Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

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Noisakran, Sansanee; Sengsai, Suchada; Thongboonkerd, Visith; Kanlaya, Rattiyaporn; Sinchaikul, Supachok; Chen, Shui-Tein; Puttikhunt, Chunya

2008-01-01

Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells

Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen.

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Alves, Rúbens Prince Dos Santos; Pereira, Lennon Ramos; Fabris, Denicar Lina Nascimento; Salvador, Felipe Scassi; Santos, Robert Andreata; Zanotto, Paolo Marinho de Andrade; Romano, Camila Malta; Amorim, Jaime Henrique; Ferreira, Luís Carlos de Souza

2016-06-01

Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Functional investigation of grass carp reovirus nonstructural protein NS80

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Shao Ling

2011-04-01

Full Text Available Abstract Background Grass Carp Reovirus (GCRV, a highly virulent agent of aquatic animals, has an eleven segmented dsRNA genome encased in a multilayered capsid shell, which encodes twelve proteins including seven structural proteins (VP1-VP7, and five nonstructural proteins (NS80, NS38, NS31, NS26, and NS16. It has been suggested that the protein NS80 plays an important role in the viral replication cycle that is similar to that of its homologous protein μNS in the genus of Orthoreovirus. Results As a step to understanding the basis of the part played by NS80 in GCRV replication and particle assembly, we used the yeast two-hybrid (Y2H system to identify NS80 interactions with proteins NS38, VP4, and VP6 as well as NS80 and NS38 self-interactions, while no interactions appeared in the four protein pairs NS38-VP4, NS38-VP6, VP4-VP4, and VP4-VP6. Bioinformatic analyses of NS80 with its corresponding proteins were performed with all currently available homologous protein sequences in ARVs (avian reoviruses and MRVs (mammalian reoviruses to predict further potential functional domains of NS80 that are related to VFLS (viral factory-like structures formation and other roles in viral replication. Two conserved regions spanning from aa (amino acid residues of 388 to 433, and 562 to 580 were discovered in this study. The second conserved region with corresponding conserved residues Tyr565, His569, Cys571, Asn573, and Glu576 located between the two coiled-coils regions (aa ~513-550 and aa ~615-690 in carboxyl-proximal terminus were supposed to be essential to form VFLS, so that aa residues ranging from 513 to 742 of NS80 was inferred to be the smallest region that is necessary for forming VFLS. The function of the first conserved region including Ala395, Gly419, Asp421, Pro422, Leu438, and Leu443 residues is unclear, but one-third of the amino-terminal region might be species specific, dominating interactions with other viral components. Conclusions Our

Computer Aided Screening of Phytochemicals from Garcinia against the Dengue NS2B/NS3 Protease.

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Qamar, Tahir Ul; Mumtaz, Arooj; Ashfaq, Usman Ali; Azhar, Samia; Fatima, Tabeer; Hassan, Muhammad; Hussain, Syed Sajid; Akram, Waheed; Idrees, Sobia

2014-01-01

Dengue virus NS2/NS3 protease because of its ability to cleave viral proteins is considered as an attractive target to screen antiviral agents. Medicinal plants contain a variety of phytochemicals that can be used as drug against different diseases and infections. Therefore, this study was designed to uncover possible phytochemical of different classes (Aromatic, Carbohydrates, Lignin, Saponins, Steroids, Tannins, Terpenoids, Xanthones) that could be used as inhibitors against the NS2B/NS3 protease of DENV. With the help of molecular docking, Garcinia phytochemicals found to be bound deeply inside the active site of DENV NS2B/NS3 protease among all tested phytochemicals and had interactions with catalytic triad (His51, Asp75, Ser135). Thus, it can be concluded from the study that these Gracinia phytochemicals could serve as important inhibitors to inhibit the viral replication inside the host cell. Further in-vitro investigations require confirming their efficacy.

Infection of Common Marmosets with GB Virus B Chimeric Virus Encoding the Major Nonstructural Proteins NS2 to NS4A of Hepatitis C Virus.

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Zhu, Shaomei; Li, Tingting; Liu, Bochao; Xu, Yuxia; Sun, Yachun; Wang, Yilin; Wang, Yuanzhan; Shuai, Lifang; Chen, Zixuan; Allain, Jean-Pierre; Li, Chengyao

2016-09-15

A lack of immunocompetent-small-primate models has been an obstacle for developing hepatitis C virus (HCV) vaccines and affordable antiviral drugs. In this study, HCV/GB virus B (GBV-B) chimeric virus carrying the major nonstructural proteins NS2 to NS4A (HCV NS2 to -4A chimera) was produced and used to infect common marmosets, since HCV NS2 to NS4A proteins are critical proteases and major antigens. Seven marmosets were inoculated intrahepatically with HCV NS2 to -4A chimera RNA for primary infection or intravenously injected with chimera-containing serum for passage infection. Three animals used as controls were injected with phosphate-buffered saline (PBS) or GBV-B, respectively. Six of seven HCV NS2 to -4A chimera-infected marmosets exhibited consistent viremia and one showed transient viremia during the course of follow-up detection. All six infected animals with persistent circulating viremia presented characteristics typical of viral hepatitis, including viral RNA and proteins in hepatocytes and histopathological changes in liver tissue. Viremia was consistently detected for 5 to 54 weeks of follow-up. FK506 immunosuppression facilitated the establishment of persistent chimera infection in marmosets. An animal with chimera infection spontaneously cleared the virus in blood 7 weeks following the first inoculation, but viral-RNA persistence, low-level viral protein, and mild necroinflammation remained in liver tissue. The specific antibody and T-cell response to HCV NS3 in this viremia-resolved marmoset was boosted by rechallenging, but no viremia was detected during 57 weeks of follow-up. The chimera-infected marmosets described can be used as a suitable small-primate animal model for studying novel antiviral drugs and T-cell-based vaccines against HCV infection. HCV infection causes approximately 70% of chronic hepatitis and is frequently associated with primary liver cancer globally. Chimpanzees have been used as a reliable primate model for HCV infection

Specific Mutations in the PB2 Protein of Influenza A Virus Compensate for the Lack of Efficient Interferon Antagonism of the NS1 Protein of Bat Influenza A-Like Viruses.

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Aydillo, Teresa; Ayllon, Juan; Pavlisin, Amzie; Martinez-Romero, Carles; Tripathi, Shashank; Mena, Ignacio; Moreira-Soto, Andrés; Vicente-Santos, Amanda; Corrales-Aguilar, Eugenia; Schwemmle, Martin; García-Sastre, Adolfo

2018-04-01

Recently, two new influenza A-like viruses have been discovered in bats, A/little yellow-shouldered bat/Guatemala/060/2010 (HL17NL10) and A/flat-faced bat/Peru/033/2010 (HL18NL11). The hemagglutinin (HA)-like (HL) and neuraminidase (NA)-like (NL) proteins of these viruses lack hemagglutination and neuraminidase activities, despite their sequence and structural homologies with the HA and NA proteins of conventional influenza A viruses. We have now investigated whether the NS1 proteins of the HL17NL10 and HL18NL11 viruses can functionally replace the NS1 protein of a conventional influenza A virus. For this purpose, we generated recombinant influenza A/Puerto Rico/8/1934 (PR8) H1N1 viruses containing the NS1 protein of the PR8 wild-type, HL17NL10, and HL18NL11 viruses. These viruses (r/NS1PR8, r/NS1HL17, and r/NS1HL18, respectively) were tested for replication in bat and nonbat mammalian cells and in mice. Our results demonstrate that the r/NS1HL17 and r/NS1HL18 viruses are attenuated in vitro and in vivo However, the bat NS1 recombinant viruses showed a phenotype similar to that of the r/NS1PR8 virus in STAT1 -/- human A549 cells and mice, both in vitro and in vivo systems being unable to respond to interferon (IFN). Interestingly, multiple mouse passages of the r/NS1HL17 and r/NS1HL18 viruses resulted in selection of mutant viruses containing single amino acid mutations in the viral PB2 protein. In contrast to the parental viruses, virulence and IFN antagonism were restored in the selected PB2 mutants. Our results indicate that the NS1 protein of bat influenza A-like viruses is less efficient than the NS1 protein of its conventional influenza A virus NS1 counterpart in antagonizing the IFN response and that this deficiency can be overcome by the influenza virus PB2 protein. IMPORTANCE Significant gaps in our understanding of the basic features of the recently discovered bat influenza A-like viruses HL17NL10 and HL18NL11 remain. The basic biology of these unique

Hepatitis C virus NS5A protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins A and B.

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Hanoulle, Xavier; Badillo, Aurélie; Wieruszeski, Jean-Michel; Verdegem, Dries; Landrieu, Isabelle; Bartenschlager, Ralf; Penin, François; Lippens, Guy

2009-05-15

We report here a biochemical and structural characterization of domain 2 of the nonstructural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy, and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to cyclosporin A resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both cyclophilins. The interaction surface on the cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved to be distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB.

Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3.

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Zheng, Fengwei; Lu, Guoliang; Li, Ling; Gong, Peng; Pan, Zishu

2017-11-01

The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å 2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis -cleavage event. Although this conformation is incompatible with protease trans -cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis -cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different

Activation of ERG2 potassium channels by the diphenylurea NS1643

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Elmedyb, Pernille; Olesen, Søren-Peter; Grunnet, Morten

2007-01-01

Three members of the ERG potassium channel family have been described (ERG1-3 or Kv 11.1-3). ERG1 is by far the best characterized subtype and it constitutes the molecular component of the cardiac I(Kr) current. All three channel subtypes are expressed in neurons but their function remains unclear....... The lack of functional information is at least partly due to the lack of specific pharmacological tools. The compound NS1643 has earlier been reported as an ERG1 channel activator. We found that NS1643 also activates the ERG2 channel; however, the molecular mechanism of the activation differs between...... the ERG1 and ERG2 channels. This is surprising since ERG1 and ERG2 channels have very similar biophysical and structural characteristics. For ERG2, NS1643 causes a left-ward shift of the activation curve, a faster time-constant of activation and a slower time-constant of inactivation as well...

Crystal structure of full-length Zika virus NS5 protein reveals a conformation similar to Japanese encephalitis virus NS5

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Upadhyay, Anup K.; Cyr, Matthew; Longenecker, Kenton; Tripathi, Rakesh; Sun, Chaohong; Kempf, Dale J. (AbbVie)

2017-02-21

The rapid spread of the recentZika virus(ZIKV) epidemic across various countries in the American continent poses a major health hazard for the unborn fetuses of pregnant women. To date, there is no effective medical intervention. The nonstructural protein 5 ofZika virus(ZIKV-NS5) is critical for ZIKV replication through the 5'-RNA capping and RNA polymerase activities present in its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent RNA polymerase (RdRp) domains, respectively. The crystal structure of the full-length ZIKV-NS5 protein has been determined at 3.05 Å resolution from a crystal belonging to space groupP2₁2₁2 and containing two protein molecules in the asymmetric unit. The structure is similar to that reported for the NS5 protein fromJapanese encephalitis virusand suggests opportunities for structure-based drug design targeting either its MTase or RdRp domain.

Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen

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Canard Bruno

2011-10-01

Full Text Available Abstract Background The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4. Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. Results We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. Conclusions We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy.

Heat shock protein 90 positively regulates Chikungunya virus replication by stabilizing viral non-structural protein nsP2 during infection.

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Indrani Das

Full Text Available BACKGROUND: The high morbidity and socio-economic loss associated with the recent massive global outbreak of Chikungunya virus (CHIKV emphasize the need to understand the biology of the virus for developing effective antiviral therapies. METHODS AND FINDINGS: In this study, an attempt was made to understand the molecular mechanism involved in Heat shock protein 90 (Hsp90 mediated regulation of CHIKV infection in mammalian cells using CHIKV prototype strain (S 27 and Indian outbreak strain of 2006 (DRDE-06. Our results showed that Hsp90 is required at a very early stage of viral replication and Hsp90 inhibitor Geldanamycin (GA can abrogate new virus particle formation more effectively in the case of S 27 than that of DRDE-06. Further analysis revealed that CHIKV nsP2 protein level is specifically reduced by GA treatment as well as HSP90-siRNA transfection; however, viral RNA remains unaltered. Immunoprecipitation analysis showed that nsP2 interacts with Hsp90 during infection; however this interaction is reduced in the presence of GA. In addition, our analysis on Hsp90 associated PI3K/Akt/mTOR signaling pathway demonstrated that CHIKV infection stabilizes Raf1 and activates Hsp90 client protein Akt, which in turn phosphorylates mTOR. Subsequently, this phosphorylation leads to the activation of two important downstream effectors, S6K and 4EBP1, which may facilitate translation of viral as well as cellular mRNAs. Hence, the data suggests that CHIKV infection is regulated by Hsp90 associated Akt phosphorylation and DRDE-06 is more efficient than S 27 in enhancing the activation of host signaling molecules for its efficient replication and virus production. CONCLUSION: Hsp90 positively regulates Chikungunya virus replication by stabilizing CHIKV-nsP2 through its interaction during infection. The study highlights the possible molecular mechanism of GA mediated inhibition of CHIKV replication and differential effect of this drug on S 27 and DRDE-06

Noroviruses Co-opt the Function of Host Proteins VAPA and VAPB for Replication via a Phenylalanine-Phenylalanine-Acidic-Tract-Motif Mimic in Nonstructural Viral Protein NS1/2.

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McCune, Broc T; Tang, Wei; Lu, Jia; Eaglesham, James B; Thorne, Lucy; Mayer, Anne E; Condiff, Emily; Nice, Timothy J; Goodfellow, Ian; Krezel, Andrzej M; Virgin, Herbert W

2017-07-11

The Norovirus genus contains important human pathogens, but the role of host pathways in norovirus replication is largely unknown. Murine noroviruses provide the opportunity to study norovirus replication in cell culture and in small animals. The human norovirus nonstructural protein NS1/2 interacts with the host protein VAMP-associated protein A (VAPA), but the significance of the NS1/2-VAPA interaction is unexplored. Here we report decreased murine norovirus replication in VAPA- and VAPB-deficient cells. We characterized the role of VAPA in detail. VAPA was required for the efficiency of a step(s) in the viral replication cycle after entry of viral RNA into the cytoplasm but before the synthesis of viral minus-sense RNA. The interaction of VAPA with viral NS1/2 proteins is conserved between murine and human noroviruses. Murine norovirus NS1/2 directly bound the major sperm protein (MSP) domain of VAPA through its NS1 domain. Mutations within NS1 that disrupted interaction with VAPA inhibited viral replication. Structural analysis revealed that the viral NS1 domain contains a mimic of the phenylalanine-phenylalanine-acidic-tract (FFAT) motif that enables host proteins to bind to the VAPA MSP domain. The NS1/2-FFAT mimic region interacted with the VAPA-MSP domain in a manner similar to that seen with bona fide host FFAT motifs. Amino acids in the FFAT mimic region of the NS1 domain that are important for viral replication are highly conserved across murine norovirus strains. Thus, VAPA interaction with a norovirus protein that functionally mimics host FFAT motifs is important for murine norovirus replication. IMPORTANCE Human noroviruses are a leading cause of gastroenteritis worldwide, but host factors involved in norovirus replication are incompletely understood. Murine noroviruses have been studied to define mechanisms of norovirus replication. Here we defined the importance of the interaction between the hitherto poorly studied NS1/2 norovirus protein and the

Poly (I:C) enhances the anti-tumor activity of canine parvovirus NS1 protein by inducing a potent anti-tumor immune response.

