood cells in patients with chronic myelogenous leukemia (CML and compared the levels of BCR-ABL mRNA between plasma/serum and peripheral blood cells. mRNA of BCR-ABL, WT1 or GAPDH (control molecule was detected by real-time RT-PCR using RNA extracted from plasma/serum of almost all the patients with CML. Copy numbers of BCR-ABL mRNA were significantly correlated between plasma/serum and peripheral blood cells. However, levels of BCR-ABL mRNA extracted from serum were low compared with those extracted with peripheral blood cells. The present findings suggest that although real-time RT-PCR of mRNA existing in plasma/serum could be used for evaluating the whole tumor cell burden in the body, it's required to establish an efficient method to quantify plasma/serum mRNA by nature without degrading during the procedure.

  18. The H4 subunit of vaccinia virus RNA polymerase is not required for transcription initiation at a viral late promoter.

    OpenAIRE

    Wright, C F; Coroneos, A M

    1995-01-01

    Chromatography of RNA polymerase purified from vaccinia virions and from vaccinia virus-infected HeLa cells resulted in the separation of populations active for early and late transcription. An RNA polymerase population immunodepleted for the vaccinia virus H4 gene peptide could support late transcription.

  19. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH interaction with 3' ends of Japanese encephalitis virus RNA and colocalization with the viral NS5 protein

    Directory of Open Access Journals (Sweden)

    Chou Shih-Jie

    2009-04-01

    Full Text Available Abstract Replication of the Japanese encephalitis virus (JEV genome depends on host factors for successfully completing their life cycles; to do this, host factors have been recruited and/or relocated to the site of viral replication. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a cellular metabolic protein, was found to colocalize with viral RNA-dependent RNA polymerase (NS5 in JEV-infected cells. Subcellular fractionation further indicated that GAPDH remained relatively constant in the cytosol, while increasing at 12 to 24 hours postinfection (hpi and decreasing at 36 hpi in the nuclear fraction of infected cells. In contrast, the redistribution patterns of GAPDH were not observed in the uninfected cells. Co-immunoprecipitation of GAPDH and JEV NS5 protein revealed no direct protein-protein interaction; instead, GAPDH binds to the 3' termini of plus- and minus-strand RNAs of JEV by electrophoretic mobility shift assays. Accordingly, GAPDH binds to the minus strand more efficiently than to the plus strand of JEV RNAs. This study highlights the findings that infection of JEV changes subcellular localization of GAPDH suggesting that this metabolic enzyme may play a role in JEV replication.

  20. RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots

    Directory of Open Access Journals (Sweden)

    Larsen Jeffrey S

    2012-05-01

    Full Text Available Abstract Background Plant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral vectors in plant tissue co-cultures. Results Replicating vectors derived from Potato virus X (PVX and Tobacco rattle virus (TRV were modified to contain the reporter gene β-glucuronidase (GUS with a plant intron to prevent bacterial expression. In cell suspensions, a minimal PVX vector retaining only the viral RNA polymerase gene yielded 6.6-fold more GUS than an analogous full-length PVX vector. Transient co-expression of the minimal PVX vector with P19 of Tomato bushy stunt virus or HC-Pro of Tobacco etch virus to suppress post-transcriptional gene silencing increased GUS expression by 44 and 83%, respectively. A non-replicating vector containing a leader sequence from Cowpea mosaic virus (CPMV-HT modified for enhanced translation led to 70% higher transient GUS expression than a control treatment. In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector. Histochemical staining for GUS in TRV-infected hairy roots revealed the capacity for achieving even higher productivity per unit biomass. Conclusions For the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions. A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens.

  1. RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots

    Science.gov (United States)

    2012-01-01

    Background Plant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral vectors in plant tissue co-cultures. Results Replicating vectors derived from Potato virus X (PVX) and Tobacco rattle virus (TRV) were modified to contain the reporter gene β-glucuronidase (GUS) with a plant intron to prevent bacterial expression. In cell suspensions, a minimal PVX vector retaining only the viral RNA polymerase gene yielded 6.6-fold more GUS than an analogous full-length PVX vector. Transient co-expression of the minimal PVX vector with P19 of Tomato bushy stunt virus or HC-Pro of Tobacco etch virus to suppress post-transcriptional gene silencing increased GUS expression by 44 and 83%, respectively. A non-replicating vector containing a leader sequence from Cowpea mosaic virus (CPMV-HT) modified for enhanced translation led to 70% higher transient GUS expression than a control treatment. In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector. Histochemical staining for GUS in TRV-infected hairy roots revealed the capacity for achieving even higher productivity per unit biomass. Conclusions For the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions. A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens. PMID:22559055

  2. Effects of a human plasma membrane-associated sialidase siRNA on prostate cancer invasion

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiaojie [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Taizhou Polytechnic College, Taizhou (China); Zhang, Ling; Shao, Yueting; Liang, Zuowen; Shao, Chen; Wang, Bo; Guo, Baofeng; Li, Na; Zhao, Xuejian [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Li, Yang, E-mail: lyang@jlu.edu.cn [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Xu, Deqi [Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD (United States)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Neu3 is as one of the sialidases and regulates cell surface functions. Black-Right-Pointing-Pointer A Neu3-specific siRNA inhibited prostrate cancer cell invasion and migration. Black-Right-Pointing-Pointer The Neu3-specific siRNA inhibited prostate cancer metastasis in mice. Black-Right-Pointing-Pointer Targeting Neu3 may have utility for gene-based therapy of human cancer metastasis. -- Abstract: Human plasma membrane-associated sialidase (Neu3) is one of several sialidases that hydrolyze sialic acids in the terminal position of the carbohydrate groups of glycolipids and glycoproteins. Neu3 is mainly localized in plasma membranes and plays crucial roles in the regulation of cell surface functions. In this study, we investigated the effects and molecular mechanisms of Neu3 on cell invasion and migration in vivo and in vitro. Initially, we found that the levels of Neu3 expression were higher in prostate cancer tissues and cell lines than in normal prostate tissues based on RT-PCR and Western blotting analyses. We then applied a Neu3 siRNA approach to block Neu3 signaling using PC-3M cells as model cells. Transwell invasion assays and wound assays showed significantly decreased invasion and migration potential in the Neu3 siRNA-transfected cells. RT-PCR and Western blotting analyses revealed that Neu3 knockdown decreased the expressions of the matrix metalloproteinases MMP-2 and MMP-9. In vivo, mice injected with PC-3M cell tumors were evaluated by SPECT/CT to determine the presence of bone metastases. Mice treated with attenuated Salmonella carrying the Neu3 siRNA developed fewer bone metastases than mice treated with attenuated Salmonella carrying a control Scramble siRNA, attenuated Salmonella alone or PBS. The results for bone metastasis detection by pathology were consistent with the data obtained by SPECT/CT. Tumor blocks were evaluated by histochemical, RT-PCR and Western blotting analyses. The results revealed

  3. A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons.

    Science.gov (United States)

    Porrero, César; Rodríguez-Moreno, Javier; Quetglas, José I; Smerdou, Cristian; Furuta, Takahiro; Clascá, Francisco

    2016-01-01

    We report a highly efficient, simple, and non-infective method for labeling individual long-range projection neurons (LRPNs) in a specific location with enough sparseness and intensity to allow complete and unambiguous reconstructions of their entire axonal tree. The method is based on the "in vivo" transfection of a large RNA construct that drives the massive expression of green fluorescent protein. The method combines two components: injection of a small volume of a hyperosmolar NaCl solution containing the Pal-eGFP-Sindbis RNA construct (Furuta et al., 2001), followed by the application of high-frequency electric current pulses through the micropipette tip. We show that, although each component alone increases transfection efficacy, compared to simple volume injections of standard RNA solution, the highest efficacy (85.7%) is achieved by the combination of both components. In contrast with the infective viral Sindbis vector, RNA transfection occurs exclusively at the position of the injection micropipette tip. This method simplifies consistently labeling one or a few isolated neurons per brain, a strategy that allows unambiguously resolving and quantifying the brain-wide and often multi-branched monosynaptic circuits created by LRPNs.

  4. Plasma micro-RNA biomarkers for diagnosis and prognosis after traumatic brain injury: A pilot study.

    Science.gov (United States)

    Mitra, Biswadev; Rau, Thomas F; Surendran, Nanda; Brennan, James H; Thaveenthiran, Prasanthan; Sorich, Edmond; Fitzgerald, Mark C; Rosenfeld, Jeffrey V; Patel, Sarjubhai A

    2017-04-01

    Prediction of post-concussive syndrome after apparent mild traumatic brain injury (TBI) and subsequent cognitive recovery remains challenging, with substantial limitations of current methods of cognitive testing. This pilot study aimed to determine if levels of micro ribonucleic acids (RNAs) circulating in plasma are altered following TBI, and if changes to levels of such biomarkers over time could assist in determination of prognosis after TBI. Patients were enrolled after TBI on presentation to the Emergency Department and allocated to three groups: A - TBI (physical trauma to the head), witnessed loss of consciousness, amnesia, GCS=15, a normal CT Brain and a recorded first pass after post-traumatic amnesia (PTA) scale; B TBI, witnessed LOC, amnesia, GCS=15, a normal CT brain and a PTA scale test fail and: C - TBI and initial GCS RNA was then assayed using a custom miRNA PCR array. Two micro-RNAs, mir142-3p and mir423-3p demonstrated potential clinical utility differentiating patients after mild head injury into those at greater risk of developing amnesia and therefore, post-concussive syndromes. In addition, these miRNA demonstrated a decrease in expression over time, possibly indicative of brain healing after the injury. Further evaluation of these identified miRNA markers with larger patient cohorts, correlation with clinical symptoms and analysis over longer time periods are essential next steps in developing objective markers of severity of TBI. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. MicroRNA signatures in vitreous humour and plasma of patients with exudative AMD

    OpenAIRE

    Ménard, Catherine; Rezende, Flavio A.; Miloudi, Khalil; Wilson, Ariel; Tétreault, Nicolas; Hardy, Pierre; SanGiovanni, John Paul; De Guire, Vincent; Sapieha, Przemyslaw

    2016-01-01

    Age-related macular degeneration (AMD) is a leading cause of blindness worldwide affecting individuals over the age of 50. The neovascular form (NV AMD) is characterized by choroidal neovascularization (CNV) and responsible for the majority of central vision impairment. Using non-biased microRNA arrays and individual TaqMan qPCRs, we profiled miRNAs in the vitreous humour and plasma of patients with NV AMD. We identified a disease-associated increase in miR-146a and a decrease in miR-106b and...

  6. Vaccine-induced measles virus-specific T cells do not prevent infection or disease but facilitate subsequent clearance of viral RNA.

    Science.gov (United States)

    Lin, Wen-Hsuan W; Pan, Chien-Hsiung; Adams, Robert J; Laube, Beth L; Griffin, Diane E

    2014-04-15

    Infection with wild-type measles virus (MeV) induces lifelong protection from reinfection, and parenteral delivery of the live attenuated measles vaccine (LAV) also provides protection from measles. The level of neutralizing antibody is a good indicator of protection, but the independent roles of MeV-specific antibody and T cells have not been identified. In this study, macaques immunized with LAV through a nebulizer and a mouthpiece developed MeV-specific T-cell responses but not neutralizing antibodies. Upon challenge with wild-type MeV, these animals developed rashes and viremias similar to those in naive animals but cleared viral RNA from blood 25 to 40 days faster. The nebulizer-immunized animals also had more robust MeV-specific CD4(+) and CD8(+) T-cell responses than the naive animals after challenge, characterized by a higher number and better durability of gamma interferon (IFN-γ)-producing cells. Induction of MeV-specific circulating CD4(+) and CD8(+) T cells capable of producing multiple cytokines correlated with clearance of viral RNA in the nebulizer-immunized macaques. These studies demonstrated that MeV-specific T-cell immunity alone did not prevent measles, but T-cell priming enhanced the magnitude, durability, and polyfunctionality of MeV-specific T cells after challenge infection and correlated with more rapid clearance of MeV RNA. IMPORTANCE The components of vaccine-induced immunity necessary for protection from infection and disease have not been clearly identified for most vaccines. Vaccine development usually focuses on induction of antibody, but T-cell-based vaccines are also under development. The live attenuated measles vaccine (LAV) given subcutaneously induces both T cells and neutralizing antibody and provides solid protection from infection. LAV delivered to the upper respiratory tract through a nebulizer and mouthpiece induced a T-cell response but no neutralizing antibody. These T-cell-primed macaques demonstrated no protection from

  7. The development of an efficient multipurpose bean pod mottle virus viral vector set for foreign gene expression and RNA silencing.