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Gupta, Shishir Kumar; Yadav, Pavan Kumar; Tiwari, A K; Gandham, Ravi Kumar; Sahoo, A P

2016-09-01

The canine parvovirus NS1 (CPV2.NS1) protein selectively induces apoptosis in the malignant cells. However, for an effective in vivo tumor treatment strategy, an oncolytic agent also needs to induce a potent anti-tumor immune response. In the present study, we used poly (I:C), a TLR3 ligand, as an adjuvant along with CPV2.NS1 to find out if the combination can enhance the oncolytic activity by inducing a potent anti-tumor immune response. The 4T1 mammary carcinoma cells were used to induce mammary tumor in Balb/c mice. The results suggested that poly (I:C), when given along with CPV2.NS1, not only significantly reduced the tumor growth but also augmented the immune response against tumor antigen(s) as indicated by the increase in blood CD4+ and CD8+ counts and infiltration of immune cells in the tumor tissue. Further, blood serum analysis of the cytokines revealed that Th1 cytokines (IFN-γ and IL-2) were significantly upregulated in the treatment group indicating activation of cell-mediated immune response. The present study reports the efficacy of CPV2.NS1 along with poly (I:C) not only in inhibiting the mammary tumor growth but also in generating an active anti-tumor immune response without any visible toxicity. The results of our study may help in developing CPV2.NS1 and poly (I: C) combination as a cancer therapeutic regime to treat various malignancies.

Epitope Sequences in Dengue Virus NS1 Protein Identified by Monoclonal Antibodies

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Leticia Barboza Rocha

2017-10-01

Full Text Available Dengue nonstructural protein 1 (NS1 is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal antibodies against recombinant NS1 protein from dengue virus serotype 2 (DENV2, which were used to map three NS1 epitopes. The sequence 193AVHADMGYWIESALNDT209 was recognized by monoclonal antibodies 2H5 and 4H1BC, which also cross-reacted with Zika virus (ZIKV protein. On the other hand, the sequence 25VHTWTEQYKFQPES38 was recognized by mAb 4F6 that did not cross react with ZIKV. Lastly, a previously unidentified DENV2 NS1-specific epitope, represented by the sequence 127ELHNQTFLIDGPETAEC143, is described in the present study after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections.

Dengue Virus NS2B/NS3 Protease Inhibitors Exploiting the Prime Side.

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Lin, Kuan-Hung; Ali, Akbar; Rusere, Linah; Soumana, Djade I; Kurt Yilmaz, Nese; Schiffer, Celia A

2017-05-15

The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity for DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue virus protease. The design was based on two aprotinin loops and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin's high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a K i value of 2.9 μM against DENV3 wild-type (WT) protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV. IMPORTANCE Viruses of the flaviviral family, including DENV and Zika virus transmitted by Aedes aegypti , continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors

Unexpected Functional Divergence of Bat Influenza Virus NS1 Proteins.

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Turkington, Hannah L; Juozapaitis, Mindaugas; Tsolakos, Nikos; Corrales-Aguilar, Eugenia; Schwemmle, Martin; Hale, Benjamin G

2018-03-01

Recently, two influenza A virus (FLUAV) genomes were identified in Central and South American bats. These sequences exhibit notable divergence from classical FLUAV counterparts, and functionally, bat FLUAV glycoproteins lack canonical receptor binding and destroying activity. Nevertheless, other features that distinguish these viruses from classical FLUAVs have yet to be explored. Here, we studied the viral nonstructural protein NS1, a virulence factor that modulates host signaling to promote efficient propagation. Like all FLUAV NS1 proteins, bat FLUAV NS1s bind double-stranded RNA and act as interferon antagonists. Unexpectedly, we found that bat FLUAV NS1s are unique in being unable to bind host p85β, a regulatory subunit of the cellular metabolism-regulating enzyme, phosphoinositide 3-kinase (PI3K). Furthermore, neither bat FLUAV NS1 alone nor infection with a chimeric bat FLUAV efficiently activates Akt, a PI3K effector. Structure-guided mutagenesis revealed that the bat FLUAV NS1-p85β interaction can be reengineered (in a strain-specific manner) by changing two to four NS1 residues (96L, 99M, 100I, and 145T), thereby creating a hydrophobic patch. Notably, ameliorated p85β-binding is insufficient for bat FLUAV NS1 to activate PI3K, and a chimeric bat FLUAV expressing NS1 with engineered hydrophobic patch mutations exhibits cell-type-dependent, but species-independent, propagation phenotypes. We hypothesize that bat FLUAV hijacking of PI3K in the natural bat host has been selected against, perhaps because genes in this metabolic pathway were differentially shaped by evolution to suit the unique energy use strategies of this flying mammal. These data expand our understanding of the enigmatic functional divergence between bat FLUAVs and classical mammalian and avian FLUAVs. IMPORTANCE The potential for novel influenza A viruses to establish infections in humans from animals is a source of continuous concern due to possible severe outbreaks or pandemics. The

Dimerization site 2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation.

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Yamanaka, Yuki; Winardhi, Ricksen S; Yamauchi, Erika; Nishiyama, So-Ichiro; Sowa, Yoshiyuki; Yan, Jie; Kawagishi, Ikuro; Ishihama, Akira; Yamamoto, Kaneyoshi

2018-06-15

The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Parvovirus B19 NS1 protein induces cell cycle arrest at G2-phase by activating the ATR-CDC25C-CDK1 pathway.

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Peng Xu

2017-03-01

Full Text Available Human parvovirus B19 (B19V infection of primary human erythroid progenitor cells (EPCs arrests infected cells at both late S-phase and G2-phase, which contain 4N DNA. B19V infection induces a DNA damage response (DDR that facilitates viral DNA replication but is dispensable for cell cycle arrest at G2-phase; however, a putative C-terminal transactivation domain (TAD2 within NS1 is responsible for G2-phase arrest. To fully understand the mechanism underlying B19V NS1-induced G2-phase arrest, we established two doxycycline-inducible B19V-permissive UT7/Epo-S1 cell lines that express NS1 or NS1mTAD2, and examined the function of the TAD2 domain during G2-phase arrest. The results confirm that the NS1 TAD2 domain plays a pivotal role in NS1-induced G2-phase arrest. Mechanistically, NS1 transactivated cellular gene expression through the TAD2 domain, which was itself responsible for ATR (ataxia-telangiectasia mutated and Rad3-related activation. Activated ATR phosphorylated CDC25C at serine 216, which in turn inactivated the cyclin B/CDK1 complex without affecting nuclear import of the complex. Importantly, we found that the ATR-CHK1-CDC25C-CDK1 pathway was activated during B19V infection of EPCs, and that ATR activation played an important role in B19V infection-induced G2-phase arrest.

Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A

DEFF Research Database (Denmark)

Gottwein, Judith M; Jensen, Tanja B; Mathiesen, Christian K

2011-01-01

to 2a(J6) tagged with EGFP, DsRed-Express2, mCherry, or Renilla luciferase (RLuc), yielding peak supernatant infectivity titers of 4 to 5 log(10) focus-forming units (FFU)/ml. 2a(J6) with ¿40 or ¿25 was fully viable in Huh7.5 cells. In human liver chimeric mice, 2a(J6)-EGFP¿40 acquired various...... deletions in EGFP, while 2a(J6)¿40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5A¿40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those...

The effect of glycosylation on cytotoxicity of Ibaraki virus nonstructural protein NS3

Science.gov (United States)

URATA, Maho; WATANABE, Rie; IWATA, Hiroyuki

2015-01-01

The cytotoxicity of Ibaraki virus nonstructural protein NS3 was confirmed, and the contribution of glycosylation to this activity was examined by using glycosylation mutants of NS3 generated by site-directed mutagenesis. The expression of NS3 resulted in leakage of lactate dehydrogenase to the culture supernatant, suggesting the cytotoxicity of this protein. The lack of glycosylation impaired the transport of NS3 to the plasma membrane and resulted in reduced cytotoxicity. Combined with the previous observation that NS3 glycosylation was specifically observed in mammalian cells (Urata et al., Virus Research 2014), it was suggested that the alteration of NS3 cytotoxicity through modulating glycosylation is one of the strategies to achieve host specific pathogenisity of Ibaraki virus between mammals and vector arthropods. PMID:26178820

HCV NS5A protein containing potential ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src family kinase Fyn in B cells.

Science.gov (United States)

Nakashima, Kenji; Takeuchi, Kenji; Chihara, Kazuyasu; Horiguchi, Tomoko; Sun, Xuedong; Deng, Lin; Shoji, Ikuo; Hotta, Hak; Sada, Kiyonao

2012-01-01

Hepatitis C virus (HCV) infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A) protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV) implied that NS5A was tyrosine phosphorylated by pervanadate (PV) treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST)-fusion proteins of various Src homology 2 (SH2) domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3) domain. Substitution of Arg(176) to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr(334) was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.

HCV NS5A protein containing potential ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src family kinase Fyn in B cells.

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Kenji Nakashima

Full Text Available Hepatitis C virus (HCV infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV implied that NS5A was tyrosine phosphorylated by pervanadate (PV treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST-fusion proteins of various Src homology 2 (SH2 domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3 domain. Substitution of Arg(176 to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr(334 was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.

Construction and analysis of a plant non-specific lipid transfer protein database (nsLTPDB).

Science.gov (United States)

Wang, Nai-Jyuan; Lee, Chi-Ching; Cheng, Chao-Sheng; Lo, Wei-Cheng; Yang, Ya-Fen; Chen, Ming-Nan; Lyu, Ping-Chiang

2012-01-01

Plant non-specific lipid transfer proteins (nsLTPs) are small and basic proteins. Recently, nsLTPs have been reported involved in many physiological functions such as mediating phospholipid transfer, participating in plant defence activity against bacterial and fungal pathogens, and enhancing cell wall extension in tobacco. However, the lipid transfer mechanism of nsLTPs is still unclear, and comprehensive information of nsLTPs is difficult to obtain. In this study, we identified 595 nsLTPs from 121 different species and constructed an nsLTPs database--nsLTPDB--which comprises the sequence information, structures, relevant literatures, and biological data of all plant nsLTPs http://nsltpdb.life.nthu.edu.tw/. Meanwhile, bioinformatics and statistics methods were implemented to develop a classification method for nsLTPs based on the patterns of the eight highly-conserved cysteine residues, and to suggest strict Prosite-styled patterns for Type I and Type II nsLTPs. The pattern of Type I is C X2 V X5-7 C [V, L, I] × Y [L, A, V] X8-13 CC × G X12 D × [Q, K, R] X2 CXC X16-21 P X2 C X13-15C, and that of Type II is C X4 L X2 C X9-11 P [S, T] X2 CC X5 Q X2-4 C[L, F]C X2 [A, L, I] × [D, N] P X10-12 [K, R] X4-5 C X3-4 P X0-2 C. Moreover, we referred the Prosite-styled patterns to the experimental mutagenesis data that previously established by our group, and found that the residues with higher conservation played an important role in the structural stability or lipid binding ability of nsLTPs. Taken together, this research has suggested potential residues that might be essential to modulate the structural and functional properties of plant nsLTPs. Finally, we proposed some biologically important sites of the nsLTPs, which are described by using a new Prosite-styled pattern that we defined.

Construction and analysis of a plant non-specific lipid transfer protein database (nsLTPDB

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Wang Nai-Jyuan

2012-01-01

Full Text Available Abstract Background Plant non-specific lipid transfer proteins (nsLTPs are small and basic proteins. Recently, nsLTPs have been reported involved in many physiological functions such as mediating phospholipid transfer, participating in plant defence activity against bacterial and fungal pathogens, and enhancing cell wall extension in tobacco. However, the lipid transfer mechanism of nsLTPs is still unclear, and comprehensive information of nsLTPs is difficult to obtain. Methods In this study, we identified 595 nsLTPs from 121 different species and constructed an nsLTPs database -- nsLTPDB -- which comprises the sequence information, structures, relevant literatures, and biological data of all plant nsLTPs http://nsltpdb.life.nthu.edu.tw/. Results Meanwhile, bioinformatics and statistics methods were implemented to develop a classification method for nsLTPs based on the patterns of the eight highly-conserved cysteine residues, and to suggest strict Prosite-styled patterns for Type I and Type II nsLTPs. The pattern of Type I is C X2 V X5-7 C [V, L, I] × Y [L, A, V] X8-13 CC × G X12 D × [Q, K, R] X2 CXC X16-21 P X2 C X13-15C, and that of Type II is C X4 L X2 C X9-11 P [S, T] X2 CC X5 Q X2-4 C[L, F]C X2 [A, L, I] × [D, N] P X10-12 [K, R] X4-5 C X3-4 P X0-2 C. Moreover, we referred the Prosite-styled patterns to the experimental mutagenesis data that previously established by our group, and found that the residues with higher conservation played an important role in the structural stability or lipid binding ability of nsLTPs. Conclusions Taken together, this research has suggested potential residues that might be essential to modulate the structural and functional properties of plant nsLTPs. Finally, we proposed some biologically important sites of the nsLTPs, which are described by using a new Prosite-styled pattern that we defined.

Influenza A H3N2 subtype virus NS1 protein targets into the nucleus and binds primarily via its C-terminal NLS2/NoLS to nucleolin and fibrillarin

Science.gov (United States)

2012-01-01

Background Influenza A virus non-structural protein 1 (NS1) is a virulence factor, which is targeted into the cell cytoplasm, nucleus and nucleolus. NS1 is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. Previously, we have shown that the NS1 protein of the H3N2 subtype influenza viruses possesses a C-terminal nuclear localization signal (NLS) that also functions as a nucleolar localization signal (NoLS) and targets the protein into the nucleolus. Results Here, we show that the NS1 protein of the human H3N2 virus subtype interacts in vitro primarily via its C-terminal NLS2/NoLS and to a minor extent via its N-terminal NLS1 with the nucleolar proteins, nucleolin and fibrillarin. Using chimeric green fluorescence protein (GFP)-NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions. Conclusion NS1 protein of the human H3N2 virus interacts primarily via the C-terminal NLS2/NoLS and to a minor extent via the N-terminal NLS1 with the main nucleolar proteins, nucleolin, B23 and fibrillarin. PMID:22909121

Human Parvovirus B19 NS1 Protein Aggravates Liver Injury in NZB/W F1 Mice

Science.gov (United States)

Tsai, Chun-Chou; Chiu, Chun-Ching; Hsu, Jeng-Dong; Hsu, Huai-Sheng; Tzang, Bor-Show; Hsu, Tsai-Ching

2013-01-01

Human parvovirus B19 (B19) has been associated with a variety of diseases. However, the influence of B19 viral proteins on hepatic injury in SLE is still obscure. To elucidate the effects of B19 viral proteins on livers in SLE, recombinant B19 NS1, VP1u or VP2 proteins were injected subcutaneously into NZB/W F1 mice, respectively. Significant expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected in NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Markedly hepatocyte disarray and lymphocyte infiltration were observed in livers from NZB/WF 1 mice receiving B19 NS1 as compared to those mice receiving PBS. Additionally, significant increases of Tumor Necrosis Factor –α (TNF-α), TNF-α receptor, IκB kinase –α (IKK-α), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) and nuclear factor-kappa B (NF-κB) were detected in livers from NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Accordingly, significant increases of matrix metalloproteinase-9 (MMP9) and U-plasminogen activator (uPA) were also detected in livers from NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Contrarily, no significant variation on livers from NZB/W F1 mice receiving B19 VP1u or VP2 was observed as compared to those mice receiving PBS. These findings firstly demonstrated the aggravated effects of B19 NS1 but not VP1u or VP2 protein on hepatic injury and provide a clue in understanding the role of B19 NS1 on hepatic injury in SLE. PMID:23555760

Proliferation of NS0 cells in protein-free medium: the role of cell-derived proteins, known growth factors and cellular receptors.

Science.gov (United States)

Spens, Erika; Häggström, Lena

2009-05-20

NS0 cells proliferate without external supply of growth factors in protein-free media. We hypothesize that the cells produce their own factors to support proliferation. Understanding the mechanisms behind this autocrine regulation of proliferation may open for the novel approaches to improve animal cell processes. The following proteins were identified in NS0 conditioned medium (CM): cyclophilin A, cyclophilin B (CypB), cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerase, macrophage migration inhibitory factor (MIF), beta(2)-microglobulin, Niemann pick type C2, secretory leukocyte protease inhibitor, thioredoxin-1, TNF-alpha, tumour protein translationally controlled 1 and ubiquitin. Further, cDNA microarray analysis indicated that the genes for IL-11, TNF receptor 6, TGF-beta receptor 1 and the IFN-gamma receptor were transcribed. CypB, IFN-alpha/beta/gamma, IL-11, IL-25, MIF, TGF-beta and TNF-alpha as well as the known growth factors EGF, IGF-I/II, IL-6, leukaemia inhibitory factor and oncostatin M (OSM) were excluded as involved in autocrine regulation of NS0 cell proliferation. The receptors for TGF-beta, IGF and OSM are however present in NS0 cell membranes since TGF-beta(1) caused cell death, and IGF-I/II and OSM improved cell growth. Even though no ligand was found, the receptor subunit gp130, active in signal transduction of the IL-6 like proteins, was shown to be essential for NS0 cells as demonstrated by siRNA gene silencing.

A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease.

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Matthew Brecher

2017-05-01

Full Text Available The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2 in vitro, with IC50 values of 1.8 μM, 11.4 μM, and 4.8 μM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV, West Nile virus (WNV, and Yellow fever virus (YFV on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 μM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and

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.

Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes.

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Suzich, J A; Tamura, J K; Palmer-Hill, F; Warrener, P; Grakoui, A; Rice, C M; Feinstone, S M; Collett, M S

1993-01-01

Sequence motifs within the nonstructural protein NS3 of members of the Flaviviridae family suggest that this protein possesses nucleoside triphosphatase (NTPase) and RNA helicase activity. The RNA-stimulated NTPase activity of this protein from prototypic members of the Pestivirus and Flavivirus genera has recently been established and enzymologically characterized. Here, we experimentally demonstrate that the NS3 protein from a member of the third genus of Flaviviridae, human hepatitis C virus (HCV), also possesses a polynucleotide-stimulated NTPase activity. Characterization of the purified HCV NTPase activity showed that it exhibited reaction condition optima with respect to pH, MgCl2, and salt identical to those of the representative pestivirus and flavivirus enzymes. However, each NTPase also possessed several unique properties when compared with one another. Notably, the profile of polynucleotide stimulation of the NTPase activity was distinct for the three enzymes. The HCV NTPase was the only one whose activity was significantly enhanced by a deoxyribopolynucleotide. Additional distinguishing features among the three enzymes relating to the kinetic properties of their NTPase activities are discussed. These studies provide a foundation for investigation of the putative RNA helicase activity of these proteins and for further study of the role of the NS3 proteins of members of the Flaviviridae in the replication cycle of these viruses. Images PMID:8396675

PBDE: Structure-Activity Studies for the Inhibition of Hepatitis C Virus NS3 Helicase

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Kazi Abdus Salam

2014-04-01

Full Text Available The helicase portion of the hepatitis C virus nonstructural protein 3 (NS3 is considered one of the most validated targets for developing direct acting antiviral agents. We isolated polybrominated diphenyl ether (PBDE 1 from a marine sponge as an NS3 helicase inhibitor. In this study, we evaluated the inhibitory effects of PBDE (1 on the essential activities of NS3 protein such as RNA helicase, ATPase, and RNA binding activities. The structure-activity relationship analysis of PBDE (1 against the HCV ATPase revealed that the biphenyl ring, bromine, and phenolic hydroxyl group on the benzene backbone might be a basic scaffold for the inhibitory potency.

Comparative study and grouping of nonstructural (NS1)proteins of influenza A viruses by the method of oligopeptide mapping

International Nuclear Information System (INIS)

Sokolov, B.P.; Rudneva, I.A.; Zhdanov, V.M.

1983-01-01

Oligopeptide mapping of 35 S-methionine labeled non-stuctural (NS1) proteins of 23 influenza A virus strains showed the presence of both common and variable oligopeptides. Analysis of the oligopeptide maps revealed at least four groups of NS1 proteins. The first group includes NS1 proteins of several human H1N1 influenza viruses (that were designated as H0N1 according to the old classification). The second group is composed of NS1 proteins of H1N1 and H2N2 viruses. The third group includes NS1 proteins of H3N2 human influenza viruses. The fourth group is composed of NS1 proteins of five avian influenza viruses and an equine (H3N8) influenza virus. Two animal influenza viruses A/equi/Prague/56 (H7N7) and A/duck/England/56 (H11N6) contain NS1 proteins that belong to the second group. (Author)

The T4 Phage DNA Mimic Protein Arn Inhibits the DNA Binding Activity of the Bacterial Histone-like Protein H-NS*

Science.gov (United States)

Ho, Chun-Han; Wang, Hao-Ching; Ko, Tzu-Ping; Chang, Yuan-Chih; Wang, Andrew H.-J.

2014-01-01

The T4 phage protein Arn (Anti restriction nuclease) was identified as an inhibitor of the restriction enzyme McrBC. However, until now its molecular mechanism remained unclear. In the present study we used structural approaches to investigate biological properties of Arn. A structural analysis of Arn revealed that its shape and negative charge distribution are similar to dsDNA, suggesting that this protein could act as a DNA mimic. In a subsequent proteomic analysis, we found that the bacterial histone-like protein H-NS interacts with Arn, implying a new function. An electrophoretic mobility shift assay showed that Arn prevents H-NS from binding to the Escherichia coli hns and T4 p8.1 promoters. In vitro gene expression and electron microscopy analyses also indicated that Arn counteracts the gene-silencing effect of H-NS on a reporter gene. Because McrBC and H-NS both participate in the host defense system, our findings suggest that T4 Arn might knock down these mechanisms using its DNA mimicking properties. PMID:25118281

Detection of antibodies against porcine parvovirus nonstructural protein NS1 may distinguish between vaccinated and infected pigs

DEFF Research Database (Denmark)

Madsen, Eva Smedegaard; Madsen, Knud Gert; Nielsen, Jens

1997-01-01

The humoral antibody response against the nonstructural protein NS1 and the structural protein VP2 of porcine parvovirus (PPV) was evaluated by immuno-peroxidase test (IPT) and enzyme linked immune sorbent assay (ELISA) using recombinant PPV antigens. The coding sequence for NS1 and VP2...... was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) genome resulting in two recombinant baculoviruses AcNPV-NS1 and AcNPV-VP2, respectively. Sf9 cells (Spodoptora frugidiperda) inoculated with AcNPV-NS1 producing recombinant nonstructural protein (rNS1) and AcNPV-VP2...... producing recombinant virion protein (rVP2) were used in IPT and ELISA to analyse serum antibodies. Pigs vaccinated with an inactivated whole virus vaccine and experimentally infected pigs were studied. Significant titers against rVP2 were obtained in both vaccinated and infected pigs. Specific antibodies...

Small-angle X-Ray analysis of macromolecular structure: the structure of protein NS2 (NEP) in solution

Science.gov (United States)

Shtykova, E. V.; Bogacheva, E. N.; Dadinova, L. A.; Jeffries, C. M.; Fedorova, N. V.; Golovko, A. O.; Baratova, L. A.; Batishchev, O. V.

2017-11-01

A complex structural analysis of nuclear export protein NS2 (NEP) of influenza virus A has been performed using bioinformatics predictive methods and small-angle X-ray scattering data. The behavior of NEP molecules in a solution (their aggregation, oligomerization, and dissociation, depending on the buffer composition) has been investigated. It was shown that stable associates are formed even in a conventional aqueous salt solution at physiological pH value. For the first time we have managed to get NEP dimers in solution, to analyze their structure, and to compare the models obtained using the method of the molecular tectonics with the spatial protein structure predicted by us using the bioinformatics methods. The results of the study provide a new insight into the structural features of nuclear export protein NS2 (NEP) of the influenza virus A, which is very important for viral infection development.

2′-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

Science.gov (United States)

Dong, Hongping; Chang, David C.; Hua, Maggie Ho Chia; Lim, Siew Pheng; Chionh, Yok Hian; Hia, Fabian; Lee, Yie Hou; Kukkaro, Petra; Lok, Shee-Mei; Dedon, Peter C.; Shi, Pei-Yong

2012-01-01

RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′-O methyltransferase activities that are required for the formation of 5′ type I cap (m7GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N6-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of

Docking, synthesis and bioassay studies of imine derivatives as potential inhibitors for dengue NS2B/ NS3 serine protease

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Neni Frimayanti

2017-11-01

Full Text Available Objective: To search imine derivatives as new active agents against dengue type 2 NS2B/NS3 using molecular docking, since there is no effective vaccine against flaviviral infections. Methods: In this research, molecular docking was performed for a series of imine derivatives and the information obtained from the docking studies was used to explore the binding modes of these imine derivatives with dengue type 2 NS2B/NS3 serine protease. A set of imine were synthesized and bioassay study of the inhibitory activities of these compounds was then performed. Results: The results indicated that MY8 and MY4 have the ability to inhibit DEN2 NS2B/NS3 proteolytic activity. Conclusions: These two compounds were chosen as the reference for the next stage in drug design as new inhibitor agents against NS2B/NS3.

Molecular and biochemical characterization of the NS1 protein of non-cultured influenza B virus strains circulating in Singapore

KAUST Repository

Jumat, Muhammad; Sugrue, Richard J.; Tan, Boon Huan; Maurer-Stroh, Sebastian; Lee, Raphael Tze Chuen; Wong, Puisan

2016-01-01

In this study we compared the NS1 protein of Influenza B/Lee/40 and several non-cultured Influenza B virus clinical strains detected in Singapore. In B/Lee/40 virus-infected cells and in cells expressing the recombinant B/Lee/40 NS1 protein a full-length 35 kDa NS1 protein and a 23 kDa NS1 protein species (p23) were detected. Mutational analysis of the NS1 gene indicated that p23 was generated by a novel cleavage event within the linker domain between an aspartic acid and proline at amino acid residues at positions 92 and 93 respectively (DP92–93), and that p23 contained the first 92 amino acids of the NS1 protein. Sequence analysis of the Singapore strains indicated the presence of either DP92–93 or NP92–93 in the NS1 protein, but protein expression analysis showed that p23 was only detected in NS1 proteins with DP92–93.. An additional adjacent proline residue at position 94 (P94) was present in some strains and correlated with increased p23 levels, suggesting that P94 has a synergistic effect on the cleavage of the NS1 protein. The first 145 amino acids of the NS1 protein are required for inhibition of ISG15-mediated ubiquitination, and our analysis showed that Influenza B viruses circulating in Singapore with DP92–93 expressed truncated NS1 proteins and may differ in their capacity to inhibit ISG15 activity. Thus, DP92–93 in the NS1 protein may confer a disadvantage to Influenza B viruses circulating in the human population and interestingly the low frequency of DP92–93detection in the NS1 protein since 2004 is consistent with this suggestion.

Molecular and biochemical characterization of the NS1 protein of non-cultured influenza B virus strains circulating in Singapore

KAUST Repository

Jumat, Muhammad Raihan

2016-08-04

In this study we compared the NS1 protein of Influenza B/Lee/40 and several non-cultured Influenza B virus clinical strains detected in Singapore. In B/Lee/40 virus-infected cells and in cells expressing the recombinant B/Lee/40 NS1 protein a full-length 35 kDa NS1 protein and a 23 kDa NS1 protein species (p23) were detected. Mutational analysis of the NS1 gene indicated that p23 was generated by a novel cleavage event within the linker domain between an aspartic acid and proline at amino acid residues at positions 92 and 93 respectively (DP92–93), and that p23 contained the first 92 amino acids of the NS1 protein. Sequence analysis of the Singapore strains indicated the presence of either DP92–93 or NP92–93 in the NS1 protein, but protein expression analysis showed that p23 was only detected in NS1 proteins with DP92–93.. An additional adjacent proline residue at position 94 (P94) was present in some strains and correlated with increased p23 levels, suggesting that P94 has a synergistic effect on the cleavage of the NS1 protein. The first 145 amino acids of the NS1 protein are required for inhibition of ISG15-mediated ubiquitination, and our analysis showed that Influenza B viruses circulating in Singapore with DP92–93 expressed truncated NS1 proteins and may differ in their capacity to inhibit ISG15 activity. Thus, DP92–93 in the NS1 protein may confer a disadvantage to Influenza B viruses circulating in the human population and interestingly the low frequency of DP92–93detection in the NS1 protein since 2004 is consistent with this suggestion.

The DNA-mimic antirestriction proteins ArdA ColIB-P9, Arn T4, and Ocr T7 as activators of H-NS-dependent gene transcription.

Science.gov (United States)

Melkina, Olga E; Goryanin, Ignatiy I; Zavilgelsky, Gennadii B

2016-11-01

The antirestriction proteins ArdA ColIb-P9, Arn T4 and Ocr T7 specifically inhibit type I and type IV restriction enzymes and belong to the family of DNA-mimic proteins because their three-dimensional structure is similar to the double-helical B-form DNA. It is proposed that the DNA-mimic proteins are able to bind nucleoid protein H-NS and alleviate H-NS-silencing of the transcription of bacterial genes. Escherichia coli lux biosensors were constructed by inserting H-NS-dependent promoters into a vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE operon. It was demonstrated that the DNA-mimic proteins ArdA, Arn and Ocr activate the transcription of H-NS-dependent promoters of the lux operon of marine luminescent bacteria (mesophilic Aliivibrio fischeri and psychrophilic Aliivibrio logei), and the dps gene from E. coli. It was also demonstrated that the ArdA antirestriction protein, the genes of which are located on transmissive plasmids ColIb-P9, R64, PK101, decreases levels of H-NS silencing of the PluxC promoter during conjugation in the recipient bacteria. Copyright © 2016 Elsevier GmbH. All rights reserved.

Identification of one B-cell epitope from NS1 protein of duck Tembusu virus with monoclonal antibodies.

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Jinfeng Ti

Full Text Available This study describes the identification of one linear B-cell epitope on TMUV NS1 protein with monoclonal antibody (mAb 3G2 by indirect enzyme-linked immunosorbent assay (ELISA. In this study, NS1 protein was expressed in prokaryotic expression system and purified. One mAb against NS1 protein was generated from Balb/c mice immunized with recombinant protein NS1. A set of 35 partially-overlapping polypeptides covering the entire NS1 protein was expressed with PGEX-6P-1 vector and screened with mAb 3G2. One polypeptide against the mAb was acquired and identified by indirect ELISA and western-blot. To map the epitope accurately, one or two amino acid residues were removed from the carboxy and amino terminal of polypeptide sequentially. A series of truncated oligopeptides were expressed and purified. The minimal determinant of the linear B cell epitope was recognized and identified with mAb 3G2. The accurate linear B-cell epitope was 269DEKEIV274 located in NS1 protein. Furthermore, sequence alignment showed that the epitope was highly conserved and specific among TMUV strains and other flavivirus respectively. The linear B-cell epitope of TMUV NS1 protein could benefit the development of new vaccines and diagnostic assays.

The NS1 Protein from Influenza Virus Stimulates Translation Initiation by Enhancing Ribosome Recruitment to mRNAs.

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Panthu, Baptiste; Terrier, Olivier; Carron, Coralie; Traversier, Aurélien; Corbin, Antoine; Balvay, Laurent; Lina, Bruno; Rosa-Calatrava, Manuel; Ohlmann, Théophile

2017-10-27

The non-structural protein NS1 of influenza A viruses exerts pleiotropic functions during infection. Among these functions, NS1 was shown to be involved in the control of both viral and cellular translation; however, the mechanism by which this occurs remains to be determined. Thus, we have revisited the role of NS1 in translation by using a combination of influenza infection, mRNA reporter transfection, and in vitro functional and biochemical assays. Our data show that the NS1 protein is able to enhance the translation of virtually all tested mRNAs with the exception of constructs bearing the Dicistroviruses Internal ribosome entry segment (IRESes) (DCV and CrPV), suggesting a role at the level of translation initiation. The domain of NS1 required for translation stimulation was mapped to the RNA binding amino-terminal motif of the protein with residues R38 and K41 being critical for activity. Although we show that NS1 can bind directly to mRNAs, it does not correlate with its ability to stimulate translation. This activity rather relies on the property of NS1 to associate with ribosomes and to recruit them to target mRNAs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Computational study on the inhibitor binding mode and allosteric regulation mechanism in hepatitis C virus NS3/4A protein.