    Science.gov (United States)

    Zhang, Chunquan; Bradshaw, Jeffrey D; Whitham, Steven A; Hill, John H

    2010-05-01

    Plant viral vectors are valuable tools for heterologous gene expression, and because of virus-induced gene silencing (VIGS), they also have important applications as reverse genetics tools for gene function studies. Viral vectors are especially useful for plants such as soybean (Glycine max) that are recalcitrant to transformation. Previously, two generations of bean pod mottle virus (BPMV; genus Comovirus) vectors have been developed for overexpressing and silencing genes in soybean. However, the design of the previous vectors imposes constraints that limit their utility. For example, VIGS target sequences must be expressed as fusion proteins in the same reading frame as the viral polyprotein. This requirement limits the design of VIGS target sequences to open reading frames. Furthermore, expression of multiple genes or simultaneous silencing of one gene and expression of another was not possible. To overcome these and other issues, a new BPMV-based vector system was developed to facilitate a variety of applications for gene function studies in soybean as well as in common bean (Phaseolus vulgaris). These vectors are designed for simultaneous expression of multiple foreign genes, insertion of noncoding/antisense sequences, and simultaneous expression and silencing. The simultaneous expression of green fluorescent protein and silencing of phytoene desaturase shows that marker gene-assisted silencing is feasible. These results demonstrate the utility of this BPMV vector set for a wide range of applications in soybean and common bean, and they have implications for improvement of other plant virus-based vector systems.

  8. Iron(II) supramolecular helicates interfere with the HIV-1 Tat–TAR RNA interaction critical for viral replication

    Science.gov (United States)

    Malina, Jaroslav; Hannon, Michael J.; Brabec, Viktor

    2016-07-01

    The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat–TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates.

  9. Efficacy of inactivation of viral contaminants in hyperimmune horse plasma against botulinum toxin by low pH alone and combined with pepsin digestion.

    Science.gov (United States)

    Torgeman, Amram; Mador, Nurit; Dorozko, Marina; Lifshitz, Aliza; Eschar, Naomi; White, Moshe D; Wolf, Dana G; Epstein, Eyal

    2017-07-01

    Assuring viral safety of horse plasma-derived products is fundamental for ethical and regulatory reasons. We previously demonstrated the ability of pepsin digestion at low pH to inactivate West Nile and Sindbis viruses in horse plasma. The present study further examined the efficiency of pepsin digestion to inactivate four additional viruses: HSV-1 and BVDV (lipid-enveloped), BPV and Reo-3 (nonenveloped). These viruses were spiked into hyperimmunized horse plasma against botulinum toxin and subjected to low pH (3.2) alone or combined with pepsin digestion (1200 units/ml). Peptic digestion inactivated the lipid-enveloped viruses, whereas the nonenveloped viruses were unaffected. Interestingly, HSV-1 was rapidly inactivated by acidic pH alone (≥4.9 ± 0.6 log10), whereas a non-robust but meaningful BVDV inactivation (2.9 ± 0.7 log10) was achieved by combined low pH and pepsin. The current study demonstrated the ability of low pH alone and in combination with pepsin digestion to inactivate enveloped viral contaminants in anti-toxin horse plasma. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  10. Successful disabling of the 5' UTR of HCV using adeno-associated viral vectors to deliver modular multimeric primary microRNA mimics.

    Science.gov (United States)

    Bourhill, Tarryn; Arbuthnot, Patrick; Ely, Abdullah

    2016-09-01

    Chronic hepatitis C virus (HCV) infection is a major health concern and is strongly associated with cirrhosis, hepatocellular carcinoma and liver-related mortality. The HCV genome is the template for both protein translation and viral replication and, being RNA, is amenable to direct genetic silencing by RNA interference (RNAi). HCV is a highly mutable virus and is capable of escaping RNAi-mediated silencing. This has highlighted the importance of developing RNAi-based therapy that simultaneously targets multiple regions of the HCV genome. To develop a multi-targeting RNAi activator, a novel approach for the generation of anti-HCV gene therapy was investigated. Five artificial primary miRNA (pri-miR) were each designed to mimic the naturally occurring monomeric pri-miR-31. Potent knockdown of an HCV reporter was seen with four of the five constructs and were processed according to the intended design. The design of the individual pri-miR mimics enabled the modular assembly into multimeric mimics of any possible conformation. Consequently the four potent pri-miR mimics were used to generate polycistronic cassettes, which showed impressive silencing of an HCV target. To further their application as a gene therapy, recombinant adeno-associated viral (rAAV) vectors that express the polycistronic pri-miR mimics were generated. All AAV-delivered anti-HCV pri-miR mimics significantly knocked down the expression of an HCV target and showed inhibition of HCV replicon replication. Here we describe a protocol for the generation of therapeutic rAAVs that express modular polycistronic pri-miR cassettes allowing for rapid alteration and generation of tailored therapeutic constructs against HCV.

  11. Role of the Hepatitis C Virus Core+1 Open Reading Frame and Core cis-Acting RNA Elements in Viral RNA Translation and Replication▿ †

    Science.gov (United States)

    Vassilaki, Niki; Friebe, Peter; Meuleman, Philipe; Kallis, Stephanie; Kaul, Artur; Paranhos-Baccalà, Glaucia; Leroux-Roels, Geert; Mavromara, Penelope; Bartenschlager, Ralf

    2008-01-01

    Four conserved RNA stem-loop structures designated SL47, SL87, SL248, and SL443 have been predicted in the hepatitis C virus (HCV) core encoding region. Moreover, alternative translation products have been detected from a reading frame overlapping the core gene (core+1/ARFP/F). To study the importance of the core+1 frame and core-RNA structures for HCV replication in cell culture and in vivo, a panel of core gene silent mutations predicted to abolish core+1 translation and affecting core-RNA stem-loops were introduced into infectious-HCV genomes of the isolate JFH1. A mutation disrupting translation of all known forms of core+1 and affecting SL248 did not alter virus production in Huh7 cells and in mice xenografted with human liver tissue. However, a combination of mutations affecting core+1 at multiple codons and at the same time, SL47, SL87, and SL248, delayed RNA replication kinetics and substantially reduced virus titers. The in vivo infectivity of this mutant was impaired, and in virus genomes recovered from inoculated mice, SL87 was restored by reversion and pseudoreversion. Mutations disrupting the integrity of this stem-loop, as well as that of SL47, were detrimental for virus viability, whereas mutations disrupting SL248 and SL443 had no effect. This phenotype was not due to impaired RNA stability but to reduced RNA translation. Thus, SL47 and SL87 are important RNA elements contributing to HCV genome translation and robust replication in cell culture and in vivo. PMID:18799568

  12. Plasma levels of microRNA in chronic kidney disease: patterns in acute and chronic exercise.

    Science.gov (United States)

    Van Craenenbroeck, Amaryllis H; Ledeganck, Kristien J; Van Ackeren, Katrijn; Jürgens, Angelika; Hoymans, Vicky Y; Fransen, Erik; Adams, Volker; De Winter, Benedicte Y; Verpooten, Gert A; Vrints, Christiaan J; Couttenye, Marie M; Van Craenenbroeck, Emeline M

    2015-12-15

    Exercise training is an effective way to improve exercise capacity in chronic kidney disease (CKD), but the underlying mechanisms are only partly understood. In healthy subjects (HS), microRNA (miRNA or miR) are dynamically regulated following exercise and have, therefore, been suggested as regulators of cardiovascular adaptation to exercise. However, these effects were not studied in CKD before. The effect of acute exercise (i.e., an acute exercise bout) was assessed in 32 patients with CKD and 12 age- and sex-matched HS (study 1). miRNA expression in response to chronic exercise (i.e., a 3-mo exercise training program) was evaluated in 40 CKD patients (study 2). In a subgroup of study 2, the acute-exercise induced effect was evaluated at baseline and at follow-up. Plasma levels of a preselected panel miRNA, involved in exercise adaptation processes such as angiogenesis (miR-126, miR-210), inflammation (miR-21, miR-146a), hypoxia/ischemia (miR-21, miR-210), and progenitor cells (miR-150), were quantified by RT-PCR. Additionally, seven miRNA involved in similar biological processes were quantified in the subgroup of study 2. Baseline, studied miRNA were comparable in CKD and HS. Following acute exercise, miR-150 levels increased in both CKD (fold change 2.12 ± 0.39, P = 0.002; and HS: fold change 2.41 ± 0.48 P = 0.018, P for interaction > 0.05). miR-146a acutely decreased in CKD (fold change 0.92 ± 0.13, P = 0.024), whereas it remained unchanged in HS. Levels of miR-21, miR-126, and miR-210 remained unaltered. Chronic exercise did not elicit a significant change in the studied miRNA levels. However, an acute exercise-induced decrease in miR-210 was observed in CKD patients, only after training (fold change 0.76 ± 0.15). The differential expression in circulating miRNA in response to acute and chronic exercise may point toward a physiological role in cardiovascular adaptation to exercise, also in CKD.

  13. Mimic Phosphorylation of a βC1 Protein Encoded by TYLCCNB Impairs Its Functions as a Viral Suppressor of RNA Silencing and a Symptom Determinant.