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Weiwei Xue

Full Text Available HCV NS3/4A protein is an attractive therapeutic target responsible for harboring serine protease and RNA helicase activities during the viral replication. Small molecules binding at the interface between the protease and helicase domains can stabilize the closed conformation of the protein and thus block the catalytic function of HCV NS3/4A protein via an allosteric regulation mechanism. But the detailed mechanism remains elusive. Here, we aimed to provide some insight into the inhibitor binding mode and allosteric regulation mechanism of HCV NS3/4A protein by using computational methods. Four simulation systems were investigated. They include: apo state of HCV NS3/4A protein, HCV NS3/4A protein in complex with an allosteric inhibitor and the truncated form of the above two systems. The molecular dynamics simulation results indicate HCV NS3/4A protein in complex with the allosteric inhibitor 4VA adopts a closed conformation (inactive state, while the truncated apo protein adopts an open conformation (active state. Further residue interaction network analysis suggests the communication of the domain-domain interface play an important role in the transition from closed to open conformation of HCV NS3/4A protein. However, the inhibitor stabilizes the closed conformation through interaction with several key residues from both the protease and helicase domains, including His57, Asp79, Asp81, Asp168, Met485, Cys525 and Asp527, which blocks the information communication between the functional domains interface. Finally, a dynamic model about the allosteric regulation and conformational changes of HCV NS3/4A protein was proposed and could provide fundamental insights into the allosteric mechanism of HCV NS3/4A protein function regulation and design of new potent inhibitors.

Production of recombinant dengue non-structural 1 (NS1) proteins from clinical virus isolates.

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Yohan, Benediktus; Wardhani, Puspa; Aryati; Trimarsanto, Hidayat; Sasmono, R Tedjo

2017-01-01

Dengue is a febrile disease caused by infection of dengue virus (DENV). Early diagnosis of dengue infection is important for better management of the disease. The DENV Non-Structural Protein 1 (NS1) antigen has been routinely used for the early dengue detection. In dengue epidemic countries such as Indonesia, clinicians are increasingly relying on the NS1 detection for confirmation of dengue infection. Various NS1 diagnostic tests are commercially available, however different sensitivities and specificities were observed in various settings. This study was aimed to generate dengue NS1 recombinant protein for the development of dengue diagnostic tests. Four Indonesian DENV isolates were used as the source of the NS1 gene cloning, expression, and purification in bacterial expression system. Recombinant NS1 proteins were successfully purified and their antigenicities were assessed. Immunization of mice with recombinant proteins observed the immunogenicity of the NS1 protein. The generated recombinant proteins can be potentially used in the development of NS1 diagnostic test. With minimal modifications, this method can be used for producing NS1 recombinant proteins from isolates obtained from other geographical regions. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification and subcellular localization of porcine deltacoronavirus accessory protein NS6

International Nuclear Information System (INIS)

Fang, Puxian; Fang, Liurong; Liu, Xiaorong; Hong, Yingying; Wang, Yongle; Dong, Nan; Ma, Panpan; Bi, Jing; Wang, Dang; Xiao, Shaobo

2016-01-01

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus. Accessory proteins are genus-specific for coronavirus, and two putative accessory proteins, NS6 and NS7, are predicted to be encoded by PDCoV; however, this remains to be confirmed experimentally. Here, we identified the leader-body junction sites of NS6 subgenomic RNA (sgRNA) and found that the actual transcription regulatory sequence (TRS) utilized by NS6 is non-canonical and is located upstream of the predicted TRS. Using the purified NS6 from an Escherichia coli expression system, we obtained two anti-NS6 monoclonal antibodies that could detect the predicted NS6 in cells infected with PDCoV or transfected with NS6-expressing plasmids. Further studies revealed that NS6 is always localized in the cytoplasm of PDCoV-infected cells, mainly co-localizing with the endoplasmic reticulum (ER) and ER-Golgi intermediate compartments, as well as partially with the Golgi apparatus. Together, our results identify the NS6 sgRNA and demonstrate its expression in PDCoV-infected cells. -- Highlights: •The leader-body fusion site of NS6 sgRNA is identified. •NS6 sgRNA uses a non-canonical transcription regulatory sequence (TRS). •NS6 can be expressed in PDCoV-infected cell. •NS6 predominantly localize to the ER complex and ER-Golgi intermediate compartment.

Identification and subcellular localization of porcine deltacoronavirus accessory protein NS6

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Fang, Puxian; Fang, Liurong; Liu, Xiaorong; Hong, Yingying; Wang, Yongle; Dong, Nan; Ma, Panpan [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China); Bi, Jing [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); Department of Immunology and Aetology, College of Basic Medicine, Hubei University of Chinese Medicine, Wuhan 430065 (China); Wang, Dang [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China); Xiao, Shaobo, E-mail: vet@mail.hzau.edu.cn [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China)

2016-12-15

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus. Accessory proteins are genus-specific for coronavirus, and two putative accessory proteins, NS6 and NS7, are predicted to be encoded by PDCoV; however, this remains to be confirmed experimentally. Here, we identified the leader-body junction sites of NS6 subgenomic RNA (sgRNA) and found that the actual transcription regulatory sequence (TRS) utilized by NS6 is non-canonical and is located upstream of the predicted TRS. Using the purified NS6 from an Escherichia coli expression system, we obtained two anti-NS6 monoclonal antibodies that could detect the predicted NS6 in cells infected with PDCoV or transfected with NS6-expressing plasmids. Further studies revealed that NS6 is always localized in the cytoplasm of PDCoV-infected cells, mainly co-localizing with the endoplasmic reticulum (ER) and ER-Golgi intermediate compartments, as well as partially with the Golgi apparatus. Together, our results identify the NS6 sgRNA and demonstrate its expression in PDCoV-infected cells. -- Highlights: •The leader-body fusion site of NS6 sgRNA is identified. •NS6 sgRNA uses a non-canonical transcription regulatory sequence (TRS). •NS6 can be expressed in PDCoV-infected cell. •NS6 predominantly localize to the ER complex and ER-Golgi intermediate compartment.

Effect of the I(to) activator NS5806 on cloned K(V)4 channels depends on the accessory protein KChIP2

DEFF Research Database (Denmark)

Lundby, Alicia; Jespersen, Thomas; Schmitt, Nicole

2010-01-01

The compound NS5806 increases the transient outward current (I(to)) in canine ventricular cardiomyocytes and slows current decay. In human and canine ventricle, I(to) is thought to be mediated by K(V)4.3 and various ancillary proteins, yet, the exact subunit composition of I(to) channels is still...... debated. Here we characterize the effect of NS5806 on heterologously expressed putative I(to) channel subunits and other potassium channels....

Hepatitis C virus NS3/4A protease inhibits complement activation by cleaving complement component 4.

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Seiichi Mawatari

Full Text Available BACKGROUND: It has been hypothesized that persistent hepatitis C virus (HCV infection is mediated in part by viral proteins that abrogate the host immune response, including the complement system, but the precise mechanisms are not well understood. We investigated whether HCV proteins are involved in the fragmentation of complement component 4 (C4, composed of subunits C4α, C4β, and C4γ, and the role of HCV proteins in complement activation. METHODS: Human C4 was incubated with HCV nonstructural (NS 3/4A protease, core, or NS5. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then subjected to peptide sequencing. The activity of the classical complement pathway was examined using an erythrocyte hemolysis assay. The cleavage pattern of C4 in NS3/4A-expressing and HCV-infected cells, respectively, was also examined. RESULTS: HCV NS3/4A protease cleaved C4γ in a concentration-dependent manner, but viral core and NS5 did not. A specific inhibitor of NS3/4A protease reduced C4γ cleavage. NS3/4A protease-mediated cleavage of C4 inhibited classical pathway activation, which was abrogated by a NS3/4A protease inhibitor. In addition, co-transfection of cells with C4 and wild-type NS3/4A, but not a catalytic-site mutant of NS3/4A, produced cleaved C4γ fragments. Such C4 processing, with a concomitant reduction in levels of full-length C4γ, was also observed in HCV-infected cells expressing C4. CONCLUSIONS: C4 is a novel cellular substrate of the HCV NS3/4A protease. Understanding disturbances in the complement system mediated by NS3/4A protease may provide new insights into the mechanisms underlying persistent HCV infection.

Analysis of the PDZ binding specificities of Influenza A Virus NS1 proteins

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Nagasaka Kazunori

2011-01-01

Full Text Available Abstract The Influenza A virus non-structural protein 1 (NS1 is a multifunctional virulence factor with several protein-protein interaction domains, involved in preventing apoptosis of the infected cell and in evading the interferon response. In addition, the majority of influenza A virus NS1 proteins have a class I PDZ-binding motif at the C-terminus, and this itself has been shown to be a virulence determinant. In the majority of human influenza NS1 proteins the consensus motif is RSxV: in avian NS1 it is ESxV. Of the few human strains that have the avian motif, all were from very high mortality outbreaks of the disease. Previous work has shown that minor differences in PDZ-binding motifs can have major effects on the spectrum of cellular proteins targeted. In this study we analyse the effect of these differences upon the binding of Influenza A virus NS1 protein to a range of cellular proteins involved in polarity and signal transduction.

In Vitro Evaluation of Novel Inhibitors against the NS2B-NS3 Protease of Dengue Fever Virus Type 4

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Thi Thanh Hanh Nguyen

2013-12-01

Full Text Available The discovery of potent therapeutic compounds against dengue virus is urgently needed. The NS2B-NS3 protease (NS2B-NS3pro of dengue fever virus carries out all enzymatic activities needed for polyprotein processing and is considered to be amenable to antiviral inhibition by analogy. Virtual screening of 300,000 compounds using Autodock 3 on the GVSS platform was conducted to identify novel inhibitors against the NS2B-NS3pro. Thirty-six compounds were selected for in vitro assay against NS2B-NS3pro expressed in Pichia pastoris. Seven novel compounds were identified as inhibitors with IC50 values of 3.9 ± 0.6–86.7 ± 3.6 μM. Three strong NS2B-NS3pro inhibitors were further confirmed as competitive inhibitors with Ki values of 4.0 ± 0.4, 4.9 ± 0.3, and 3.4 ± 0.1 μM, respectively. Hydrophobic and hydrogen bond interactions between amino acid residues in the NS3pro active site with inhibition compounds were also identified.

The Influenza NS1 Protein: What Do We Know in Equine Influenza Virus Pathogenesis?

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Marta Barba

2016-08-01

Full Text Available Equine influenza virus remains a serious health and potential economic problem throughout most parts of the world, despite intensive vaccination programs in some horse populations. The influenza non-structural protein 1 (NS1 has multiple functions involved in the regulation of several cellular and viral processes during influenza infection. We review the strategies that NS1 uses to facilitate virus replication and inhibit antiviral responses in the host, including sequestering of double-stranded RNA, direct modulation of protein kinase R activity and inhibition of transcription and translation of host antiviral response genes such as type I interferon. Details are provided regarding what it is known about NS1 in equine influenza, especially concerning C-terminal truncation. Further research is needed to determine the role of NS1 in equine influenza infection, which will help to understand the pathophysiology of complicated cases related to cytokine imbalance and secondary bacterial infection, and to investigate new therapeutic and vaccination strategies.

In Silico Screening, Alanine Mutation, and DFT Approaches for Identification of NS2B/NS3 Protease Inhibitors

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R. Balajee

2016-01-01

Full Text Available To identify the ligand that binds to a target protein with high affinity is a nontrivial task in computer-assisted approaches. Antiviral drugs have been identified for NS2B/NS3 protease enzyme on the mechanism to cleave the viral protein using the computational tools. The consequence of the molecular docking, free energy calculations, and simulation protocols explores the better ligand. It provides in-depth structural insights with the catalytic triad of His51, Asp75, Ser135, and Gly133. The MD simulation was employed here to predict the stability of the complex. The alanine mutation has been performed and its stability was monitored by using the molecular dynamics simulation. The minimal RMSD value suggests that the derived complexes are close to equilibrium. The DFT outcome reveals that the HOMO-LUMO gap of Ligand19 is 2.86 kcal/mol. Among the considered ligands, Ligand19 shows the lowest gap and it is suggested that the HOMO of Ligand19 may transfer the electrons to the LUMO in the active regions. The calculated binding energy of Ligand19 using the DFT method is in good agreement with the docking studies. The pharmacological activity of ligand was performed and satisfies Lipinski rule of 5. Moreover, the computational results are compared with the available IC50 values of experimental results.

Gelatin nanoparticles enhance delivery of hepatitis C virus recombinant NS2 gene.

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Sabet, Salwa; George, Marina A; El-Shorbagy, Haidan M; Bassiony, Heba; Farroh, Khaled Y; Youssef, Tareq; Salaheldin, Taher A

2017-01-01

Development of an effective non-viral vaccine against hepatitis C virus infection is of a great importance. Gelatin nanoparticles (Gel.NPs) have an attention and promising approach as a viable carrier for delivery of vaccine, gene, drug and other biomolecules in the body. The present study aimed to develop stable Gel.NPs conjugated with nonstructural protein 2 (NS2) gene of Hepatitis C Virus genotype 4a (HCV4a) as a safe and an efficient vaccine delivery system. Gel.NPs were synthesized and characterized (size: 150±2 nm and zeta potential +17.6 mv). NS2 gene was successfully cloned and expressed into E. coli M15 using pQE-30 vector. Antigenicity of the recombinant NS2 protein was confirmed by Western blotting to verify the efficiency of NS2 as a possible vaccine. Then NS2 gene was conjugated to gelatin nanoparticles and a successful conjugation was confirmed by labeling and imaging using Confocal Laser Scanning Microscope (CLSM). Interestingly, the transformation of the conjugated NS2/Gel.NPs complex into E. coli DH5-α was 50% more efficient than transformation with the gene alone. In addition, conjugated NS2/Gel.NPs with ratio 1:100 (w/w) showed higher transformation efficiency into E. coli DH5-α than the other ratios (1:50 and 2:50). Gel.NPs effectively enhanced the gene delivery in bacterial cells without affecting the structure of NS2 gene and could be used as a safe, easy, rapid, cost-effective and non-viral vaccine delivery system for HCV.

Rab5 Enhances Classical Swine Fever Virus Proliferation and Interacts with Viral NS4B Protein to Facilitate Formation of NS4B Related Complex

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Jihui Lin

2017-08-01

Full Text Available Classical swine fever virus (CSFV is a fatal pig pestivirus and causes serious financial losses to the pig industry. CSFV NS4B protein is one of the most important viral replicase proteins. Rab5, a member of the small Rab GTPase family, is involved in infection and replication of numerous viruses including hepatitis C virus and dengue virus. Until now, the effects of Rab5 on the proliferation of CSFV are poorly defined. In the present study, we showed that Rab5 could enhance CSFV proliferation by utilizing lentivirus-mediated constitutive overexpression and eukaryotic plasmid transient overexpression approaches. On the other hand, lentivirus-mediated short hairpin RNA knockdown of Rab5 dramatically inhibited virus production. Co-immunoprecipitation, glutathione S-transferase pulldown and laser confocal microscopy assays further confirmed the interaction between Rab5 and CSFV NS4B protein. In addition, intracellular distribution of NS4B-Red presented many granular fluorescent signals (GFS in CSFV infected PK-15 cells. Inhibition of basal Rab5 function with Rab5 dominant negative mutant Rab5S34N resulted in disruption of the GFS. These results indicate that Rab5 plays a critical role in facilitating the formation of the NS4B related complexes. Furthermore, it was observed that NS4B co-localized with viral NS3 and NS5A proteins in the cytoplasm, suggesting that NS3 and NS5A might be components of the NS4B related complex. Taken together, these results demonstrate that Rab5 positively modulates CSFV propagation and interacts with NS4B protein to facilitate the NS4B related complexes formation.

Virtual Screening for Potential Inhibitors of NS3 Protein of Zika Virus

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Maheswata Sahoo

2016-09-01

Full Text Available Zika virus (ZIKV is a mosquito borne pathogen, belongs to Flaviviridae family having a positive-sense single-stranded RNA genome, currently known for causing large epidemics in Brazil. Its infection can cause microcephaly, a serious birth defect during pregnancy. The recent outbreak of ZIKV in February 2016 in Brazil realized it as a major health risk, demands an enhanced surveillance and a need to develop novel drugs against ZIKV. Amodiaquine, prochlorperazine, quinacrine, and berberine are few promising drugs approved by Food and Drug Administration against dengue virus which also belong to Flaviviridae family. In this study, we performed molecular docking analysis of these drugs against nonstructural 3 (NS3 protein of ZIKV. The protease activity of NS3 is necessary for viral replication and its prohibition could be considered as a strategy for treatment of ZIKV infection. Amongst these four drugs, berberine has shown highest binding affinity of –5.8 kcal/mol and it is binding around the active site region of the receptor. Based on the properties of berberine, more similar compounds were retrieved from ZINC database and a structure-based virtual screening was carried out by AutoDock Vina in PyRx 0.8. Best 10 novel drug-like compounds were identified and amongst them ZINC53047591 (2-(benzylsulfanyl-3-cyclohexyl-3H-spiro[benzo[h]quinazoline-5,1'-cyclopentan]-4(6H-one was found to interact with NS3 protein with binding energy of –7.1 kcal/mol and formed H-bonds with Ser135 and Asn152 amino acid residues. Observations made in this study may extend an assuring platform for developing anti-viral competitive inhibitors against ZIKV infection.

Introduction to Network Simulator NS2

CERN Document Server

Issariyakul, Teerawat

2012-01-01

"Introduction to Network Simulator NS2" is a primer providing materials for NS2 beginners, whether students, professors, or researchers for understanding the architecture of Network Simulator 2 (NS2) and for incorporating simulation modules into NS2. The authors discuss the simulation architecture and the key components of NS2 including simulation-related objects, network objects, packet-related objects, and helper objects. The NS2 modules included within are nodes, links, SimpleLink objects, packets, agents, and applications. Further, the book covers three helper modules: timers, ra

Effects of indomethacin, NS-398 (a selective prostaglandin H synthase-2 inhibitor) and protein synthesis inhibitors on prostaglandin production by the guinea-pig placenta.

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Aitken, H; Poyser, N L

2001-01-01

The outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)were similar from the day 22 guinea-pig placenta and sub-placenta in culture, except for PGE2 output from the sub-placenta which was lower. Between days 22 and 29 of pregnancy, the outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)during the initial 2 h culture period increased 6.9-, 1.1- and 3.2-fold, respectively, from the placenta, and 2.1-, 1.4- and 2.2-fold, respectively, from the sub-placenta. Therefore, there was a relatively specific increase in PGF(2 alpha)production by the guinea-pig placenta between days 22 and 29 of pregnancy. The output of PGFM from the cultured placenta also increased between days 22 and 29, indicating that the increase in PGF(2 alpha)output was due to increased synthesis rather than to decreased metabolism. By comparing the amounts of prostaglandins produced by tissue homogenates during a 1 h incubation period, it appears that there is approximately a 2-fold increase in the amount of prostaglandin H synthase (PGHS) present in the guinea-pig placenta between days 22 and 29. NS-398 (a specific inhibitor of PGHS-2) and indomethacin (an inhibitor of both PGHS-1 and PGHS-2) both inhibited prostaglandin production by homogenates of day 22 and day 29 placenta. Indomethacin was more effective than NS-398, except for their actions on PGF(2 alpha)production by the day 29 placenta where indomethacin and NS-398 were equiactive. Indomethacin and NS-398 were both very effective at inhibiting the outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)from the day 22 and day 29 placenta and sub-placenta in culture, indicating that prostaglandin production by the guinea-pig placenta and sub-placenta in culture is largely dependent upon the activity of PGHS-2. The high production of PGF(2 alpha)by the day 29 placenta is not dependent on the continual synthesis of fresh protein(s), as inhibitors of protein synthesis did not reduce PGF(2 alpha)output from the day 29 guinea-pig placenta in culture

Detergent-resistant membrane association of NS2 and E2 during hepatitis C virus replication.

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Shanmugam, Saravanabalaji; Saravanabalaji, Dhanaranjani; Yi, MinKyung

2015-04-01

Previously, we demonstrated that the efficiency of hepatitis C virus (HCV) E2-p7 processing regulates p7-dependent NS2 localization to putative virus assembly sites near lipid droplets (LD). In this study, we have employed subcellular fractionations and membrane flotation assays to demonstrate that NS2 associates with detergent-resistant membranes (DRM) in a p7-dependent manner. However, p7 likely plays an indirect role in this process, since only the background level of p7 was detectable in the DRM fractions. Our data also suggest that the p7-NS2 precursor is not involved in NS2 recruitment to the DRM, despite its apparent targeting to this location. Deletion of NS2 specifically inhibited E2 localization to the DRM, indicating that NS2 regulates this process. Treatment of cells with methyl-β-cyclodextrin (MβCD) significantly reduced the DRM association of Core, NS2, and E2 and reduced infectious HCV production. Since disruption of the DRM localization of NS2 and E2, either due to p7 and NS2 defects, respectively, or by MβCD treatment, inhibited infectious HCV production, these proteins' associations with the DRM likely play an important role during HCV assembly. Interestingly, we detected the HCV replication-dependent accumulation of ApoE in the DRM fractions. Taking into consideration the facts that ApoE was shown to be a major determinant for infectious HCV particle production at the postenvelopment step and that the HCV Core protein strongly associates with the DRM, recruitment of E2 and ApoE to the DRM may allow the efficient coordination of Core particle envelopment and postenvelopment events at the DRM to generate infectious HCV production. The biochemical nature of HCV assembly sites is currently unknown. In this study, we investigated the correlation between NS2 and E2 localization to the detergent-resistant membranes (DRM) and HCV particle assembly. We determined that although NS2's DRM localization is dependent on p7, p7 was not targeted to these

NS309 decreases rat detrusor smooth muscle membrane potential and phasic contractions by activating SK3 channels

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Parajuli, Shankar P; Hristov, Kiril L; Soder, Rupal P; Kellett, Whitney F; Petkov, Georgi V

2013-01-01

Background and Purpose Overactive bladder (OAB) is often associated with abnormally increased detrusor smooth muscle (DSM) contractions. We used NS309, a selective and potent opener of the small or intermediate conductance Ca2+-activated K+ (SK or IK, respectively) channels, to evaluate how SK/IK channel activation modulates DSM function. Experimental Approach We employed single-cell RT-PCR, immunocytochemistry, whole cell patch-clamp in freshly isolated rat DSM cells and isometric tension recordings of isolated DSM strips to explore how the pharmacological activation of SK/IK channels with NS309 modulates DSM function. Key Results We detected SK3 but not SK1, SK2 or IK channels expression at both mRNA and protein levels by RT-PCR and immunocytochemistry in DSM single cells. NS309 (10 μM) significantly increased the whole cell SK currents and hyperpolarized DSM cell resting membrane potential. The NS309 hyperpolarizing effect was blocked by apamin, a selective SK channel inhibitor. NS309 inhibited the spontaneous phasic contraction amplitude, force, frequency, duration and tone of isolated DSM strips in a concentration-dependent manner. The inhibitory effect of NS309 on spontaneous phasic contractions was blocked by apamin but not by TRAM-34, indicating no functional role of the IK channels in rat DSM. NS309 also significantly inhibited the pharmacologically and electrical field stimulation-induced DSM contractions. Conclusions and Implications Our data reveal that SK3 channel is the main SK/IK subtype in rat DSM. Pharmacological activation of SK3 channels with NS309 decreases rat DSM cell excitability and contractility, suggesting that SK3 channels might be potential therapeutic targets to control OAB associated with detrusor overactivity. PMID:23145946

Identification of specific regions in hepatitis C virus core, NS2 and NS5A that genetically interact with p7 and co-ordinate infectious virus production.

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Gouklani, H; Beyer, C; Drummer, H; Gowans, E J; Netter, H J; Haqshenas, G

2013-04-01

The p7 protein of hepatitis C virus (HCV) is a small, integral membrane protein that plays a critical role in virus replication. Recently, we reported two intergenotypic JFH1 chimeric viruses encoding the partial or full-length p7 protein of the HCV-A strain of genotype 1b (GT1b; Virology; 2007; 360:134). In this study, we determined the consensus sequences of the entire polyprotein coding regions of the wild-type JFH1 and the revertant chimeric viruses and identified predominant amino acid substitutions in core (K74M), NS2 (T23N, H99P) and NS5A (D251G). Forward genetic analysis demonstrated that all single mutations restored the infectivity of the defective chimeric genomes suggesting that the infectious virus production involves the association of p7 with specific regions in core, NS2 and NS5A. In addition, it was demonstrated that the NS2 T23N facilitated the generation of infectious intergenotypic chimeric virus encoding p7 from GT6 of HCV. © 2012 Blackwell Publishing Ltd.

Engineered toxins "zymoxins" are activated by the HCV NS3 protease by removal of an inhibitory protein domain.

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Assaf Shapira

Full Text Available The synthesis of inactive enzyme precursors, also known as "zymogens," serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV as a model, we designed two HCV NS3 protease-activated "zymogenized" chimeric toxins (which we denote "zymoxins". In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA and Ricin A chain (RTA, respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the "zymoxin" approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.

The N-Terminal of Aquareovirus NS80 Is Required for Interacting with Viral Proteins and Viral Replication.

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Jie Zhang

Full Text Available Reovirus replication and assembly occurs within viral inclusion bodies that formed in specific intracellular compartments of cytoplasm in infected cells. Previous study indicated that aquareovirus NS80 is able to form inclusion bodies, and also can retain viral proteins within its inclusions. To better understand how NS80 performed in viral replication and assembly, the functional regions of NS80 associated with other viral proteins in aquareovirus replication were investigated in this study. Deletion mutational analysis and rotavirus NSP5-based protein association platform were used to detect association regions. Immunofluorescence images indicated that different N-terminal regions of NS80 could associate with viral proteins VP1, VP4, VP6 and NS38. Further co-immunoprecipitation analysis confirmed the interaction between VP1, VP4, VP6 or NS38 with different regions covering the N-terminal amino acid (aa, 1-471 of NS80, respectively. Moreover, removal of NS80 N-terminal sequences required for interaction with proteins VP1, VP4, VP6 or NS38 not only prevented the capacity of NS80 to support viral replication in NS80 shRNA-based replication complementation assays, but also inhibited the expression of aquareovirus proteins, suggesting that N-terminal regions of NS80 are necessary for viral replication. These results provided a foundational basis for further understanding the role of NS80 in viral replication and assembly during aquareovirus infection.

A crystal structure of the Dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication.

Directory of Open Access Journals (Sweden)

Yongqian Zhao

2015-03-01

Full Text Available Flavivirus RNA replication occurs within a replication complex (RC that assembles on ER membranes and comprises both non-structural (NS viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase and C-terminal RNA-dependent-RNA polymerase (RdRp domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3 at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV, the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.

Identification of novel small molecule inhibitors against NS2B/NS3 serine protease from Zika virus

Energy Technology Data Exchange (ETDEWEB)

Lee, Hyun; Ren, Jinhong; Nocadello, Salvatore; Rice, Amy J.; Ojeda, Isabel; Light, Samuel; Minasov, George; Vargas, Jason; Nagarathnam, Dhanapalan; Anderson, Wayne F.; Johnson, Michael E. (UIC); (NWU); (Novalex); (DNSK)

2016-12-26

Zika flavivirus infection during pregnancy appears to produce higher risk of microcephaly, and also causes multiple neurological problems such as Guillain–Barré syndrome. The Zika virus is now widespread in Central and South America, and is anticipated to become an increasing risk in the southern United States. With continuing global travel and the spread of the mosquito vector, the exposure is expected to accelerate, but there are no currently approved treatments against the Zika virus. The Zika NS2B/NS3 protease is an attractive drug target due to its essential role in viral replication. Our studies have identified several compounds with inhibitory activity (IC50) and binding affinity (KD) of ~5–10 μM against the Zika NS2B-NS3 protease from testing 71 HCV NS3/NS4A inhibitors that were initially discovered by high-throughput screening of 40,967 compounds. Competition surface plasmon resonance studies and mechanism of inhibition analyses by enzyme kinetics subsequently determined the best compound to be a competitive inhibitor with a Ki value of 9.5 μM. We also determined the X-ray structure of the Zika NS2B-NS3 protease in a “pre-open conformation”, a conformation never observed before for any flavivirus proteases. This provides the foundation for new structure-based inhibitor design.

Characterization of the expression and immunogenicity of the ns4b protein of human coronavirus 229E

DEFF Research Database (Denmark)

Chagnon, F; Lamarre, A; Lachance, C

1998-01-01

to demonstrate the expression of ns4b in HCV-229E-infected cells using flow cytometry. Given a previously reported contiguous five amino acid shared region between ns4b and myelin basic protein, a purified recombinant histidine-tagged ns4b protein and (or) human myelin basic protein were injected into mice......Sequencing of complementary DNAs prepared from various coronaviruses has revealed open reading frames encoding putative proteins that are yet to be characterized and are so far only described as nonstructural (ns). As a first step in the elucidation of its function, we characterized the expression...... and immunogenicity of the ns4b gene product from strain 229E of human coronavirus (HCV-229E), a respiratory virus with a neurotropic potential. The gene was cloned and expressed in bacteria. A fusion protein of ns4b with maltose-binding protein was injected into rabbits to generate specific antibodies that were used...

Engineered Toxins “Zymoxins” Are Activated by the HCV NS3 Protease by Removal of an Inhibitory Protein Domain

Science.gov (United States)

Shapira, Assaf; Gal-Tanamy, Meital; Nahary, Limor; Litvak-Greenfeld, Dana; Zemel, Romy; Tur-Kaspa, Ran; Benhar, Itai

2011-01-01

The synthesis of inactive enzyme precursors, also known as “zymogens,” serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated “zymogenized” chimeric toxins (which we denote “zymoxins”). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the “zymoxin” approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected. PMID:21264238

Advances in animal cell recombinant protein production: GS-NS0 expression system.

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Barnes, L M; Bentley, C M; Dickson, A J

2000-02-01

The production of recombinant proteins using mammalian cell expression systems is of growing importance within biotechnology, largely due to the ability of specific mammalian cells to carry out post-translational modifications of the correct fidelity. The Glutamine Synthetase-NS0 system is now one such industrially important expression system.Glutamine synthetase catalyses the formation ofglutamine from glutamate and ammonia. NS0 cellscontain extremely low levels of endogenous glutaminesynthetase activity, therefore exogenous glutaminesynthetase can be used efficiently as a selectablemarker to identify successful transfectants in theabsence of glutamine in the media. In addition, theinclusion of methionine sulphoximine, an inhibitor ofglutamine synthetase activity, enables furtherselection of those clones producing relatively highlevels of transfected glutamine synthetase and henceany heterologous gene which is coupled to it. Theglutamine synthetase system technology has been usedfor research and development purposes during thisdecade and its importance is clearly demonstrated nowthat two therapeutic products produced using thissystem have reached the market place.

The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization

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Lalime, Erin N.; Pekosz, Andrew, E-mail: apekosz@jhsph.edu

2014-06-15

The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 in addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization. - Highlights: • Mutations were introduced into influenza NS1 NLS1. • NS1 R37A, R38A, K41A viruses had minimal changes in replication and NS1 localization. • Viruses from NS1 R35A rescue all contained additional mutations at D39. • NS1 R35A D39X mutations recover dimerization lost in NS1 R35A mutations. • These results reaffirm the importance of dimerization for NS1 protein function.