    Science.gov (United States)

    Zhong, Xueting; Wang, Zhan Qi; Xiao, Ruyuan; Cao, Linge; Wang, Yaqin; Xie, Yan; Zhou, Xueping

    2017-08-15

    Phosphorylation of the βC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNB-βC1) by SNF1-related protein kinase 1 (SnRK1) plays a critical role in defense of host plants against geminivirus infection in Nicotiana benthamiana However, how phosphorylation of TYLCCNB-βC1 impacts its pathogenic functions during viral infection remains elusive. In this study, we identified two additional tyrosine residues in TYLCCNB-βC1 that are phosphorylated by SnRK1. The effects of TYLCCNB-βC1 phosphorylation on its functions as a viral suppressor of RNA silencing (VSR) and a symptom determinant were investigated via phosphorylation mimic mutants in N. benthamiana plants. Mutations that mimic phosphorylation of TYLCCNB-βC1 at tyrosine 5 and tyrosine 110 attenuated disease symptoms during viral infection. The phosphorylation mimics weakened the ability of TYLCCNB-βC1 to reverse transcriptional gene silencing and to suppress posttranscriptional gene silencing and abolished its interaction with N. benthamiana ASYMMETRIC LEAVES 1 in N. benthamiana leaves. The mimic phosphorylation of TYLCCNB-βC1 had no impact on its protein stability, subcellular localization, or self-association. Our data establish an inhibitory effect of phosphorylation of TYLCCNB-βC1 on its pathogenic functions as a VSR and a symptom determinant and provide a mechanistic explanation of how SnRK1 functions as a host defense factor.IMPORTANCE Tomato yellow leaf curl China virus (TYLCCNV), which causes a severe yellow leaf curl disease in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). TYLCCNB encodes a single pathogenicity protein, βC1 (TYLCCNB-βC1), which functions as both a viral suppressor of RNA silencing (VSR) and a symptom determinant. Here, we show that mimicking phosphorylation of TYLCCNB-βC1 weakens its ability to reverse transcriptional gene silencing, to suppress posttranscriptional gene silencing, and to interact with N

  14. Plasma microRNA-186 and proteinuria in focal segmental glomerulosclerosis.

    Science.gov (United States)

    Zhang, Changming; Zhang, Wanfen; Chen, Hui-Mei; Liu, Chunbei; Wu, Junnan; Shi, Shaolin; Liu, Zhi-Hong

    2015-02-01

    MicroRNAs (miRNAs) are stable in circulation, and their unique expression profiles can serve as fingerprints for various diseases. This study explored whether plasma miRNAs could be used as biomarkers to evaluate disease activity in patients with focal segmental glomerulosclerosis (FSGS). Retrospective and prospective cohorts. 78 patients with FSGS with nephrotic proteinuria (protein excretion > 3.5g/24 h), 35 patients with FSGS in complete remission, 63 patients with membranous nephropathy, 59 patients with diabetic nephropathy, and 69 apparently healthy controls were recruited. Plasma samples from 51 other patients with FSGS with nephrotic proteinuria were collected prospectively before and after steroid treatment. Plasma miRNA concentration. Complete remission (protein excretion 3.5g/24 h after 8 weeks of steroid treatment). Quantitative reverse transcription-polymerase chain reaction analysis of plasma miRNAs. Increases in miR-125b, miR-186, and miR-193a-3p levels were identified in a pooled plasma sample of 9 patients with FSGS compared with that of 9 healthy controls and were confirmed with individual samples from patients with FSGS (n=32) and healthy controls (n=30). Areas under the receiver operating characteristic curves of miR-125b, miR-186, miR-193a-3p, and the 3 miRNAs in combination were 0.882, 0.789, 0.910, and 0.963, respectively. miR-125b and miR-186 concentrations were significantly lower in patients with FSGS in complete remission (n=35) than those with nephrotic proteinuria (n=37). In a prospective study, miR-125b and miR-186 levels declined markedly in patients with FSGS with complete remission (n=29), but not those with no response (n=22), after steroid treatment. Plasma miR-125b and miR-186 levels were not elevated in patients with membranous nephropathy (n=63) and diabetic nephropathy (n=59) regardless of degree of proteinuria. Last, plasma miR-186, but not miR-125b, level was correlated with degree of proteinuria in patients with FSGS (151

  15. Comparison Analysis of Dysregulated LncRNA Profile in Mouse Plasma and Liver after Hepatic Ischemia/Reperfusion Injury.

    Science.gov (United States)

    Chen, Zhenzhen; Luo, Yanjin; Yang, Weili; Ding, Liwei; Wang, Junpei; Tu, Jian; Geng, Bin; Cui, Qinghua; Yang, Jichun

    2015-01-01

    Long noncoding RNAs (LncRNAs) have been believed to be the major transcripts in various tissues and organs, and may play important roles in regulation of many biological processes. The current study determined the LncRNA profile in mouse plasma after liver ischemia/reperfusion injury (IRI) using microarray technology. Microarray assays revealed that 64 LncRNAs were upregulated, and 244 LncRNAs were downregulated in the plasma of liver IRI mouse. Among these dysregulated plasma LncRNAs, 59-61% were intergenic, 22-25% were antisense overlap, 8-12% were sense overlap and 6-7% were bidirectional. Ten dysregulated plasma LncRNAs were validated by quantitative PCR assays, confirming the accuracy of microarray analysis result. Comparison analysis between dysregulated plasma and liver LncRNA profile after liver IRI revealed that among the 308 dysregulated plasma LncRNAs, 245 LncRNAs were present in the liver, but remained unchanged. In contrast, among the 98 dysregulated liver LncRNAs after IRI, only 19 were present in the plasma, but remained unchanged. LncRNA AK139328 had been previously reported to be upregulated in the liver after IRI, and silencing of hepatic AK139328 ameliorated liver IRI. Both microarray and RT-PCR analyses failed to detect the presence of AK139328 in mouse plasma. In summary, the current study compared the difference between dysregulated LncRNA profile in mouse plasma and liver after liver IRI, and suggested that a group of dysregulated plasma LncRNAs have the potential of becoming novel biomarkers for evaluation of ischemic liver injury.

  16. Effectiveness of first-line antiretroviral therapy based on NNRTIs vs ritonavir-boosted PIs in HIV-1 infected patients with high plasma viral load

    Directory of Open Access Journals (Sweden)

    A Imaz

    2012-11-01

    Full Text Available Purpose of the study: Few clinical trials have compared non-nucleoside reverse transcriptase inhibitors (NNRTI and ritonavir-boosted protease inhibitors (PI/r as initial combined antiretroviral therapy (cART for HIV-1-infected patients with high plasma viral load (pVL, and non-conclusive results have been reported. We compared the effectiveness between NNRTI and PI/r as first-line cART for HIV-1-infected patients with high pVL. Methods: Observational retrospective study of 664 consecutive treatment-naïve HIV-1-infected patients with pVL (HIV-1 RNA >100,000 copies/mL who initiated NNRTI or PI/r-based cART between 2000–2010 in three University hospitals. Only currently preferred or alternative regimens in clinical guidelines were included. Primary endpoint: percentage of therapeutic failures at week 48. Virologic failure was defined as: a lack of virologic response (<1 log RNA HIV-1 decrease in first 3 months; b RNA HIV-1 >50 c/mL at week 48; c confirmed rebound >50 c/ml after a previous value <50 c/mL. Intent-to-treat (ITT noncompleter=failure and on-treatment (OT analyses were performed. Results: 62% of patients initiated NNRTI-regimens (83% efavirenz and 38% PI/r-regimens (62% lopinavir/. Baseline characteristics: male 83%; median age 39 yrs; median CD4 count: 212/µL (NNRTI 232 vs PI/r 177, p=0.028; pVL 5.83 log10 c/mL (NNRTI 5.43 vs PI/r 5.55, p=0.007; AIDS 24% (NNRTI 21% vs PI/r 29%, p=0.015. NRTI backbones were tenofovir plus 3TC or FTC in 72%. The percentage of therapeutic failure was higher in the PI/r group (ITT NC=F 26% vs 18%, p=0.012 with no differences in virologic failures (PI/r 5%, NNRTI 6%, p=0.688. The rate of treatment changes due to toxicity and/or voluntary discontinuations was higher in the PI/r group (15% vs 8%, p=0.008. A multivariate analysis adjusted for age, gender, CD4 count, VL and AIDS showed NNRTI vs PI/r as the only variable associated with treatment response (OR 0.61, 95% CI 0.41–0.88. Median pVL and rate of

  17. Viral markers in HIV infection and AIDS.

    Science.gov (United States)

    Cunningham, A L; Dwyer, D E; Dowton, D N

    1993-01-01

    Viral and immune markers are used for monitoring either progression of human immunodeficiency virus (HIV) disease or response to antiviral therapy. Ideal properties of viral markers are that they are present in all HIV-infected persons at all stages of disease, that they are related to disease pathogenesis, that they can be easily quantitated, that this quantitation correlates rapidly and predictably with both disease stage and response to antivirals, and that they can be developed into rapid, reproducible automated tests. Currently available viral markers include HIV p24 antigenemia (after acid glycine dissociation), anti-p24 antibody titres, quantitative DNA and RNA polymerase chain reaction performed on cells and plasma, and HIV isolate phenotype. In Australia, these markers have been studied in acute HIV seroconversion, in neonatal infection, in body fluids other than blood, and in monitoring of response to antiviral drug therapy.

  18. Bunyaviridae RNA polymerases (L-protein have an N-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription.

    Directory of Open Access Journals (Sweden)

    Juan Reguera

    Full Text Available Bunyaviruses are a large family of segmented RNA viruses which, like influenza virus, use a cap-snatching mechanism for transcription whereby short capped primers derived by endonucleolytic cleavage of host mRNAs are used by the viral RNA-dependent RNA polymerase (L-protein to transcribe viral mRNAs. It was recently shown that the cap-snatching endonuclease of influenza virus resides in a discrete N-terminal domain of the PA polymerase subunit. Here we structurally and functionally characterize a similar endonuclease in La Crosse orthobunyavirus (LACV L-protein. We expressed N-terminal fragments of the LACV L-protein and found that residues 1-180 have metal binding and divalent cation dependent nuclease activity analogous to that of influenza virus endonuclease. The 2.2 A resolution X-ray crystal structure of the domain confirms that LACV and influenza endonucleases have similar overall folds and identical two metal binding active sites. The in vitro activity of the LACV endonuclease could be abolished by point mutations in the active site or by binding 2,4-dioxo-4-phenylbutanoic acid (DPBA, a known influenza virus endonuclease inhibitor. A crystal structure with bound DPBA shows the inhibitor chelating two active site manganese ions. The essential role of this endonuclease in cap-dependent transcription was demonstrated by the loss of transcriptional activity in a RNP reconstitution system in cells upon making the same point mutations in the context of the full-length LACV L-protein. Using structure based sequence alignments we show that a similar endonuclease almost certainly exists at the N-terminus of L-proteins or PA polymerase subunits of essentially all known negative strand and cap-snatching segmented RNA viruses including arenaviruses (2 segments, bunyaviruses (3 segments, tenuiviruses (4-6 segments, and orthomyxoviruses (6-8 segments. This correspondence, together with the well-known mapping of the conserved polymerase motifs to the

  19. Can non-viral technologies knockdown the barriers to siRNA delivery and achieve the next generation of cancer therapeutics?

    Science.gov (United States)

    Guo, Jianfeng; Bourre, Ludovic; Soden, Declan M; O'Sullivan, Gerald C; O'Driscoll, Caitriona

    2011-01-01

    Cancer is one of the most wide-spread diseases of modern times, with an estimated increase in the number of patients diagnosed worldwide, from 11.3 million in 2007 to 15.5 million in 2030 (www.who.int). In many cases, due to the delay in diagnosis and high increase of relapse, survival rates are low. Current therapies, including surgery, radiation and chemotherapy, have made significant progress, but they have many limitations and are far from ideal. Although immunotherapy has recently offered great promise as a new approach in cancer treatment, it is still very much in its infancy and more information on this approach is required before it can be widely applied. For these reasons effective, safe and patient-acceptable cancer therapy is still largely an unmet clinical need. Recent knowledge of the genetic basis of the disease opens up the potential for cancer gene therapeutics based on siRNA. However, the future of such gene-based therapeutics is dependent on achieving successful delivery. Extensive research is ongoing regarding the design and assessment of non-viral delivery technologies for siRNA to treat a wide range of cancers. Preliminary results on the first human Phase I trial for solid tumours, using a targeted non-viral vector, illustrate the enormous therapeutic benefits once the issue of delivery is resolved. In this review the genes regulating cancer will be discussed and potential therapeutic targets will be identified. The physiological and biochemical changes caused by tumours, and the potential to exploit this knowledge to produce bio-responsive 'smart' delivery systems, will be evaluated. This review will also provide a critical and comprehensive overview of the different non-viral formulation strategies under investigation for siRNA delivery, with particular emphasis on those designed to exploit the physiological environment of the disease site. In addition, a section of the review will be dedicated to pre-clinical animal models used to evaluate

  20. Double-Stranded RNA-Binding Protein 4 Is Required for Resistance Signaling against Viral and Bacterial Pathogens

    Directory of Open Access Journals (Sweden)

    Shifeng Zhu

    2013-09-01

    Full Text Available Plant viruses often encode suppressors of host RNA silencing machinery, which occasionally function as avirulence factors that are recognized by host resistance (R proteins. For example, the Arabidopsis R protein, hypersensitive response to TCV (HRT, recognizes the turnip crinkle virus (TCV coat protein (CP. HRT-mediated resistance requires the RNA-silencing component double-stranded RNA-binding protein 4 (DRB4 even though it neither is associated with the accumulation of TCV-specific small RNA nor requires the RNA silencing suppressor function of CP. HRT interacts with the cytosolic fraction of DRB4. Interestingly, TCV infection both increases the cytosolic DRB4 pool and inhibits the HRT-DRB4 interaction. The virulent R8A CP derivative, which induces a subset of HRT-derived responses, also disrupts this interaction. The differential localization of DRB4 in the presence of wild-type and R8A CP implies the importance of subcellular compartmentalization of DRB4. The requirement of DRB4 in resistance to bacterial infection suggests a universal role in R-mediated defense signaling.

  1. RT-Bst: an integrated approach for reverse transcription and enrichment of cDNA from viral RNA.

    Science.gov (United States)

    Kabir, M S; Clements, M O; Kimmitt, P T

    2015-01-01

    The synthesis of cDNA from RNA is challenging due to the inefficiency of reverse transcription (RT). In order to address this, an RT-Bst method was developed for sequential RT of RNA and Bst DNA polymerase amplification for enrichment of cDNA in a single-tube reaction. Using genomic RNA from bacteriophage MS2, the yield of cDNA produced by RT alone and RT-Bst were compared by analysis of polymerase chain reaction (PCR)-amplified products. A superior performance was observed when amplifying MS2 cDNA with random primers following RT-Bst compared to RT alone, indicating greater quantities of cDNA were present after RT-Bst. RT-Bst was also compared with RT alone for their relative ability to produce sufficient cDNA to amplify eight target regions spanning the respiratory syncytial virus (RSV) genome. Six out of eight targets were amplified consistently by PCR subsequent to RT-Bst amplification, whereas only three out of eight targets could be amplified after RT alone. The RSV sequences were selectively amplified using RSV-specific primers from a mixed template containing an excess of MS2 RNA without amplifying MS2 sequences. This suggests that RT-Bst can be used to amplify RNA sequences non-specifically using random primers and specifically using sequence-specific primers, and enhances the yield of cDNA when compared to RT alone.

  2. The development of a viral mediated CRISPR/Cas9 system with doxycycline dependent gRNA expression for inducible in vitro and in vivo genome editing.

    Directory of Open Access Journals (Sweden)

    Christopher A. de Solis

    2016-08-01

    Full Text Available The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV. Specifically, we developed an inducible gRNA (gRNAi AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as one day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e. Cas9 mouse, CRISPRi, etc., and therefore it likely can be used to render these systems inducible as well.

  3. Destabilization of PDK1 by Hsp90 inactivation suppresses hepatitis C virus replication through inhibition of PRK2-mediated viral RNA polymerase phosphorylation.

    Science.gov (United States)

    Kim, Mi-Gyeong; Moon, Jae-Su; Kim, Eun-Jung; Lee, Seung-Hoon; Oh, Jong-Won

    2012-04-27

    Heat shock protein 90 (Hsp90), which chaperones multiple client proteins, has been shown to be implicated in HCV replication. Pharmacological inhibitors of Hsp90 display an anti-HCV activity. However, little is known about the mechanisms of regulation of HCV replication by Hsp90. Here, we show that Hsp90 inhibition by 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) destabilizes phosphoinositide-dependent kinase-1 (PDK1), an upstream kinase of the protein kinase C-related kinase 2 (PRK2) responsible for phosphorylation of HCV RNA polymerase, through the proteosome pathway. Destabilization of PDK1 led to inhibition of phosphorylation of the viral RNA polymerase through a decrease in the abundance of active form PRK2 level. Consequently, Hsp90 inhibition resulted in suppression of HCV replication both in human hepatoma Huh7 cells harboring an HCV subgenomic replicon and in HCV-infected cells. 17-DMAG treatment did not interfere with HCV internal ribosome entry site-mediated translation and the cell cycle in Huh7 cells. Co-treatment of 17-DMAG with interferon-α or HA1077, an inhibitor of PRK2, enhanced the anti-HCV activity of 17-DMAG. Taken together, these findings suggest that Hsp90 plays a critical role in the regulation of HCV RNA polymerase phosphorylation via the PDK1-PRK2 signaling pathway.

  4. Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells

    Science.gov (United States)

    Dixit, Updesh; Pandey, Ashutosh K.; Mishra, Priya; Sengupta, Amitabha; Pandey, Virendra N.

    2016-01-01

    Persistent hepatitis C virus (HCV) infection leads to chronic hepatitis C (CHC), which often progresses to liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The molecular mechanisms that establish CHC and cause its subsequent development into LC and HCC are poorly understood. We have identified a cytoplasmic double-stranded RNA binding protein, Stau1, which is crucial for HCV replication. In this study, Stau1 specifically interacted with the variable-stem-loop region in the 3′ NTR and domain IIId of the HCV-IRES in the 5′ NTR, and promoted HCV replication and translation. Stau1 coimmunoprecipitates HCV NS5B and a cell factor, protein kinase R (PKR), which is critical for interferon-induced cellular antiviral and antiproliferative responses. Like Stau1, PKR displayed binding specificity to domain IIId of HCV-IRES. Stau1 binds to PKR and strongly inhibits PKR-autophosphorylation. We demonstrated that the transport of HCV RNA on the polysomes is Stau1-dependent, being mainly localized in the monosome fractions when Stau1 is downregulated and exclusively localized in the polysomes when Stau1 is overexpressed. Our findings suggest that HCV may appropriate Stau1 to its advantage to prevent PKR-mediated inhibition of eIF2α, which is required for the synthesis of HCV proteins for translocation of viral RNA genome to the polysomes for efficient translation and replication. PMID:27106056

  5. Rescue of foot-and-mouth disease viruses that are pathogenic for cattle from preserved viral RNA samples

    DEFF Research Database (Denmark)

    Belsham, Graham; Jamal, Syed Muhammad; Tjørnehøj, Kirsten;

    2011-01-01

    Background: Foot and mouth disease is an economically important disease of cloven-hoofed animals including cattle, sheep and pigs. It is caused by a picornavirus, foot-and-mouth disease virus (FMDV), which has a positive sense RNA genome which, when introduced into cells, can initiate virus...... replication. Principal Findings: A system has been developed to rescue infectious FMDV from RNA preparations generated from clinical samples obtained under experimental conditions and then applied to samples collected in the ‘‘field’’. Clinical samples from suspect cases of foot-and-mouth disease (FMD) were...

  6. Detection of Peste des Petits Ruminants Viral RNA in Fecal Samples of Goats after an Outbreak in Punjab Province of Pakistan: A Longitudinal Study

    Science.gov (United States)

    Bin Zahur, Aamer; Latif, Asma; Iqbal Dasti, Javid; Irshad, Hamid; Afzal, Muhammad; Rasheed, Tahir; Rashid Malik, Adnan; Qureshi, Zafar-ul-Ahsan

    2016-01-01

    Peste des petits ruminants (PPR) is a highly contagious viral disease of domestic and wild small ruminants and thus has serious socioeconomic implications. In Pakistan, during the year 2012-2013, estimated losses due to PPR were worth Rs. 31.51 billions. Close contact between infected and susceptible animals is an important route of transmission of PPR. Therefore, carrier animals play an important role in unnoticed transmission of PPR. The objective of the study was to investigate the detection of PPR virus in goats recovered from PPR. A suspected PPR outbreak was investigated and confirmed as PPR after analysing appropriate samples collected from infected animals using rRT-PCR. A longitudinal study was conducted over the period of 16 weeks to ascertain the detection of PPR virus (PPRV) in faecal samples of recovered goats. Ninety-six (96) faecal samples from each sampling were collected at 4, 8, 12, and 16 weeks after the outbreak. Faecal samples were analysed using rRT-PCR. Of 96 from each sampling a total of 46, 37, 29, and 25 samples were positive for PPR viral genome at 4, 8, 12, and 16 weeks, respectively, after recovery. Attempts were made for the isolation of PPR virus on Vero cells, but results were negative. These results indicated the detection of PPR viral RNA up to 16 weeks after infection. Therefore, these results may help in the future epidemiology of PPR virus shedding and possible role as source of silent infection for healthy animals especially when there is no history of any outbreak in nearby flock or area. PMID:27597951

  7. Detection of Peste des Petits Ruminants Viral RNA in Fecal Samples of Goats after an Outbreak in Punjab Province of Pakistan: A Longitudinal Study

    Directory of Open Access Journals (Sweden)

    Riasat Wasee Ullah

    2016-01-01

    Full Text Available Peste des petits ruminants (PPR is a highly contagious viral disease of domestic and wild small ruminants and thus has serious socioeconomic implications. In Pakistan, during the year 2012-2013, estimated losses due to PPR were worth Rs. 31.51 billions. Close contact between infected and susceptible animals is an important route of transmission of PPR. Therefore, carrier animals play an important role in unnoticed transmission of PPR. The objective of the study was to investigate the detection of PPR virus in goats recovered from PPR. A suspected PPR outbreak was investigated and confirmed as PPR after analysing appropriate samples collected from infected animals using rRT-PCR. A longitudinal study was conducted over the period of 16 weeks to ascertain the detection of PPR virus (PPRV in faecal samples of recovered goats. Ninety-six (96 faecal samples from each sampling were collected at 4, 8, 12, and 16 weeks after the outbreak. Faecal samples were analysed using rRT-PCR. Of 96 from each sampling a total of 46, 37, 29, and 25 samples were positive for PPR viral genome at 4, 8, 12, and 16 weeks, respectively, after recovery. Attempts were made for the isolation of PPR virus on Vero cells, but results were negative. These results indicated the detection of PPR viral RNA up to 16 weeks after infection. Therefore, these results may help in the future epidemiology of PPR virus shedding and possible role as source of silent infection for healthy animals especially when there is no history of any outbreak in nearby flock or area.

  8. Detection of Peste des Petits Ruminants Viral RNA in Fecal Samples of Goats after an Outbreak in Punjab Province of Pakistan: A Longitudinal Study.

    Science.gov (United States)

    Wasee Ullah, Riasat; Bin Zahur, Aamer; Latif, Asma; Iqbal Dasti, Javid; Irshad, Hamid; Afzal, Muhammad; Rasheed, Tahir; Rashid Malik, Adnan; Qureshi, Zafar-Ul-Ahsan

    2016-01-01

    Peste des petits ruminants (PPR) is a highly contagious viral disease of domestic and wild small ruminants and thus has serious socioeconomic implications. In Pakistan, during the year 2012-2013, estimated losses due to PPR were worth Rs. 31.51 billions. Close contact between infected and susceptible animals is an important route of transmission of PPR. Therefore, carrier animals play an important role in unnoticed transmission of PPR. The objective of the study was to investigate the detection of PPR virus in goats recovered from PPR. A suspected PPR outbreak was investigated and confirmed as PPR after analysing appropriate samples collected from infected animals using rRT-PCR. A longitudinal study was conducted over the period of 16 weeks to ascertain the detection of PPR virus (PPRV) in faecal samples of recovered goats. Ninety-six (96) faecal samples from each sampling were collected at 4, 8, 12, and 16 weeks after the outbreak. Faecal samples were analysed using rRT-PCR. Of 96 from each sampling a total of 46, 37, 29, and 25 samples were positive for PPR viral genome at 4, 8, 12, and 16 weeks, respectively, after recovery. Attempts were made for the isolation of PPR virus on Vero cells, but results were negative. These results indicated the detection of PPR viral RNA up to 16 weeks after infection. Therefore, these results may help in the future epidemiology of PPR virus shedding and possible role as source of silent infection for healthy animals especially when there is no history of any outbreak in nearby flock or area.