The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization

International Nuclear Information System (INIS)

Lalime, Erin N.; Pekosz, Andrew

2014-01-01

The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 in addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization. - Highlights: • Mutations were introduced into influenza NS1 NLS1. • NS1 R37A, R38A, K41A viruses had minimal changes in replication and NS1 localization. • Viruses from NS1 R35A rescue all contained additional mutations at D39. • NS1 R35A D39X mutations recover dimerization lost in NS1 R35A mutations. • These results reaffirm the importance of dimerization for NS1 protein function

Sensitive luminescent reporter viruses reveal appreciable release of hepatitis C virus NS5A protein into the extracellular environment.

Science.gov (United States)

Eyre, Nicholas S; Aloia, Amanda L; Joyce, Michael A; Chulanetra, Monrat; Tyrrell, D Lorne; Beard, Michael R

2017-07-01

The HCV NS5A protein is essential for viral RNA replication and virus particle assembly. To study the viral replication cycle and NS5A biology we generated an infectious HCV construct with a NanoLuciferase (NLuc) insertion within NS5A. Surprisingly, beyond its utility as a sensitive reporter of cytoplasmic viral RNA replication, we also observed strong luminescence in cell culture fluids. Further analysis using assembly-defective viruses and subgenomic replicons revealed that infectious virus production was not required for extracellular NS5A-NLuc activity but was associated with enrichment of extracellular NS5A-NLuc in intermediate-density fractions similar to those of exosomes and virus particles. Additionally, BRET analysis indicated that intracellular and extracellular forms of NS5A may adopt differing conformations. Importantly, infection studies using a human liver chimeric mouse model confirmed robust infection in vivo and ready detection of NLuc activity in serum. We hypothesise that the presence of NS5A in extracellular fluids contributes to HCV pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Naringenin and quercetin--potential anti-HCV agents for NS2 protease targets.

Science.gov (United States)

Lulu, S Sajitha; Thabitha, A; Vino, S; Priya, A Mohana; Rout, Madhusmita

2016-01-01

Nonstructural proteins of hepatitis C virus had drawn much attention for the scientific fraternity in drug discovery due to its important role in the disease. 3D structure of the protein was predicted using molecular modelling protocol. Docking studies of 10 medicinal plant compounds and three drugs available in the market (control) with NS2 protease were employed by using rigid docking approach of AutoDock 4.2. Among the molecules tested for docking study, naringenin and quercetin revealed minimum binding energy of - 7.97 and - 7.95 kcal/mol with NS2 protease. All the ligands were docked deeply within the binding pocket region of the protein. The docking study results showed that these compounds are potential inhibitors of the target; and also all these docked compounds have good inhibition constant, vdW+Hbond+desolv energy with best RMSD value.

Activation of human ether-a-go-go-related gene potassium channels by the diphenylurea 1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643)

DEFF Research Database (Denmark)

Hansen, Rie Schultz; Diness, Thomas Goldin; Christ, Torsten

2005-01-01

The cardiac action potential is generated by a concerted action of different ion channels and transporters. Dysfunction of any of these membrane proteins can give rise to cardiac arrhythmias, which is particularly true for the repolarizing potassium channels. We suggest that an increased repolari......The cardiac action potential is generated by a concerted action of different ion channels and transporters. Dysfunction of any of these membrane proteins can give rise to cardiac arrhythmias, which is particularly true for the repolarizing potassium channels. We suggest that an increased......M. Application of NS1643 also resulted in a prolonged postrepolarization refractory time. Finally, cardiomyocytes exposed to NS1643 resisted reactivation by small depolarizing currents mimicking early afterdepolarizations. In conclusion, HERG channel activation by small molecules such as NS1643 increases...

Activation of human IK and SK Ca2+ -activated K+ channels by NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime)

DEFF Research Database (Denmark)

Strøbaek, Dorte; Teuber, Lene; Jørgensen, Tino D

2004-01-01

We have identified and characterized the compound NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime) as a potent activator of human Ca2+ -activated K+ channels of SK and IK types, whereas it is devoid of effect on BK type channels. IK- and SK-channels have previously been reported to be activated...

A Novel Benzodiazepine Compound Inhibits Yellow Fever Virus Infection by Specifically Targeting NS4B Protein.

Science.gov (United States)

Guo, Fang; Wu, Shuo; Julander, Justin; Ma, Julia; Zhang, Xuexiang; Kulp, John; Cuconati, Andrea; Block, Timothy M; Du, Yanming; Guo, Ju-Tao; Chang, Jinhong

2016-09-21

Although a highly effective vaccine is available, the number of yellow fever cases has increased over the past two decades, which highlights the pressing need for antiviral therapeutics. In a high throughput screening campaign, we identified an acetic acid benzodiazepine (BDAA) compound, which potently inhibits yellow fever virus (YFV). Interestingly, while treatment of YFV infected cultures with 2 μM of BDAA reduced the virion production by greater than 2 logs, the compound is not active against 21 other viruses from 14 different viral families. Selection and genetic analysis of drug resistant viruses revealed that substitution of proline at amino acid 219 (P219) of the nonstructural protein 4B (NS4B) with serine, threonine or alanine confers YFV resistance to BDAA without apparent loss of replication fitness in cultured mammalian cells. However, substitution of P219 with glycine confers BDAA resistance with significant loss of replication ability. Bioinformatics analysis predicts that the P219 localizes at the endoplasmic reticulum lumen side of the fifth putative trans-membrane domain of NS4B and the mutation may render the viral protein incapable of interacting with BDAA. Our studies thus revealed important role and structural basis for NS4B protein in supporting YFV replication. Moreover, in YFV-infected hamsters, oral administration of BDAA protected 90% of the animals from death, significantly reduced viral load by greater than 2 logs and attenuated viral infection-induced liver injury and body weight loss. The encouraging preclinical results thus warrant further development of BDAA or its derivatives as antiviral agents to treat yellow fever. Yellow fever is an acute viral hemorrhagic disease which threatens approximately one billion people living in tropical areas of Africa and Latin America. Although a highly effective yellow fever vaccine has been available for more than seven decades, the low vaccination rate fails to prevent outbreaks in at

NMR analysis of the dynamic exchange of the NS2B cofactor between open and closed conformations of the West Nile virus NS2B-NS3 protease.

Directory of Open Access Journals (Sweden)

Xun-Cheng Su

Full Text Available BACKGROUND: The two-component NS2B-NS3 proteases of West Nile and dengue viruses are essential for viral replication and established targets for drug development. In all crystal structures of the proteases to date, the NS2B cofactor is located far from the substrate binding site (open conformation in the absence of inhibitor and lining the substrate binding site (closed conformation in the presence of an inhibitor. METHODS: In this work, nuclear magnetic resonance (NMR spectroscopy of isotope and spin-labeled samples of the West Nile virus protease was used to investigate the occurrence of equilibria between open and closed conformations in solution. FINDINGS: In solution, the closed form of the West Nile virus protease is the predominant conformation irrespective of the presence or absence of inhibitors. Nonetheless, dissociation of the C-terminal part of the NS2B cofactor from the NS3 protease (open conformation occurs in both the presence and the absence of inhibitors. Low-molecular-weight inhibitors can shift the conformational exchange equilibria so that over 90% of the West Nile virus protease molecules assume the closed conformation. The West Nile virus protease differs from the dengue virus protease, where the open conformation is the predominant form in the absence of inhibitors. CONCLUSION: Partial dissociation of NS2B from NS3 has implications for the way in which the NS3 protease can be positioned with respect to the host cell membrane when NS2B is membrane associated via N- and C-terminal segments present in the polyprotein. In the case of the West Nile virus protease, discovery of low-molecular-weight inhibitors that act by breaking the association of the NS2B cofactor with the NS3 protease is impeded by the natural affinity of the cofactor to the NS3 protease. The same strategy can be more successful in the case of the dengue virus NS2B-NS3 protease.

Conserved amino acids within the N-terminus of the West Nile virus NS4A protein contribute to virus replication, protein stability and membrane proliferation

International Nuclear Information System (INIS)

Ambrose, R.L.; Mackenzie, J.M.

2015-01-01

The West Nile virus strain Kunjin virus (WNV KUN ) NS4A protein is a multifunctional protein involved in many aspects of the virus life-cycle and is a major component of the WNV KUN replication complex (RC). Previously we identified a conserved region in the C-terminus of NS4A regulating proteolytic processing and RC assembly, and now investigate key conserved residues in the N-terminus of NS4A and their contribution to WNV KUN replication. Mutation of P13 completely ablated replication, whereas, mutation of P48 and D49, near the first transmembrane helix, and G66 within the helix, showed variable defects in replication, virion secretion and membrane proliferation. Intriguingly, the P48 and G66 NS4A mutants resulted in specific proteasome depletion of NS4A that could in part be rescued with a proteasome inhibitor. Our results suggest that the N-terminus of NS4A contributes to correct folding and stability, essential for facilitating the essential roles of NS4A during replication. - Highlights: • Mutation of Proline13 of the WNV NS4A protein is lethal to replication. • 1st TMB helix of NS4A contributes to protein stability and membrane remodelling. • Unstable mutants of NS4A can be rescued with a proteasome inhibitor. • This study (and of others) contributes to a functional mapping of the NS4A protein

A thiophene-modified screen printed electrode for detection of dengue virus NS1 protein.

Science.gov (United States)

Silva, M M S; Dias, A C M S; Cordeiro, M T; Marques, E; Goulart, M O F; Dutra, R F

2014-10-01

A thiophene-modified screen printed electrode (SPE) for detection of the Dengue virus non-structural protein 1 (NS1), an important marker for acute phase diagnosis, is described. A sulfur-containing heterocyclic compound, the thiophene was incorporated to a carbon ink to prepare reproducible screen printed electrodes. After cured, the thiophene SPE was coated by gold nanoparticles conjugated to Protein A to form a nanostrutured surface. The Anti-NS1 antibodies immobilized via their Fc portions via Protein A, leaving their antigen specific sites free circumventing the problem of a random antibodies immobilization. Amperometric responses to the NS1 protein of dengue virus were obtained by cyclic voltammetries performed in presence of ferrocyanide/ferricyanide as redox probe. The calibration curve of immunosensor showed a linear response from 0.04 µg mL(-1) to 0.6 µg mL(-1) of NS1 with a good linear correlation (r=0.991, pink enhanced the electroanalytical properties of the SPEs, increasing their reproducibility and sensitivity. This point-of-care testing represents a great potential for use in epidemic situations, facilitating the early diagnosis in acute phase of dengue virus. Copyright © 2014 Elsevier B.V. All rights reserved.

[Advances in Parvovirus Non-structural Protein NS1 Induced Apoptosis].

Science.gov (United States)

Tu, Mengyu; Liu, Fei; Chen, Shun; Wang, Mingshu; Cheng, Anchun

2015-11-01

Until now, more than seventeen parvovirus have been reported which can infect mammals and poultries. The infected cells appeared different properties of apoptosis and death, present a typical cytopathic effect. NS1 is a major nonstructural protein of parvovirus, with a conservative structure and function, which plays an important role in the viral life cycle. In addition to the influence on viral replication, the NS1 also participates in apoptosis induced by viruses. Parvovirus induced apoptosis which is mainly mediated by mitochondrial pathway, this review summarized the latest research progresses of parvovirus induced apoptosis.

Effects of the small molecule HERG activator NS1643 on Kv11.3 channels.

Directory of Open Access Journals (Sweden)

Arne Bilet

Full Text Available NS1643 is one of the small molecule HERG (Kv11.1 channel activators and has also been found to increase erg2 (Kv11.2 currents. We now investigated whether NS1643 is also able to act as an activator of Kv11.3 (erg3 channels expressed in CHO cells. Activation of rat Kv11.3 current occurred in a dose-dependent manner and maximal current increasing effects were obtained with 10 µM NS1643. At this concentration, steady-state outward current increased by about 80% and the current increase was associated with a significant shift in the voltage dependence of activation to more negative potentials by about 15 mV. In addition, activation kinetics were accelerated, whereas deactivation was slowed. There was no significant effect on the kinetics of inactivation and recovery from inactivation. The strong current-activating agonistic effect of NS1643 did not result from a shift in the voltage dependence of Kv11.3 channel inactivation and was independent from external Na(+ or Ca(2+. At the higher concentration of 20 µM, NS1643 induced clearly less current increase. The left shift in the voltage dependence of activation reversed and the voltage sensitivity of activation dramatically decreased along with a slowing of Kv11.3 channel activation. These data show that, in comparison to other Kv11 family members, NS1643 exerts distinct effects on Kv11.3 channels with especially pronounced partial antagonistic effects at higher concentration.

NS19504

DEFF Research Database (Denmark)

Nausch, Bernhard; Rode, Frederik; Jørgensen, Susanne

2014-01-01

channel activators and identified a small-molecule positive modulator, NS19504 (5-[(4-bromophenyl)methyl]-1,3-thiazol-2-amine), which activated the BK channel with an EC50 value of 11.0 ± 1.4 µM. Hit validation was performed using high-throughput electrophysiology (QPatch), and further characterization......19504 activated BK channels in native smooth muscle cells from guinea pig urinary bladder. In guinea pig urinary bladder strips, NS19504 (1 µM) reduced spontaneous phasic contractions, an effect that was significantly inhibited by the specific BK channel blocker iberiotoxin. In contrast, NS19504 (1 µ......M) only modestly inhibited nerve-evoked contractions and had no effect on contractions induced by a high K(+) concentration consistent with a K(+) channel-mediated action. Collectively, these results show that NS19504 is a positive modulator of BK channels and provide support for the role of BK channels...

Structural insight and flexible features of NS5 proteins from all four serotypes of Dengue virus in solution

Energy Technology Data Exchange (ETDEWEB)

Saw, Wuan Geok; Tria, Giancarlo; Grüber, Ardina; Subramanian Manimekalai, Malathy Sony; Zhao, Yongqian; Chandramohan, Arun; Srinivasan Anand, Ganesh; Matsui, Tsutomu; Weiss, Thomas M.; Vasudevan, Subhash G.; Grüber, Gerhard

2015-10-31

Infection by the four serotypes ofDengue virus(DENV-1 to DENV-4) causes an important arthropod-borne viral disease in humans. The multifunctional DENV nonstructural protein 5 (NS5) is essential for capping and replication of the viral RNA and harbours a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain. In this study, insights into the overall structure and flexibility of the entire NS5 of all fourDengue virusserotypes in solution are presented for the first time. The solution models derived revealed an arrangement of the full-length NS5 (NS5FL) proteins with the MTase domain positioned at the top of the RdRP domain. The DENV-1 to DENV-4 NS5 forms are elongated and flexible in solution, with DENV-4 NS5 being more compact relative to NS5 from DENV-1, DENV-2 and DENV-3. Solution studies of the individual MTase and RdRp domains show the compactness of the RdRp domain as well as the contribution of the MTase domain and the ten-residue linker region to the flexibility of the entire NS5. Swapping the ten-residue linker between DENV-4 NS5FL and DENV-3 NS5FL demonstrated its importance in MTase–RdRp communication and in concerted interaction with viral and host proteins, as probed by amide hydrogen/deuterium mass spectrometry. Conformational alterations owing to RNA binding are presented.

The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis.

Science.gov (United States)

Aoubala, M; Holt, J; Clegg, R A; Rowlands, D J; Harris, M

2001-07-01

Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system--under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.

Folding Proteins at 500 ns/hour with Work Queue.