  9. Measuring fate and rate of single-molecule competition of amplification and restriction digestion, and its use for rapid genotyping tested with hepatitis C viral RNA.

    Science.gov (United States)

    Sun, Bing; Rodriguez-Manzano, Jesus; Selck, David A; Khorosheva, Eugenia; Karymov, Mikhail A; Ismagilov, Rustem F

    2014-07-28

    We experimentally monitored, at the single-molecule level, the competition among reverse transcription, exponential amplification (RT-LAMP), and linear degradation (restriction enzymes) starting with hepatitis C viral RNA molecules. We found significant heterogeneity in the rate of single-molecule amplification; introduction of the restriction enzymes affected both the rate and the "fate" (the binary outcome) of single-molecule amplification. While end-point digital measurements were primarily sensitive to changes in fate, the bulk real-time kinetic measurements were dominated by the rate of amplification of the earliest molecules, and were not sensitive to fate of the rest of the molecules. We show how this competition of reactions can be used for rapid HCV genotyping with either digital or bulk readout. This work advances our understanding of single-molecule dynamics in reaction networks and may help bring genotyping capabilities out of clinical labs and into limited-resource settings.

  10. In Situ Dynamics of F-Specific RNA Bacteriophages in a Small River: New Way to Assess Viral Propagation in Water Quality Studies.

    Science.gov (United States)

    Fauvel, Blandine; Gantzer, Christophe; Cauchie, Henry-Michel; Ogorzaly, Leslie

    2017-03-01

    The occurrence and propagation of enteric viruses in rivers constitute a major public health issue. However, little information is available on the in situ transport and spread of viruses in surface water. In this study, an original in situ experimental approach using the residence time of the river water mass was developed to accurately follow the propagation of F-specific RNA bacteriophages (FRNAPHs) along a 3-km studied river. Surface water and sediment of 9 sampling campaigns were collected and analyzed using both infectivity and RT-qPCR assays. In parallel, some physico-chemical variables such as flow rate, water temperature, conductivity and total suspended solids were measured to investigate the impact of environmental conditions on phage propagation. For campaigns with low flow rate and high temperature, the results highlight a decrease of infectious phage concentration along the river, which was successfully modelled according to a first-order negative exponential decay. The monitoring of infectious FRNAPHs belonging mainly to the genogroup II was confirmed with direct phage genotyping and total phage particle quantification. Reported k decay coefficients according to exponential models allowed for the determination of the actual in situ distance and time necessary for removing 90 % of infectious phage particles. This present work provides a new way to assess the true in situ viral propagation along a small river. These findings can be highly useful in water quality and risk assessment studies to determine the viral contamination spread from a point contamination source to the nearest recreational areas.

  11. Viral and cellular mRNA-specific activators harness PABP and eIF4G to promote translation initiation downstream of cap binding.

    Science.gov (United States)

    Smith, Richard W P; Anderson, Ross C; Larralde, Osmany; Smith, Joel W S; Gorgoni, Barbara; Richardson, William A; Malik, Poonam; Graham, Sheila V; Gray, Nicola K

    2017-06-13

    Regulation of mRNA translation is a major control point for gene expression and is critical for life. Of central importance is the complex between cap-bound eukaryotic initiation factor 4E (eIF4E), eIF4G, and poly(A) tail-binding protein (PABP) that circularizes mRNAs, promoting translation and stability. This complex is often targeted to regulate overall translation rates, and also by mRNA-specific translational repressors. However, the mechanisms of mRNA-specific translational activation by RNA-binding proteins remain poorly understood. Here, we address this deficit, focusing on a herpes simplex virus-1 protein, ICP27. We reveal a direct interaction with PABP that is sufficient to promote PABP recruitment and necessary for ICP27-mediated activation. PABP binds several translation factors but is primarily considered to activate translation initiation as part of the PABP-eIF4G-eIF4E complex that stimulates the initial cap-binding step. Importantly, we find that ICP27-PABP forms a complex with, and requires the activity of, eIF4G. Surprisingly, ICP27-PABP-eIF4G complexes act independently of the effects of PABP-eIF4G on cap binding to promote small ribosomal subunit recruitment. Moreover, we find that a cellular mRNA-specific regulator, Deleted in Azoospermia-like (Dazl), also employs the PABP-eIF4G interaction in a similar manner. We propose a mechanism whereby diverse RNA-binding proteins directly recruit PABP, in a non-poly(A) tail-dependent manner, to stimulate the small subunit recruitment step. This strategy may be particularly relevant to biological conditions associated with hypoadenylated mRNAs (e.g., germ cells/neurons) and/or limiting cytoplasmic PABP (e.g., viral infection, cell stress). This mechanism adds significant insight into our knowledge of mRNA-specific translational activation and the function of the PABP-eIF4G complex in translation initiation.

  12. Effect of temperature on the pathogenesis, accumulation of viral and satellite RNAs and on plant proteome in peanut stunt virus and satellite RNA-infected plants

    Directory of Open Access Journals (Sweden)

    Aleksandra eObrępalska-Stęplowska

    2015-10-01

    Full Text Available Temperature is an important environmental factor influencing plant development in natural and diseased conditions. The growth rate of plants grown at 27°C is more rapid than for plants grown at 21°C. Thus, temperature affects the rate of pathogenesis progression in individual plants. We have analyzed the effect of temperature conditions (either 21°C or 27°C during the day on the accumulation rate of the virus and satellite RNA (satRNA in Nicotiana benthamiana plants infected by peanut stunt virus (PSV with and without its satRNA, at four time points. In addition, we extracted proteins from PSV and PSV+satRNA-infected plants harvested at 21 dpi, when disease symptoms began to appear on plants grown at 21°C and were well developed on those grown at 27°C, to assess the proteome profile in infected plants compared to mock-inoculated plants grown at these two temperatures, using 2D-gel electrophoresis and mass spectrometry approaches. The accumulation rate of the viral RNAs and satRNA was more rapid at 27°C at the beginning of the infection and then rapidly decreased in PSV-infected plants. At 21 dpi, PSV and satRNA accumulation was higher at 21°C and had a tendency to increase further. In all studied plants grown at 27°C, we observed a significant drop in the identified proteins participating in photosynthesis and carbohydrate metabolism at the proteome level, in comparison to plants maintained at 21°C. On the other hand, the proteins involved in protein metabolic processes were all more abundant in plants grown at 27°C. This was especially evident when PSV-infected plants were analyzed, where increase in abundance of proteins involved in protein synthesis, degradation, and folding was revealed. In mock-inoculated and PSV-infected plants we found an increase in abundance of the majority of stress-related differently-regulated proteins and those associated with protein metabolism. In contrast, in PSV+satRNA-infected plants the shift in the

  13. Specific Induction of TSLP by the Viral RNA Analogue Poly(I:C in Primary Epithelial Cells Derived from Nasal Polyps.

    Directory of Open Access Journals (Sweden)

    Korneliusz Golebski

    Full Text Available Chronic rhinosinusitis with nasal polyposis is an inflammatory disease that, although not directly linked to allergy, often displays a Th2-skewed inflammation characterized by elevated local IgE and IL-5 levels. The nasal cavity is constantly exposed to bacteria and viruses that may trigger epithelial inflammatory responses. To gain more insight into mechanisms by which such a biased inflammation might arise, we have investigated the epithelial expression of the Th2 skewing mediators (TSLP, IL-25, and IL-33 in relationship to disease and microbial triggers.Epithelial cells were obtained from polyp tissues of nasal polyposis patients and from inferior turbinates of non-diseased controls. Cells were exposed to various TLR-specific triggers to study the effect on mRNA and protein expression level of TSLP, IL-25, and IL-33 and the potential regulatory mechanisms through the expression profile the transcription factors ATF-3, DUSP-1, EGR-1, and NFKB-1.The TLR3 agonist and viral analogue poly(I:C induced TSLP mRNA 13.0 ± 3.1 fold (p < 0.05 and protein expression by 12.1 ± 2.3-fold (p < 0.05 higher in epithelium isolated from nasal polyposis patients than in epithelium form healthy controls. This enhanced induction of TSLP may be a consequence of a down-regulated expression of DUSP-1 in polyp epithelium.The TLR3 induced expression of TSLP introduces a mechanism by which the Th2-skewed tissue environment might arise in nasal polyps and invites a further evaluation of the potential contribution of current or past viral infections to polyposis pathogenesis.

  14. Cell-Associated Viral Burden Provides Evidence of Ongoing Viral Replication in Aviremic HIV-2-Infected Patients▿

    Science.gov (United States)

    Soares, Rui S.; Tendeiro, Rita; Foxall, Russell B.; Baptista, António P.; Cavaleiro, Rita; Gomes, Perpétua; Camacho, Ricardo; Valadas, Emília; Doroana, Manuela; Lucas, Margarida; Antunes, Francisco; Victorino, Rui M. M.; Sousa, Ana E.

    2011-01-01

    Viremia is significantly lower in HIV-2 than in HIV-1 infection, irrespective of disease stage. Nevertheless, the comparable proviral DNA burdens observed for these two infections indicate similar numbers of infected cells. Here we investigated this apparent paradox by assessing cell-associated viral replication. We found that untreated HIV-1-positive (HIV-1+) and HIV-2+ individuals, matched for CD4 T cell depletion, exhibited similar gag mRNA levels, indicating that significant viral transcription is occurring in untreated HIV-2+ patients, despite the reduced viremia (undetectable to 2.6 × 104 RNA copies/ml). However, tat mRNA transcripts were observed at significantly lower levels in HIV-2+ patients, suggesting that the rate of de novo infection is decreased in these patients. Our data also reveal a direct relationship of gag and tat transcripts with CD4 and CD8 T cell activation, respectively. Antiretroviral therapy (ART)-treated HIV-2+ patients showed persistent viral replication, irrespective of plasma viremia, possibly contributing to the emergence of drug resistance mutations, persistent hyperimmune activation, and poor CD4 T cell recovery that we observed with these individuals. In conclusion, we provide here evidence of significant ongoing viral replication in HIV-2+ patients, further emphasizing the dichotomy between amount of plasma virus and cell-associated viral burden and stressing the need for antiretroviral trials and the definition of therapeutic guidelines for HIV-2 infection. PMID:21159859

  15. PP2A targeting by viral proteins: a widespread biological strategy from DNA/RNA tumor viruses to HIV-1.

    Science.gov (United States)

    Guergnon, Julien; Godet, Angélique N; Galioot, Amandine; Falanga, Pierre Barthélémy; Colle, Jean-Hervé; Cayla, Xavier; Garcia, Alphonse

    2011-11-01

    Protein phosphatase 2A (PP2A) is a large family of holoenzymes that comprises 1% of total cellular proteins and accounts for the majority of Ser/Thr phosphatase activity in eukaryotic cells. Although initially viewed as constitutive housekeeping enzymes, it is now well established that PP2A proteins represent a family of highly and sophistically regulated phosphatases. The past decade, multiple complementary studies have improved our knowledge about structural and functional regulation of PP2A holoenzymes. In this regard, after summarizing major cellular regulation, this review will mainly focus on discussing a particulate biological strategy, used by various viruses, which is based on the targeting of PP2A enzymes by viral proteins in order to specifically deregulate, for their own benefit, cellular pathways of their hosts. The impact of such PP2A targeting for research in human diseases, and in further therapeutic developments, is also discussed.

  16. A plasma microRNA signature as a biomarker for acquired aplastic anemia.

    Science.gov (United States)

    Hosokawa, Kohei; Kajigaya, Sachiko; Feng, Xingmin; Desierto, Marie J; Fernandez Ibanez, Maria Del Pilar; Rios, Olga; Weinstein, Barbara; Scheinberg, Phillip; Townsley, Danielle M; Young, Neal S

    2017-01-01

    Aplastic anemia is an acquired bone marrow failure characterized by marrow hypoplasia, a paucity of hematopoietic stem and progenitor cells, and pancytopenia of the peripheral blood, due to immune attack on the bone marrow. In aplastic anemia, a major challenge is to develop immune biomarkers to monitor the disease. We measured circulating microRNAs in plasma samples of aplastic anemia patients in order to identify disease-specific microRNAs. A total of 179 microRNAs were analyzed in 35 plasma samples from 13 aplastic anemia patients, 11 myelodysplastic syndrome patients, and 11 healthy controls using the Serum/Plasma Focus microRNA Polymerase Chain Reaction Panel. Subsequently, 19 microRNAs from the discovery set were investigated in the 108 plasma samples from 41 aplastic anemia patients, 24 myelodysplastic syndrome patients, and 43 healthy controls for validation, confirming that 3 microRNAs could be validated as dysregulated (>1.5-fold change) in aplastic anemia, compared to healthy controls. MiR-150-5p (induction of T-cell differentiation) and miR-146b-5p (involvement in the feedback regulation of innate immune response) were elevated in aplastic anemia plasma, whereas miR-1 was decreased in aplastic anemia. By receiver operating characteristic curve analysis, we developed a logistic model with these 3 microRNAs that enabled us to predict the probability of a diagnosis of aplastic anemia with an area under the curve of 0.86. Dysregulated expression levels of the microRNAs became normal after immunosuppressive therapy at 6 months. Specifically, miR-150-5p expression was significantly reduced after successful immunosuppressive therapy, but did not change in non-responders. We propose 3 novel plasma biomarkers in aplastic anemia, in which miR-150-5p, miR-146b-5p, and miR-1 can serve for diagnosis and miR-150-5p for disease monitoring. Clinicaltrials.gov identifiers:00260689, 00217594, 00961064.

  17. Pre-storage centrifugation conditions have significant impact on measured microRNA levels in biobanked EDTA plasma samples

    DEFF Research Database (Denmark)

    Binderup, Helle Glud; Houlind, Kim; Madsen, Jonna Skov;

    2016-01-01

    was significantly higher in the re-centrifuged biobanked plasma compared to PPP, even when the platelet count was reduced to 0-1×109/L. Conclusion: We found, that pre-storage centrifugation conditions have a significant impact on the measured EDTA plasma level of miRNAs known to be present in platelets. Even......Background: In the past few years, an increasing number of studies have reported the potential use of microRNAs (miRNA) as circulating biomarkers for diagnosis or prognosis of a wide variety of diseases. There is, however, a lack of reproducibility between studies. Due to the high miRNA content...

  18. Distinct genetic loci control plasma HIV-RNA and cellular HIV-DNA levels in HIV-1 infection: the ANRS Genome Wide Association 01 study.

    Directory of Open Access Journals (Sweden)

    Cyril Dalmasso

    Full Text Available Previous studies of the HIV-1 disease have shown that HLA and Chemokine receptor genetic variants influence disease progression and early viral load. We performed a Genome Wide Association study in a cohort of 605 HIV-1-infected seroconverters for detection of novel genetic factors that influence plasma HIV-RNA and cellular HIV-DNA levels. Most of the SNPs strongly associated with HIV-RNA levels were localised in the 6p21 major histocompatibility complex (MHC region and were in the vicinity of class I and III genes. Moreover, protective alleles for four disease-associated SNPs in the MHC locus (rs2395029, rs13199524, rs12198173 and rs3093662 were strikingly over-represented among forty-five Long Term HIV controllers. Furthermore, we show that the HIV-DNA levels (reflecting the HIV reservoir are associated with the same four SNPs, but also with two additional SNPs on chromosome 17 (rs6503919; intergenic region flanked by the DDX40 and YPEL2 genes and chromosome 8 (rs2575735; within the Syndecan 2 gene. Our data provide evidence that the MHC controls both HIV replication and HIV reservoir. They also indicate that two additional genomic loci may influence the HIV reservoir.

  19. Seed-borne viral dsRNA elements in three cultivated Raphanus and Brassica plants suggest three cryptoviruses.

    Science.gov (United States)

    Li, Liqiang; Liu, Jianning; Zhang, Qiong; Fu, Runying; Zhu, Xiwu; Li, Chao; Chen, Jishuang

    2016-04-01

    Since the 1970s, several dsRNA viruses, including Radish yellow edge virus, Raphanus sativus virus 1, Raphanus sativus virus 2, and Raphanus sativus virus 3, have been identified and reported as infecting radish. In the present study, in conjunction with a survey of seed-borne viruses in cultivated Brassica and Raphanus using the dsRNA diagnostic method, we discovered 3 novel cryptoviruses that infect Brassica and Raphanus: Raphanus sativus partitivirus 1, which infects radish (Raphanus sativus); Sinapis alba cryptic virus 1, which infects Sinapis alba; and Brassica rapa cryptic virus 1 (BrCV1), which infects Brassica rapa. The genomic organization of these cryptoviruses was analyzed and characterized. BrCV1 might represent the first plant partitivirus found in Gammapartitivirus. Additionally, the evolutionary relationships among all of the partitiviruses reported in Raphanus and Brassica were analyzed.

  20. A functional selection of viral genetic elements in cultured cells to identify hepatitis C virus RNA translation inhibitors †

    OpenAIRE

    Jaffrelo, Loic; Chabas, Sandrine; Reigadas, Sandrine; Pflieger, Aude; Wychowski, Czeslaw; Rumi, Julie; Ventura, Michel; Toulmé, Jean-Jacques; Staedel, Cathy

    2008-01-01

    We developed a functional selection system based on randomized genetic elements (GE) to identify potential regulators of hepatitis C virus (HCV) RNA translation, a process initiated by an internal ribosomal entry site (IRES). A retroviral HCV GE library was introduced into HepG2 cells, stably expressing the Herpes simplex virus thymidine kinase (HSV-TK) under the control of the HCV IRES. Cells that expressed transduced GEs inhibiting HSV-TK were selected via their resistance to ganciclovir. S...

  1. EKSPRESI PROTEIN COAT DAN mRNA VIRAL NERVOUS NECROSIS YANG DIKENDALIKAN OLEH PROMOTER β-AKTIN IKAN MEDAKA DAN KERATIN IKAN FLOUNDER JEPANG

    Directory of Open Access Journals (Sweden)

    Wiwien Mukti Andriyani

    2014-03-01

    Full Text Available Kemampuan promoter dalam mengatur ekspresi gen penyandi protein imunogenik sangat menentukan efikasi suatu vaksin DNA. Penelitian ini bertujuan untuk mengukur tingkat ekspresi protein dan mRNA RNA2 penyandi coat protein (CP virus viral nervous necrosis (VNN yang dikendalikan oleh dua promoter berbeda, yaitu promoter β-aktin ikan medaka (mBA, dan keratin ikan flounder Jepang (JfKer. Uji ekspresi CP dilakukan menggunakan embrio ikan lele dumbo (Clarias sp. sebagai model, sedangkan analisis mRNA dilakukan menggunakan ikan kerapu tikus. Konstruksi vektor ekspresi pmBA-CP dan pJKer-CP dengan konsentrasi 50 ng/μL KCl 1 M disuntikkan ke embrio ikan lele dumbo fase 1-2 sel. Sebanyak 30 embrio ikan lele dumbo diambil pada jam ke-6, 8, 10, 12, 14, dan 16 pascainjeksi untuk analisis protein. Hasil SDS-PAGE menunjukkan adanya protein berukuran sekitar 42 kDa, dan analisis western blot menggunakan antibodi (Ab poliklonal anti-VNN membuktikan bahwa protein tersebut adalah CP. Keberhasilan deteksi protein spesifik menggunakan Ab anti-VNN tersebut menunjukkan bahwa embrio ikan lele dapat digunakan untuk menguji potensi produksi protein imunogenik yang dikendalikan oleh promoter berbeda. Pengujian ini juga menunjukkan bahwa, aktivitas promoter mBA lebih tinggi daripada promoter JfKer, sehingga uji ekspresi mRNA dilakukan menggunakan konstruksi pmBA-CP. Benih ikan kerapu tikus (panjang badan sekitar 5 cm diinjeksi dengan pmBA-CP secara intramuskular dengan dosis 12,5 μg/ekor. Total RNA diekstraksi dari daging pada waktu 6, 12, dan 24 jam pascainjeksi. Hasil RT-PCR menunjukkan adanya ekspresi mRNA CP pada 24 jam pascainjeksi. Hal tersebut menunjukkan bahwa promotor mBA aktif mengendalikan ekspresi CP pada ikan kerapu tikus, dan pmBA-CP berpotensi digunakan sebagai vaksin DNA untuk menginduksi kekebalan ikan kerapu terhadap infeksi VNN.

  2. MicroRNA-19b Downregulates Gap Junction Protein Alpha1 and Synergizes with MicroRNA-1 in Viral Myocarditis.

    Science.gov (United States)

    Lin, Junyi; Xue, Aimin; Li, Liliang; Li, Beixu; Li, Yuhua; Shen, Yiwen; Sun, Ning; Chen, Ruizhen; Xu, Hongfei; Zhao, Ziqin

    2016-05-18

    Viral myocarditis (VMC) is a life-threatening disease that leads to heart failure or cardiac arrhythmia. A large number of researches have revealed that mircroRNAs (miRNAs) participate in the pathological processes of VMC. We previously reported that miR-1 repressed the expression of gap junction protein α1 (GJA1) in VMC. In this study, miR-19b was found to be significantly upregulated using the microarray analysis in a mouse model of VMC, and overexpression of miR-19b led to irregular beating pattern in human cardiomyocytes derived from the induced pluripotent stem cells (hiPSCs-CMs). The upregulation of miR-19b was associated with decreased GJA1 in vivo. Furthermore, a miR-19b inhibitor increased, while its mimics suppressed the expression of GJA1 in HL-1 cells. When GJA1 was overexpressed, the miR-19b mimics-mediated irregular beating was reversed in hiPSCs-CMs. In addition, the effect of miR-19b on GJA1 was enhanced by miR-1 in a dose-dependent manner. These data suggest miR-19b contributes to irregular beating through regulation of GJA1 by cooperating with miR-1. Based on the present and our previous studies, it could be indicated that miR-19b and miR-1 might be critically involved in cardiac arrhythmia associated with VMC.

  3. Transfection of the Giardia lamblia double-stranded RNA virus into giardia lamblia by electroporation of a single-stranded RNA copy of the viral genome.

    OpenAIRE

    1990-01-01

    The development of a genetic vector for protozoan parasites is a major hurdle yet to be crossed in the study of the molecular and cellular biology of these parasites. We have identified and isolated a double-stranded RNA virus (G. lamblia virus [GLV]) from certain strains of the intestinal parasitic protozoan Giardia lamblia (A. L. Wang and C. C. Wang, Mol. Biochem. Parasitol. 21:269-276, 1986), which is capable of infecting other virus-free strains of G. lamblia (R. L. Miller, A. L. Wang, an...