Science.gov (United States)

Abdul-Wahid, Badi'; Yu, Li; Rajan, Dinesh; Feng, Haoyun; Darve, Eric; Thain, Douglas; Izaguirre, Jesús A

2012-10-01

Molecular modeling is a field that traditionally has large computational costs. Until recently, most simulation techniques relied on long trajectories, which inherently have poor scalability. A new class of methods is proposed that requires only a large number of short calculations, and for which minimal communication between computer nodes is required. We considered one of the more accurate variants called Accelerated Weighted Ensemble Dynamics (AWE) and for which distributed computing can be made efficient. We implemented AWE using the Work Queue framework for task management and applied it to an all atom protein model (Fip35 WW domain). We can run with excellent scalability by simultaneously utilizing heterogeneous resources from multiple computing platforms such as clouds (Amazon EC2, Microsoft Azure), dedicated clusters, grids, on multiple architectures (CPU/GPU, 32/64bit), and in a dynamic environment in which processes are regularly added or removed from the pool. This has allowed us to achieve an aggregate sampling rate of over 500 ns/hour. As a comparison, a single process typically achieves 0.1 ns/hour.

Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus.

Science.gov (United States)

Mutso, Margit; Morro, Ainhoa Moliner; Smedberg, Cecilia; Kasvandik, Sergo; Aquilimeba, Muriel; Teppor, Mona; Tarve, Liisi; Lulla, Aleksei; Lulla, Valeria; Saul, Sirle; Thaa, Bastian; McInerney, Gerald M; Merits, Andres; Varjak, Margus

2018-04-27

Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD) of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV) harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3.

Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

Energy Technology Data Exchange (ETDEWEB)

Xiang, Zhonghua; Qiao, Ling; Zhou, Yan [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada); Babiuk, Lorne A. [University of Alberta, Edmonton, Alberta (Canada); Liu, Qiang, E-mail: qiang.liu@usask.ca [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada)

2010-11-19

Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

International Nuclear Information System (INIS)

Xiang, Zhonghua; Qiao, Ling; Zhou, Yan; Babiuk, Lorne A.; Liu, Qiang

2010-01-01

Research highlights: → A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. → HCV-3a NS5A increases mature SREBP-1c protein level. → HCV-3a NS5A activates SREBP-1c transcription. → Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. → Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus

Directory of Open Access Journals (Sweden)

Margit Mutso

2018-04-01

Full Text Available Infection by Chikungunya virus (CHIKV of the Old World alphaviruses (family Togaviridae in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP (nsP1, nsp2, nsP3 and nsP4 that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3.

Characterization of purified Sindbis virus nsP4 RNA-dependent RNA polymerase activity in vitro

International Nuclear Information System (INIS)

Rubach, Jon K.; Wasik, Brian R.; Rupp, Jonathan C.; Kuhn, Richard J.; Hardy, Richard W.; Smith, Janet L.

2009-01-01

The Sindbis virus RNA-dependent RNA polymerase (nsP4) is responsible for the replication of the viral RNA genome. In infected cells, nsP4 is localized in a replication complex along with the other viral non-structural proteins. nsP4 has been difficult to homogenously purify from infected cells due to its interactions with the other replication proteins and the fact that its N-terminal residue, a tyrosine, causes the protein to be rapidly turned over in cells. We report the successful expression and purification of Sindbis nsP4 in a bacterial system, in which nsP4 is expressed as an N-terminal SUMO fusion protein. After purification the SUMO tag is removed, resulting in the isolation of full-length nsP4 possessing the authentic N-terminal tyrosine. This purified enzyme is able to produce minus-strand RNA de novo from plus-strand templates, as well as terminally add adenosine residues to the 3' end of an RNA substrate. In the presence of the partially processed viral replicase polyprotein, P123, purified nsP4 is able to synthesize discrete template length minus-strand RNA products. Mutations in the 3' CSE or poly(A) tail of viral template RNA prevent RNA synthesis by the replicase complex containing purified nsP4, consistent with previously reported template requirements for minus-strand RNA synthesis. Optimal reaction conditions were determined by investigating the effects of time, pH, and the concentrations of nsP4, P123 and magnesium on the synthesis of RNA

2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1.

Science.gov (United States)

Dhar, Jayeeta; Cuevas, Rolando A; Goswami, Ramansu; Zhu, Jianzhong; Sarkar, Saumendra N; Barik, Sailen

2015-10-01

2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Transactivation of a cellular promoter by the NS1 protein of the parvovirus minute virus of mice through a putative hormone-responsive element.

Science.gov (United States)

Vanacker, J M; Corbau, R; Adelmant, G; Perros, M; Laudet, V; Rommelaere, J

1996-01-01

The promoter of the thyroid hormone receptor alpha gene (c-erbA-1) is activated by the nonstructural protein 1 (NS1) of parvovirus minute virus of mice (prototype strain [MVMp]) in ras-transformed FREJ4 cells that are permissive for lytic MVMp replication. This stimulation may be related to the sensitivity of host cells to MVMp, as it does not take place in parental FR3T3 cells, which are resistant to the parvovirus killing effect. The analysis of a series of deletion and point mutants of the c-erbA-1 promoter led to the identification of an upstream region that is necessary for NS1-driven transactivation. This sequence harbors a putative hormone-responsive element and is sufficient to render a minimal promoter NS1 inducible in FREJ4 but not in FR3T3 cells, and it is involved in distinct interactions with proteins from the respective cell lines. The NS1-responsive element of the c-erbA-1 promoter bears no homology with sequences that were previously reported to be necessary for NS1 DNA binding and transactivation. Altogether, our data point to a novel, cell-specific mechanism of promoter activation by NS1. PMID:8642664

Phosphorylation states of cell cycle and DNA repair proteins can be altered by the nsSNPs

International Nuclear Information System (INIS)

Savas, Sevtap; Ozcelik, Hilmi

2005-01-01

Phosphorylation is a reversible post-translational modification that affects the intrinsic properties of proteins, such as structure and function. Non-synonymous single nucleotide polymorphisms (nsSNPs) result in the substitution of the encoded amino acids and thus are likely to alter the phosphorylation motifs in the proteins. In this study, we used the web-based NetPhos tool to predict candidate nsSNPs that either introduce or remove putative phosphorylation sites in proteins that act in DNA repair and cell cycle pathways. Our results demonstrated that a total of 15 nsSNPs (16.9%) were likely to alter the putative phosphorylation patterns of 14 proteins. Three of these SNPs (CDKN1A-S31R, OGG1-S326C, and XRCC3-T241M) have already found to be associated with altered cancer risk. We believe that this set of nsSNPs constitutes an excellent resource for further molecular and genetic analyses. The novel systematic approach used in this study will accelerate the understanding of how naturally occurring human SNPs may alter protein function through the modification of phosphorylation mechanisms and contribute to disease susceptibility

Inhibition of HCV replication by oxysterol-binding protein-related protein 4 (ORP4 through interaction with HCV NS5B and alteration of lipid droplet formation.

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In-Woo Park

Full Text Available Hepatitis C virus (HCV RNA replication involves complex interactions among the 3'x RNA element within the HCV 3' untranslated region, viral and host proteins. However, many of the host proteins remain unknown. In this study, we devised an RNA affinity chromatography /2D/MASS proteomics strategy and identified nine putative 3' X-associated host proteins; among them is oxysterol-binding protein-related protein 4 (ORP4, a cytoplasmic receptor for oxysterols. We determined the relationship between ORP4 expression and HCV replication. A very low level of constitutive ORP4 expression was detected in hepatocytes. Ectopically expressed ORP4 was detected in the endoplasmic reticulum and inhibited luciferase reporter gene expression in HCV subgenomic replicon cells and HCV core expression in JFH-1-infected cells. Expression of ORP4S, an ORP4 variant that lacked the N-terminal pleckstrin-homology domain but contained the C-terminal oxysterol-binding domain also inhibited HCV replication, pointing to an important role of the oxysterol-binding domain in ORP4-mediated inhibition of HCV replication. ORP4 was found to associate with HCV NS5B and its expression led to inhibition of the NS5B activity. ORP4 expression had little effect on intracellular lipid synthesis and secretion, but it induced lipid droplet formation in the context of HCV replication. Taken together, these results demonstrate that ORP4 is a negative regulator of HCV replication, likely via interaction with HCV NS5B in the replication complex and regulation of intracellular lipid homeostasis. This work supports the important role of lipids and their metabolism in HCV replication and pathogenesis.

Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

Science.gov (United States)

Fang, Jung-Da; Lee, Sheau-Ling

2014-07-01

Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation. Copyright © 2014. Published by Elsevier B.V.

Localisation system in wireless sensor networks using ns-2

CSIR Research Space (South Africa)

Abu-Mahfouz, Adnan M

2012-04-01

Full Text Available -1 /************************************************************************** ********** * * File: readme.asn * * Author: Adnan Abu-Mahfouz * * Date: March 2012 * * Description: Localisation system in wireless sensor networks using ns-2... *************************************************************************** *********/ /************************************************************************** *************************************************************************** *****/ 1. Introduction: ns-2 contains several flexible features that encourage researchers to use ns-2 to investigate the characteristics of wireless sensor networks (WSNs). However, to implement and evaluate localisation algorithms, the current ns- 2...

Characterization of a new antifungal non-specific lipid transfer protein (nsLTP) from sugar beet leaves

DEFF Research Database (Denmark)

Kristensen, A K; Brunstedt, J; Madsen, M T

2000-01-01

A novel protein (IWF5) comprising 92 amino acids has been purified from the intercellular washing fluid of sugar beet leaves using cation exchange chromatography and reversed phase high performance liquid chromatography. Based on amino acid sequence homology, including the presence of eight...... cysteines at conserved positions, the protein can be classified as a member of the plant family of non-specific lipid transfer proteins (nsLTPs). The protein is 47% identical to IWF1, an antifungal nsLTP previously isolated from leaves of sugar beet. A potential site for N-linked glycosylation present...

X-ray structure of the pestivirus NS3 helicase and its conformation in solution.

Science.gov (United States)

Tortorici, M Alejandra; Duquerroy, Stéphane; Kwok, Jane; Vonrhein, Clemens; Perez, Javier; Lamp, Benjamin; Bricogne, Gerard; Rümenapf, Till; Vachette, Patrice; Rey, Félix A

2015-04-01

Pestiviruses form a genus in the Flaviviridae family of small enveloped viruses with a positive-sense single-stranded RNA genome. Viral replication in this family requires the activity of a superfamily 2 RNA helicase contained in the C-terminal domain of nonstructural protein 3 (NS3). NS3 features two conserved RecA-like domains (D1 and D2) with ATPase activity, plus a third domain (D3) that is important for unwinding nucleic acid duplexes. We report here the X-ray structure of the pestivirus NS3 helicase domain (pNS3h) at a 2.5-Å resolution. The structure deviates significantly from that of NS3 of other genera in the Flaviviridae family in D3, as it contains two important insertions that result in a narrower nucleic acid binding groove. We also show that mutations in pNS3h that rescue viruses from which the core protein is deleted map to D3, suggesting that this domain may be involved in interactions that facilitate particle assembly. Finally, structural comparisons of the enzyme in different crystalline environments, together with the findings of small-angle X-ray-scattering studies in solution, show that D2 is mobile with respect to the rest of the enzyme, oscillating between closed and open conformations. Binding of a nonhydrolyzable ATP analog locks pNS3h in a conformation that is more compact than the closest apo-form in our crystals. Together, our results provide new insight and bring up new questions about pNS3h function during pestivirus replication. Although pestivirus infections impose an important toll on the livestock industry worldwide, little information is available about the nonstructural proteins essential for viral replication, such as the NS3 helicase. We provide here a comparative structural and functional analysis of pNS3h with respect to its orthologs in other viruses of the same family, the flaviviruses and hepatitis C virus. Our studies reveal differences in the nucleic acid binding groove that could have implications for understanding the

Activated protein C (APC) can increase bone anabolism via a protease-activated receptor (PAR)1/2 dependent mechanism.

Science.gov (United States)

Shen, Kaitlin; Murphy, Ciara M; Chan, Ben; Kolind, Mille; Cheng, Tegan L; Mikulec, Kathy; Peacock, Lauren; Xue, Meilang; Park, Sang-Youel; Little, David G; Jackson, Chris J; Schindeler, Aaron

2014-12-01

Activated Protein C (APC) is an anticoagulant with strong cytoprotective properties that has been shown to promote wound healing. In this study APC was investigated for its potential orthopedic application using a Bone Morphogenetic Protein 2 (rhBMP-2) induced ectopic bone formation model. Local co-administration of 10 µg rhBMP-2 with 10 µg or 25 µg APC increased bone volume at 3 weeks by 32% (N.S.) and 74% (pAPC are largely mediated by its receptors endothelial protein C receptor (EPCR) and protease-activated receptors (PARs). Cultured pre-osteoblasts and bone nodule tissue sections were shown to express PAR1/2 and EPCR. When pre-osteoblasts were treated with APC, cell viability and phosphorylation of ERK1/2, Akt, and p38 were increased. Inhibition with PAR1 and sometimes PAR2 antagonists, but not with EPCR blocking antibodies, ameliorated the effects of APC on cell viability and kinase phosphorylation. These data indicate that APC can affect osteoblast viability and signaling, and may have in vivo applications with rhBMP-2 for bone repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Green fluorescent protein (GFP color reporter gene visualizes parvovirus B19 non-structural segment 1 (NS1 transfected endothelial modification.

Directory of Open Access Journals (Sweden)

Thomas Wurster

Full Text Available BACKGROUND: Human Parvovirus B19 (PVB19 has been associated with myocarditis putative due to endothelial infection. Whether PVB19 infects endothelial cells and causes a modification of endothelial function and inflammation and, thus, disturbance of microcirculation has not been elucidated and could not be visualized so far. METHODS AND FINDINGS: To examine the PVB19-induced endothelial modification, we used green fluorescent protein (GFP color reporter gene in the non-structural segment 1 (NS1 of PVB19. NS1-GFP-PVB19 or GFP plasmid as control were transfected in an endothelial-like cell line (ECV304. The endothelial surface expression of intercellular-adhesion molecule-1 (CD54/ICAM-1 and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147 were evaluated by flow cytometry after NS-1-GFP or control-GFP transfection. To evaluate platelet adhesion on NS-1 transfected ECs, we performed a dynamic adhesion assay (flow chamber. NS-1 transfection causes endothelial activation and enhanced expression of ICAM-1 (CD54: mean ± standard deviation: NS1-GFP vs. control-GFP: 85.3 ± 11.2 vs. 61.6 ± 8.1; P<0.05 and induces endothelial expression of EMMPRIN/CD147 (CD147: mean ± SEM: NS1-GFP vs. control-GFP: 114 ± 15.3 vs. 80 ± 0.91; P<0.05 compared to control-GFP transfected cells. Dynamic adhesion assays showed that adhesion of platelets is significantly enhanced on NS1 transfected ECs when compared to control-GFP (P<0.05. The transfection of ECs was verified simultaneously through flow cytometry, immunofluorescence microscopy and polymerase chain reaction (PCR analysis. CONCLUSIONS: GFP color reporter gene shows transfection of ECs and may help to visualize NS1-PVB19 induced endothelial activation and platelet adhesion as well as an enhanced monocyte adhesion directly, providing in vitro evidence of possible microcirculatory dysfunction in PVB19-induced myocarditis and, thus, myocardial tissue damage.

Structural Basis for dsRNA Recognition by NS1 Protein of Influenza A Virus

Energy Technology Data Exchange (ETDEWEB)

Cheng, A.; Wong, S; Yuan, Y

2009-01-01

Influenza A viruses are important human pathogens causing periodic pandemic threats. Nonstructural protein 1 (NS1) protein of influenza A virus (NS1A) shields the virus against host defense. Here, we report the crystal structure of NS1A RNA-binding domain (RBD) bound to a double-stranded RNA (dsRNA) at 1.7A. NS1A RBD forms a homodimer to recognize the major groove of A-form dsRNA in a length-independent mode by its conserved concave surface formed by dimeric anti-parallel alpha-helices. dsRNA is anchored by a pair of invariable arginines (Arg38) from both monomers by extensive hydrogen bonds. In accordance with the structural observation, isothermal titration calorimetry assay shows that the unique Arg38-Arg38 pair and two Arg35-Arg46 pairs are crucial for dsRNA binding, and that Ser42 and Thr49 are also important for dsRNA binding. Agrobacterium co-infiltration assay further supports that the unique Arg38 pair plays important roles in dsRNA binding in vivo.