  4. Comparative evaluation of the performance of the Abbott RealTime HIV-1 assay for measurement of HIV-1 plasma viral load on genetically diverse samples from Greece

    Directory of Open Access Journals (Sweden)

    Paraskevis Dimitrios

    2011-01-01

    Full Text Available Abstract Background HIV-1 is characterized by increased genetic heterogeneity which tends to hinder the reliability of detection and accuracy of HIV-1 RNA quantitation assays. Methods In this study, the Abbott RealTime HIV-1 (Abbott RealTime assay was compared to the Roche Cobas TaqMan HIV-1 (Cobas TaqMan and the Siemens Versant HIV-1 RNA 3.0 (bDNA 3.0 assays, using clinical samples of various viral load levels and subtypes from Greece, where the recent epidemiology of HIV-1 infection has been characterized by increasing genetic diversity and a marked increase in subtype A genetic strains among newly diagnosed infections. Results A high correlation was observed between the quantitative results obtained by the Abbott RealTime and the Cobas TaqMan assays. Viral load values quantified by the Abbott RealTime were on average lower than those obtained by the Cobas TaqMan, with a mean (SD difference of -0.206 (0.298 log10 copies/ml. The mean differences according to HIV-1 subtypes between the two techniques for samples of subtype A, B, and non-A/non-B were 0.089, -0.262, and -0.298 log10 copies/ml, respectively. Overall, differences were less than 0.5 log10 for 85% of the samples, and >1 log10 in only one subtype B sample. Similarly, Abbott RealTime and bDNA 3.0 assays yielded a very good correlation of quantitative results, whereas viral load values assessed by the Abbott RealTime were on average higher (mean (SD difference: 0.160 (0.287 log10 copies/ml. The mean differences according to HIV-1 subtypes between the two techniques for subtype A, B and non-A/non-B samples were 0.438, 0.105 and 0.191 log10 copies/ml, respectively. Overall, the majority of samples (86% differed by less than 0.5 log10, while none of the samples showed a deviation of more than 1.0 log10. Conclusions In an area of changing HIV-1 subtype pattern, the Abbott RealTime assay showed a high correlation and good agreement of results when compared both to the Cobas TaqMan and bDNA 3

  5. Haploid genetic screens identify an essential role for PLP2 in the downregulation of novel plasma membrane targets by viral E3 ubiquitin ligases.

    Directory of Open Access Journals (Sweden)

    Richard T Timms

    Full Text Available The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2, a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.

  6. Haploid genetic screens identify an essential role for PLP2 in the downregulation of novel plasma membrane targets by viral E3 ubiquitin ligases.

    Directory of Open Access Journals (Sweden)

    Richard T Timms

    Full Text Available The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2, a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.

  7. viRome: an R package for the visualization and analysis of viral small RNA sequence datasets

    OpenAIRE

    Watson, Mick; Schnettler, Esther; Kohl, Alain

    2013-01-01

    SUMMARY: RNA interference (RNAi) is known to play an important part in defense against viruses in a range of species. Second-generation sequencing technologies allow us to assay these systems and the small RNAs that play a key role with unprecedented depth. However, scientists need access to tools which can condense, analyse and display the resulting data. Here we present viRome, a package for R that takes aligned sequence data and produces a range of essential plots and reports.Availability ...

  8. Multicenter comparison of Roche COBAS AMPLICOR MONITOR version 1.5, Organon Teknika NucliSens QT with Extractor, and Bayer Quantiplex version 3.0 for quantification of human immunodeficiency virus type 1 RNA in plasma.

    Science.gov (United States)

    Murphy, D G; Côté, L; Fauvel, M; René, P; Vincelette, J

    2000-11-01

    The performance and characteristics of Roche COBAS AMPLICOR HIV-1 MONITOR version 1.5 (CA MONITOR 1.5) UltraSensitive (usCA MONITOR 1. 5) and Standard (stCA MONITOR 1.5) procedures, Organon Teknika NucliSens HIV-1 RNA QT with Extractor (NucliSens), and Bayer Quantiplex HIV RNA version 3.0 (bDNA 3.0) were compared in a multicenter trial. Samples used in this study included 460 plasma specimens from human immunodeficiency virus (HIV) type 1 (HIV-1)-infected persons, 100 plasma specimens from HIV antibody (anti-HIV)-negative persons, and culture supernatants of HIV-1 subtype A to E isolates diluted in anti-HIV-negative plasma. Overall, bDNA 3.0 showed the least variation in RNA measures upon repeat testing. For the Roche assays, usCA MONITOR 1.5 displayed less variation in RNA measures than stCA MONITOR 1.5. NucliSens, at an input volume of 2 ml, showed the best sensitivity. Deming regression analysis indicated that the results of all three assays were significantly correlated (P < 0.0001). However, the mean difference in values between CA MONITOR 1.5 and bDNA 3.0 (0.274 log(10) RNA copies/ml; 95% confidence interval, 0.192 to 0.356) was significantly different from 0, indicating that CA MONITOR 1.5 values were regularly higher than bDNA 3.0 values. Upon testing of 100 anti-HIV-negative plasma specimens, usCA MONITOR 1.5 and NucliSens displayed 100% specificity, while bDNA 3.0 showed 98% specificity. NucliSens quantified 2 of 10 non-subtype B viral isolates at 1 log(10) lower than both CA MONITOR 1.5 and bDNA 3.0. For NucliSens, testing of specimens with greater than 1,000 RNA copies/ml at input volumes of 0.1, 0.2, and 2.0 ml did not affect the quality of results. Additional factors differing between assays included specimen throughput and volume requirements, limit of detection, ease of execution, instrument work space, and costs of disposal. These characteristics, along with assay performance, should be considered when one is selecting a viral load assay.

  9. MicroRNA-19b Downregulates Gap Junction Protein Alpha1 and Synergizes with MicroRNA-1 in Viral Myocarditis

    Directory of Open Access Journals (Sweden)

    Junyi Lin

    2016-05-01

    Full Text Available Viral myocarditis (VMC is a life-threatening disease that leads to heart failure or cardiac arrhythmia. A large number of researches have revealed that mircroRNAs (miRNAs participate in the pathological processes of VMC. We previously reported that miR-1 repressed the expression of gap junction protein α1 (GJA1 in VMC. In this study, miR-19b was found to be significantly upregulated using the microarray analysis in a mouse model of VMC, and overexpression of miR-19b led to irregular beating pattern in human cardiomyocytes derived from the induced pluripotent stem cells (hiPSCs-CMs. The upregulation of miR-19b was associated with decreased GJA1 in vivo. Furthermore, a miR-19b inhibitor increased, while its mimics suppressed the expression of GJA1 in HL-1 cells. When GJA1 was overexpressed, the miR-19b mimics-mediated irregular beating was reversed in hiPSCs-CMs. In addition, the effect of miR-19b on GJA1 was enhanced by miR-1 in a dose-dependent manner. These data suggest miR-19b contributes to irregular beating through regulation of GJA1 by cooperating with miR-1. Based on the present and our previous studies, it could be indicated that miR-19b and miR-1 might be critically involved in cardiac arrhythmia associated with VMC.

  10. The dual CCR5 and CCR2 inhibitor cenicriviroc does not redistribute HIV into extracellular space: implications for plasma viral load and intracellular DNA decline.

    Science.gov (United States)

    Kramer, Victor G; Hassounah, Said; Colby-Germinario, Susan P; Oliveira, Maureen; Lefebvre, Eric; Mesplède, Thibault; Wainberg, Mark A

    2015-03-01

    Cenicriviroc is a potent antagonist of the chemokine coreceptors 5 and 2 (CCR5/CCR2) and blocks HIV-1 entry. The CCR5 inhibitor maraviroc has been shown in tissue culture to be able to repel cell-free virions from the cell surface into extracellular space. We hypothesized that cenicriviroc might exhibit a similar effect, and tested this using clinical samples from the Phase IIb study 652-2-202, by measuring rates of intracellular DNA decline. We also monitored viral RNA levels in culture fluids. We infected PM-1 cells with CCR5-tropic HIV-1 BaL in the presence or absence of inhibitory concentrations of cenicriviroc (20 nM) or maraviroc (50 nM) or controls. Viral load levels and p24 were measured by ELISA, quantitative PCR and quantitative real-time reverse transcription PCR at 4 h post-infection. Frozen PBMC DNA samples from 30 patients with virological success in the Phase IIb study were studied, as were early and late reverse transcript levels. Docking studies compared binding between cenicriviroc/CCR5 and maraviroc/CCR5. Unlike maraviroc, cenicriviroc did not cause an increase in the amount of virus present in culture fluids at 4 h compared with baseline. The use of cenicriviroc did, however, result in lower levels of intracellular viral DNA after 4 h. Structural modelling indicates that cenicriviroc binds more deeply than maraviroc to the hydrophobic pocket of CCR5, providing an explanation for the absence of viral rebound with cenicriviroc. In contrast to maraviroc, cenicriviroc does not repel virus back into extracellular space. Differences in results may be due to superior binding of cenicriviroc to CCR5 compared with maraviroc. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  11. Knockdown of cellular RNA helicase DDX3 by short hairpin RNAs suppresses HIV-1 viral replication without inducing apoptosis.

    Science.gov (United States)

    Ishaq, Musarat; Hu, Jiajie; Wu, Xiaoyun; Fu, Qiong; Yang, Yalin; Liu, Qingzhen; Guo, Deyin

    2008-07-01

    The targeting of a cellular co-factor, rather than the HIV-1-specific RNAs, by small interfering RNAs holds promise as the rapid mutational ability of the HIV-1 genome may obviate the potential clinical use of RNAi against this virus. The DEAD-box RNA helicase DDX3 is an essential Rev co-factor in the CRM1-Rev-RRE complex that promotes the export of unspliced and single-spliced HIV-1 RNAs from the nucleus to cytoplasm. In this report, human DDX3 was targeted by specific short hairpin RNAs, and the down-regulation of cell's endogenous DDX3 suppressed the nuclear export of unspliced HIV-1 RNAs but did not affect the cell viability. We further showed that the knockdown of cellular DDX3 could effectively inhibit the replication of HIV-1. Therefore, the current results suggest that the RNA helicase DDX3 may become a potential target by RNAi for future genetic therapy of HIV/AIDS.

  12. Viral precursor protein P3 and its processed products perform discrete and essential functions in the poliovirus RNA replication complex.

    Science.gov (United States)

    Spear, Allyn; Ogram, Sushma A; Morasco, B Joan; Smerage, Lucia Eisner; Flanegan, James B

    2015-11-01

    The differential use of protein precursors and their products is a key strategy used during poliovirus replication. To characterize the role of protein precursors during replication, we examined the complementation profiles of mutants that inhibited 3D polymerase or 3C-RNA binding activity. We showed that 3D entered the replication complex in the form of its precursor, P3 (or 3CD), and was cleaved to release active 3D polymerase. Furthermore, our results showed that P3 is the preferred precursor that binds to the 5'CL. Using reciprocal complementation assays, we showed that one molecule of P3 binds the 5'CL and that a second molecule of P3 provides 3D. In addition, we showed that a second molecule of P3 served as the VPg provider. These results support a model in which P3 binds to the 5'CL and recruits additional molecules of P3, which are cleaved to release either 3D or VPg to initiate RNA replication.

  13. Dual role of TRBP in HIV replication and RNA interference: viral diversion of a cellular pathway or evasion from antiviral immunity?

    Directory of Open Access Journals (Sweden)

    Clerzius Guerline

    2005-10-01

    Full Text Available Abstract Increasing evidence indicates that RNA interference (RNAi may be used to provide antiviral immunity in mammalian cells. Human micro (miRNAs can inhibit the replication of a primate virus, whereas a virally-encoded miRNA from HIV inhibits its own replication. Indirect proof comes from RNAi suppressors encoded by mammalian viruses. Influenza NS1 and Vaccinia E3L proteins can inhibit RNAi in plants, insects and worms. HIV-1 Tat protein and Adenovirus VA RNAs act as RNAi suppressors in mammalian cells. Surprisingly, many RNAi suppressors are also inhibitors of the interferon (IFN-induced protein kinase R (PKR but the potential overlap between the RNAi and the IFN pathways remains to be determined. The link between RNAi as an immune response and the IFN pathway may be formed by a cellular protein, TRBP, which has a dual role in HIV replication and RNAi. TRBP has been isolated as an HIV-1 TAR RNA binding protein that increases HIV expression and replication by inhibiting PKR and by increasing translation of structured RNAs. A recent report published in the Journal of Virology shows that the poor replication of HIV in astrocytes is mainly due to a heightened PKR response that can be overcome by supplying TRBP exogenously. In two recent papers published in Nature and EMBO Reports, TRBP is now shown to interact with Dicer and to be required for RNAi mediated by small interfering (si and micro (miRNAs. The apparent discrepancy between TRBP requirement in RNAi and in HIV replication opens the hypotheses that RNAi may be beneficial for HIV-1 replication or that HIV-1 may evade the RNAi restriction by diverting TRBP from Dicer and use it for its own benefit.

  14. Plasma microRNA profiling of pediatric patients with immune thrombocytopenic purpura.

    Science.gov (United States)

    Bay, Ali; Coskun, Enes; Oztuzcu, Serdar; Ergun, Sercan; Yilmaz, Fatih; Aktekin, Elif

    2014-06-01

    Immune thrombocytopenic purpura (ITP) is a commonly acquired autoimmune bleeding disorder in children. MicroRNAs (miRNAs) are small RNAs which are found in cells and circulation, and play a role in protein synthesis and regulation. In this study, we aimed to determine a biomarker for childhood ITP comparing the plasma miRNA levels of children having ITP with healthy children. A total of 86 patients with ITP and 56 healthy children followed up by the Department of Pediatric Hematology and Oncology in University of Gaziantep since July 2011 were enrolled in the study. The 86 patients with ITP were evaluated in two groups as 43 acute ITP (aITP) and 43 chronic ITP (cITP) patients. Plasma expression levels of 379 miRNAs were investigated by RT-PCR (quantitative RT-PCR) technique and they were compared between aITP, cITP, and control groups. For all miRNAs, the average of raw quantification cycle values of three groups separately in the analysis chip was accepted as the reference gene value, and normalization was done according to this value. Statistically significant differences were detected in seven miRNAs (miR-302c-3p, miR-483-5p, miR-410, miR-544a, miR-302a-3p, miR-223-3p, and miR-597) investigated between the groups with respect to the expression levels. The expression rates were found to be over 95% in miR-302c-3p and miR-483-5p, over 75% in miR-410, and over 40% in miR-544, miR-302a-3p, and miR-223-3p in all three groups. The detection of significant differences between plasma miRNA levels of aITP and cITP patients and healthy children may provide useful information in the prediction of the course of disease, determination of disease etiopathogenesis, and the development of new therapeutic modalities.

  15. Preferential CTL targeting of Gag is associated with relative viral control in long-term surviving HIV-1 infected former plasma donors from China

    Institute of Scientific and Technical Information of China (English)

    Mingming Jia; Quanbi Zhao; Dan Li; Hong Peng; Marcus Altfeld; Bruce D Walker; Xu G Yu; Yiming Shao; Kunxue Hong; Jianping Chen; Yuhua Ruan; Zhe Wang; Bing Su; Guoliang Ren; Xiaoqing Zhang; Zhen Liu

    2012-01-01

    It is generally believed that CD8+ cytotoxic T lymphocytes (CTLs) play a critical role in limiting the replication of human immunodeficiency virus type 1 (HIV-1) and in determining the outcome of the infection,and this effect may partly depend on which HIV product is preferentially targeted.To address the correlation between HIV-1-specific CTL responses and virus replication in a cohort of former plasma donors (FPDs),143 antiretroviral therapy naive FPDs infected with HIV-1 clade B' strains were assessed for HIV-1-specific CTL responses with an IFN-γElispot assay at single peptide level by using overlapping peptides (OLPs) covering the whole consensus clade B proteome.By using a Spearman's rank correlation analysis,we found that the proportion of Gag-specific CTL responses among the total virus-specific CTL activity was inversely correlated with viral loads while being positively correlated to CD4 counts,as opposed to Pol- and Env-specific responses that were associated with increased viral loads and decreased CD4 counts,In addition,Vpr-specifc CTL responses showed a similar protective effect with Gag responses,but with a much lower frequency of recognition.Significantly,we also observed an association between HLA-A*30/B*13/Cw*06 haplotype and lower viral loads that was probably due to restricted Gag-specific CTL responses.Thus,our data demonstrate the prominent role of Gag-specific CTL responses in disease control.The advantage of HLA-A*30/B*13/Cw*06 haplotype in viral control may be associated with the contribution of Gag-specific CTL responses in the studied individuals.

  16. Assessment of six commercial plasma small RNA isolation kits using qRT-PCR and electrophoretic separation

    DEFF Research Database (Denmark)

    Meerson, Ari; Ploug, Thorkil

    2016-01-01

    of specific miRNAs in different samples varied considerably between the tested extraction methods. Of all kits tested, the QIAGEN miRNeasy kits (Mini and Serum/Plasma kits) and the Macherey-Nagel NucleoSpin kit produced the highest RNA yields. The QIAGEN Exo kit produced lesser yields than what could...

  17. Plasma cytokines do not reflect expression of pro- and anti-inflammatory cytokine mRNA at organ level after cardiopulmonary bypass in neonatal pigs

    DEFF Research Database (Denmark)

    Brix-Christensen, V.; Vestergaard, C.; Chew, M.

    2003-01-01

    Background: Plasma concentrations of inflammatory markers are increased in response to the trauma of cardiac surgery and cardiopulmonary bypass (CPB). It is, however, unknown whether the plasma cytokine levels and cytokine mRNA expression at organ level reflect each other. Methods: Twenty-six pig...... poorly reflected mRNA expression of the pro- and anti-inflammatory cytokines....

  18. Hepatitis E virus ORF2 protein over-expressed by baculovirus in hepatoma cells, efficiently encapsidates and transmits the viral RNA to naïve cells

    Directory of Open Access Journals (Sweden)

    Emerson Suzanne U

    2011-04-01

    Full Text Available Abstract A recombinant baculovirus(vBacORF2 that expressed the full-length ORF2 capsid protein of a genotype 1 strain of hepatitis E virus(HEV was constructed. Transduction of S10-3 human hepatoma cells with this baculovirus led to large amounts of ORF2 protein production in ~50% of the cells as determined by immune fluorescence microscopy. The majority of the ORF2 protein detected by Western blot was 72 kDa, the size expected for the full-length protein. To determine if the exogenously-supplied ORF2 protein could transencapsidate viral genomes, S10-3 cell cultures that had been transfected the previous day with an HEV replicon of genotype 1 that contained the gene for green fluorescent protein(GFP, in place of that for ORF2 protein, were transduced with the vBacORF2 virus. Cell lysates were prepared 5 days later and tested for the ability to deliver the GFP gene to HepG2/C3A cells, another human hepatoma cell line. FACS analysis indicated that lysates from cell cultures receiving only the GFP replicon were incapable of introducing the replicon into the HepG2/C3A cells whereas ~2% of the HepG2/C3A cells that received lysate from cultures that had received both the replicon and the baculovirus produced GFP. Therefore, the baculovirus-expressed ORF2 protein was able to trans-encapsidate the viral replicon and form a particle that could infect naïve HepG2/C3A cells. This ex vivo RNA packaging system should be useful for studying many aspects of HEV molecular biology.

  19. Extracting viral RNAs from plant protoplasts.

    Science.gov (United States)

    Fabian, Marc R; Andrew White, K

    2007-08-01

    The analysis of viral RNA is a fundamental aspect of plant RNA virus research. Studies that focus on viral RNAs often involve virus infections of plant protoplasts (see UNITS 16D.1-16D.4). Protoplast offer the advantage of simultaneous initiation of infections, which allows for superior temporal and quantitative analyses of viral RNAs. The efficient isolation of intact viral RNA is key to any such investigations. This unit describes two basic protocols for extracting viral RNAs from plant protoplasts. An approach for preparing double-stranded viral RNA from total RNA pools is also provided. The viral RNA prepared by using these techniques can be used for further analyses such as primer extension, reverse transcription-PCR, and northern blotting.

  20. Predictors of undetectable plasma viral load in HIV-positive adults receiving antiretroviral therapy in Southern Brazil

    Directory of Open Access Journals (Sweden)

    Marysabel Pinto Telis Silveira

    2002-08-01

    Full Text Available Factors associated with undetectable viral load ( or = 95% of adherence (CI 95% 1,80-13,28; CI 95% 1,73-9,53, compared with less than 60% adherence; it was greater for less than 6 months in treatment (OR = 3.37; CI 95% 1.09-10.46; and smaller for viral load previous to adherence measurement > or = 5.2 log10 (OR = 0.19; CI95% 0.06-0.58, adjusted for these variables and sex, age, clinical status, current immune status, group of drugs and interval between the two measurements of viral load. The crude odds were lower for age 16-24 years and use of Nucleoside Analog Reverse Transcriptase Inhibitors only, but these effects were not significant in the multivariate model. There was no evidence of effect of sex, clinical status, current immune status, and changes in treatment regimen. Treatment adherence gave the largest effect. Motivational interventions directed at adherence may improve treatment effectiveness.

  1. Viral marketing

    OpenAIRE

    Král, Jiří

    2015-01-01

    Bachelor's Thesis deals with effective promotional tools called viral marketing. The main contribution of the thesis is the definition and history of viral marketing, making analysis of process of viral marketing, progresses definition and rules for creating a viral campaign. And also aspects are necessary for a successful viral spread. There are analysis of the characteristics of social media which are dividing according to the orientation and marketing tactics. Thesis is especially about so...

  2. Signature motifs of GDP polyribonucleotidyltransferase, a non-segmented negative strand RNA viral mRNA capping enzyme, domain in the L protein are required for covalent enzyme-pRNA intermediate formation.

    Science.gov (United States)

    Neubauer, Julie; Ogino, Minako; Green, Todd J; Ogino, Tomoaki

    2016-01-01

    The unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase; block V) domain in RNA polymerase L proteins of non-segmented negative strand (NNS) RNA viruses (e.g. rabies, measles, Ebola) contains five collinear sequence elements, Rx(3)Wx(3-8)ΦxGxζx(P/A) (motif A; Φ, hydrophobic; ζ, hydrophilic), (Y/W)ΦGSxT (motif B), W (motif C), HR (motif D) and ζxxΦx(F/Y)QxxΦ (motif E). We performed site-directed mutagenesis of the L protein of vesicular stomatitis virus (VSV, a prototypic NNS RNA virus) to examine participation of these motifs in mRNA capping. Similar to the catalytic residues in motif D, G1100 in motif A, T1157 in motif B, W1188 in motif C, and F1269 and Q1270 in motif E were found to be essential or important for the PRNTase activity in the step of the covalent L-pRNA intermediate formation, but not for the GTPase activity that generates GDP (pRNA acceptor). Cap defective mutations in these residues induced termination of mRNA synthesis at position +40 followed by aberrant stop-start transcription, and abolished virus gene expression in host cells. These results suggest that the conserved motifs constitute the active site of the PRNTase domain and the L-pRNA intermediate formation followed by the cap formation is essential for successful synthesis of full-length mRNAs.

  3. Altered expression of circulating microRNA in plasma of patients with primary osteoarthritis and in silico analysis of their pathways.

    Directory of Open Access Journals (Sweden)

    Verónica M Borgonio Cuadra

    Full Text