Development of a dual-protective live attenuated vaccine against H5N1 and H9N2 avian influenza viruses by modifying the NS1 gene.

Science.gov (United States)

Choi, Eun-hye; Song, Min-Suk; Park, Su-Jin; Pascua, Philippe Noriel Q; Baek, Yun Hee; Kwon, Hyeok-il; Kim, Eun-Ha; Kim, Semi; Jang, Hyung-Kwan; Poo, Haryoung; Kim, Chul-Joong; Choi, Young Ki

2015-07-01

An increasing number of outbreaks of avian influenza H5N1 and H9N2 viruses in poultry have caused serious economic losses and raised concerns for human health due to the risk of zoonotic transmission. However, licensed H5N1 and H9N2 vaccines for animals and humans have not been developed. Thus, to develop a dual H5N1 and H9N2 live-attenuated influenza vaccine (LAIV), the HA and NA genes from a virulent mouse-adapted avian H5N2 (A/WB/Korea/ma81/06) virus and a recently isolated chicken H9N2 (A/CK/Korea/116/06) virus, respectively, were introduced into the A/Puerto Rico/8/34 backbone expressing truncated NS1 proteins (NS1-73, NS1-86, NS1-101, NS1-122) but still possessing a full-length NS gene. Two H5N2/NS1-LAIV viruses (H5N2/NS1-86 and H5N2/NS1-101) were highly attenuated compared with the full-length and remaining H5N2/NS-LAIV viruses in a mouse model. Furthermore, viruses containing NS1 modifications were found to induce more IFN-β activation than viruses with full-length NS1 proteins and were correspondingly attenuated in mice. Intranasal vaccination with a single dose (10(4.0) PFU/ml) of these viruses completely protected mice from a lethal challenge with the homologous A/WB/Korea/ma81/06 (H5N2), heterologous highly pathogenic A/EM/Korea/W149/06 (H5N1), and heterosubtypic highly virulent mouse-adapted H9N2 viruses. This study clearly demonstrates that the modified H5N2/NS1-LAIV viruses attenuated through the introduction of mutations in the NS1 coding region display characteristics that are desirable for live attenuated vaccines and hold potential as vaccine candidates for mammalian hosts.

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

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Markus M. Knodel

2018-01-01

Full Text Available Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface.

Science.gov (United States)

Knodel, Markus M; Nägel, Arne; Reiter, Sebastian; Vogel, Andreas; Targett-Adams, Paul; McLauchlan, John; Herrmann, Eva; Wittum, Gabriel

2018-01-08

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

Science.gov (United States)

Nägel, Arne; Reiter, Sebastian; Vogel, Andreas; McLauchlan, John; Herrmann, Eva; Wittum, Gabriel

2018-01-01

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles. PMID:29316722

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

KAUST Repository

Knodel, Markus

2018-01-08

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

Nonstructural protein 2 (nsP2) of Chikungunya virus (CHIKV) enhances protective immunity mediated by a CHIKV envelope protein expressing DNA Vaccine.

Science.gov (United States)

Bao, Huihui; Ramanathan, Aarti A; Kawalakar, Omkar; Sundaram, Senthil G; Tingey, Colleen; Bian, Charoran B; Muruganandam, Nagarajan; Vijayachari, Paluru; Sardesai, Niranjan Y; Weiner, David B; Ugen, Kenneth E; Muthumani, Karuppiah

2013-02-01

Chikungunya virus (CHIKV) is an important emerging mosquito-borne alphavirus, indigenous to tropical Africa and Asia. It can cause epidemic fever and acute illness characterized by fever and arthralgias. The epidemic cycle of this infection is similar to dengue and urban yellow fever viral infections. The generation of an efficient vaccine against CHIKV is necessary to prevent and/or control the disease manifestations of the infection. In this report, we studied immune response against a CHIKV-envelope DNA vaccine (pEnv) and the role of the CHIKV nonstructural gene 2 (nsP2) as an adjuvant for the induction of protective immune responses in a relevant mouse challenge model. When injected with the CHIKV pEnv alone, 70% of the immunized mice survived CHIKV challenge, whereas when co-injected with pEnv+pnsP2, 90% of the mice survived viral challenge. Mice also exhibited a delayed onset signs of illness, and a marked decrease in morbidity, suggesting a nsP2 mediated adjuvant effect. Co-injection of the pnsP2 adjuvant with pEnv also qualitatively and quantitatively increased antigen specific neutralizing antibody responses compared to vaccination with pEnv alone. In sum, these novel data imply that the addition of nsP2 to the pEnv vaccine enhances anti-CHIKV-Env immune responses and maybe useful to include in future CHIKV clinical vaccination strategies.

Structure-based discovery of clinically approved drugs as Zika virus NS2B-NS3 protease inhibitors that potently inhibit Zika virus infection in vitro and in vivo.

Science.gov (United States)

Yuan, Shuofeng; Chan, Jasper Fuk-Woo; den-Haan, Helena; Chik, Kenn Ka-Heng; Zhang, Anna Jinxia; Chan, Chris Chung-Sing; Poon, Vincent Kwok-Man; Yip, Cyril Chik-Yan; Mak, Winger Wing-Nga; Zhu, Zheng; Zou, Zijiao; Tee, Kah-Meng; Cai, Jian-Piao; Chan, Kwok-Hung; de la Peña, Jorge; Pérez-Sánchez, Horacio; Cerón-Carrasco, José Pedro; Yuen, Kwok-Yung

2017-09-01

Zika virus (ZIKV) infection may be associated with severe complications in fetuses and adults, but treatment options are limited. We performed an in silico structure-based screening of a large chemical library to identify potential ZIKV NS2B-NS3 protease inhibitors. Clinically approved drugs belonging to different drug classes were selected among the 100 primary hit compounds with the highest predicted binding affinities to ZIKV NS2B-NS3-protease for validation studies. ZIKV NS2B-NS3 protease inhibitory activity was validated in most of the selected drugs and in vitro anti-ZIKV activity was identified in two of them (novobiocin and lopinavir-ritonavir). Molecular docking and molecular dynamics simulations predicted that novobiocin bound to ZIKV NS2B-NS3-protease with high stability. Dexamethasone-immunosuppressed mice with disseminated ZIKV infection and novobiocin treatment had significantly (P < 0.05) higher survival rate (100% vs 0%), lower mean blood and tissue viral loads, and less severe histopathological changes than untreated controls. This structure-based drug discovery platform should facilitate the identification of additional enzyme inhibitors of ZIKV. Copyright © 2017 Elsevier B.V. All rights reserved.

Evolution of Bacterial Global Modulators: Role of a Novel H-NS Paralogue in the Enteroaggregative Escherichia coli Strain 042.

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Prieto, A; Bernabeu, M; Aznar, S; Ruiz-Cruz, S; Bravo, A; Queiroz, M H; Juárez, A

2018-01-01

Bacterial genomes sometimes contain genes that code for homologues of global regulators, the function of which is unclear. In members of the family Enterobacteriaceae , cells express the global regulator H-NS and its paralogue StpA. In Escherichia coli , out of providing a molecular backup for H-NS, the role of StpA is poorly characterized. The enteroaggregative E. coli strain 042 carries, in addition to the hns and stpA genes, a third gene encoding an hns paralogue ( hns2 ). We present in this paper information about its biological function. Transcriptomic analysis has shown that the H-NS2 protein targets a subset of the genes targeted by H-NS. Genes targeted by H-NS2 correspond mainly with horizontally transferred (HGT) genes and are also targeted by the Hha protein, a fine-tuner of H-NS activity. Compared with H-NS, H-NS2 expression levels are lower. In addition, H-NS2 expression exhibits specific features: it is sensitive to the growth temperature and to the nature of the culture medium. This novel H-NS paralogue is widespread within the Enterobacteriaceae . IMPORTANCE Global regulators such as H-NS play key relevant roles enabling bacterial cells to adapt to a changing environment. H-NS modulates both core and horizontally transferred (HGT) genes, but the mechanism by which H-NS can differentially regulate these genes remains to be elucidated. There are several instances of bacterial cells carrying genes that encode homologues of the global regulators. The question is what the roles of these proteins are. We noticed that the enteroaggregative E. coli strain 042 carries a new hitherto uncharacterized copy of the hns gene. We decided to investigate why this pathogenic E. coli strain requires an extra H-NS paralogue, termed H-NS2. In our work, we show that H-NS2 displays specific expression and regulatory properties. H-NS2 targets a subset of H-NS-specific genes and may help to differentially modulate core and HGT genes by the H-NS cellular pool.

Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses.

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Clark, Amelia M; Nogales, Aitor; Martinez-Sobrido, Luis; Topham, David J; DeDiego, Marta L

2017-09-01

In 2009, a novel H1N1 influenza virus emerged in humans, causing a global pandemic. It was previously shown that the NS1 protein from this human 2009 pandemic H1N1 (pH1N1) virus was an effective interferon (IFN) antagonist but could not inhibit general host gene expression, unlike other NS1 proteins from seasonal human H1N1 and H3N2 viruses. Here we show that the NS1 protein from currently circulating pH1N1 viruses has evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) with respect to the original protein. Notably, these 6 residue changes restore the ability of pH1N1 NS1 to inhibit general host gene expression, mainly by their ability to restore binding to the cellular factor CPSF30. This is the first report describing the ability of the pH1N1 NS1 protein to naturally acquire mutations that restore this function. Importantly, a recombinant pH1N1 virus containing these 6 amino acid changes in the NS1 protein (pH1N1/NSs-6mut) inhibited host IFN and proinflammatory responses to a greater extent than that with the parental virus (pH1N1/NS1-wt), yet virus titers were not significantly increased in cell cultures or in mouse lungs, and the disease was partially attenuated. The pH1N1/NSs-6mut virus grew similarly to pH1N1/NSs-wt in mouse lungs, but infection with pH1N1/NSs-6mut induced lower levels of proinflammatory cytokines, likely due to a general inhibition of gene expression mediated by the mutated NS1 protein. This lower level of inflammation induced by the pH1N1/NSs-6mut virus likely accounts for the attenuated disease phenotype and may represent a host-virus adaptation affecting influenza virus pathogenesis. IMPORTANCE Seasonal influenza A viruses (IAVs) are among the most common causes of respiratory infections in humans. In addition, occasional pandemics are caused when IAVs circulating in other species emerge in the human population. In 2009, a swine-origin H1N1 IAV (pH1N1) was transmitted to humans, infecting people then and up

Interference of transcription across H-NS binding sites and repression by H-NS.

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Rangarajan, Aathmaja Anandhi; Schnetz, Karin

2018-05-01

Nucleoid-associated protein H-NS represses transcription by forming extended DNA-H-NS complexes. Repression by H-NS operates mostly at the level of transcription initiation. Less is known about how DNA-H-NS complexes interfere with transcription elongation. In vitro H-NS has been shown to enhance RNA polymerase pausing and to promote Rho-dependent termination, while in vivo inhibition of Rho resulted in a decrease of the genome occupancy by H-NS. Here we show that transcription directed across H-NS binding regions relieves H-NS (and H-NS/StpA) mediated repression of promoters in these regions. Further, we observed a correlation of transcription across the H-NS-bound region and de-repression. The data suggest that the transcribing RNA polymerase is able to remodel the H-NS complex and/or dislodge H-NS from the DNA and thus relieve repression. Such an interference of transcription and H-NS mediated repression may imply that poorly transcribed AT-rich loci are prone to be repressed by H-NS, while efficiently transcribed loci escape repression. © 2018 John Wiley & Sons Ltd.

Purification and crystallization of dengue and West Nile virus NS2B–NS3 complexes

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D’Arcy, Allan, E-mail: allan.darcy@novartis.com; Chaillet, Maxime; Schiering, Nikolaus; Villard, Frederic [Novartis Institutes of Biomedical Research, Protease Platform, Klybeckstrasse 144, CH 4002 Basel (Switzerland); Lim, Siew Pheng [Novartis Institutes of Tropical Diseases (Singapore); Lefeuvre, Peggy [Novartis Institutes of Biomedical Research, Protease Platform, Klybeckstrasse 144, CH 4002 Basel (Switzerland); Erbel, Paul [Novartis Institutes of Biomedical Research, Protease Platform, Klybeckstrasse 144, CH 4002 Basel (Switzerland); Novartis Institutes of Tropical Diseases (Singapore)

2006-02-01

Crystals of dengue serotype 2 and West Nile virus NS2B–NS3 protease complexes have been obtained and the crystals of both diffract to useful resolution. Sample homogeneity was essential for obtaining X-ray-quality crystals of the dengue protease. Controlled proteolysis produced a crystallizable fragment of the apo West Nile virus NS2B–NS3 and crystals were also obtained in the presence of a peptidic inhibitor. Both dengue and West Nile virus infections are an increasing risk to humans, not only in tropical and subtropical areas, but also in North America and parts of Europe. These viral infections are generally transmitted by mosquitoes, but may also be tick-borne. Infection usually results in mild flu-like symptoms, but can also cause encephalitis and fatalities. Approximately 2799 severe West Nile virus cases were reported this year in the United States, resulting in 102 fatalities. With this alarming increase in the number of West Nile virus infections in western countries and the fact that dengue virus already affects millions of people per year in tropical and subtropical climates, there is a real need for effective medicines. A possible therapeutic target to combat these viruses is the protease, which is essential for virus replication. In order to provide structural information to help to guide a lead identification and optimization program, crystallizations of the NS2B–NS3 protease complexes from both dengue and West Nile viruses have been initiated. Crystals that diffract to high resolution, suitable for three-dimensional structure determinations, have been obtained.

Purification and crystallization of dengue and West Nile virus NS2B–NS3 complexes

International Nuclear Information System (INIS)

D’Arcy, Allan; Chaillet, Maxime; Schiering, Nikolaus; Villard, Frederic; Lim, Siew Pheng; Lefeuvre, Peggy; Erbel, Paul

2006-01-01

Crystals of dengue serotype 2 and West Nile virus NS2B–NS3 protease complexes have been obtained and the crystals of both diffract to useful resolution. Sample homogeneity was essential for obtaining X-ray-quality crystals of the dengue protease. Controlled proteolysis produced a crystallizable fragment of the apo West Nile virus NS2B–NS3 and crystals were also obtained in the presence of a peptidic inhibitor. Both dengue and West Nile virus infections are an increasing risk to humans, not only in tropical and subtropical areas, but also in North America and parts of Europe. These viral infections are generally transmitted by mosquitoes, but may also be tick-borne. Infection usually results in mild flu-like symptoms, but can also cause encephalitis and fatalities. Approximately 2799 severe West Nile virus cases were reported this year in the United States, resulting in 102 fatalities. With this alarming increase in the number of West Nile virus infections in western countries and the fact that dengue virus already affects millions of people per year in tropical and subtropical climates, there is a real need for effective medicines. A possible therapeutic target to combat these viruses is the protease, which is essential for virus replication. In order to provide structural information to help to guide a lead identification and optimization program, crystallizations of the NS2B–NS3 protease complexes from both dengue and West Nile viruses have been initiated. Crystals that diffract to high resolution, suitable for three-dimensional structure determinations, have been obtained

Hepatitis C virus non-structural protein 3 interacts with cytosolic 5'(3'-deoxyribonucleotidase and partially inhibits its activity.

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Chiu-Ping Fang

Full Text Available Infection with hepatitis C virus (HCV is etiologically involved in liver cirrhosis, hepatocellular carcinoma and B-cell lymphomas. It has been demonstrated previously that HCV non-structural protein 3 (NS3 is involved in cell transformation. In this study, a yeast two-hybrid screening experiment was conducted to identify cellular proteins interacting with HCV NS3 protein. Cytosolic 5'(3'-deoxyribonucleotidase (cdN, dNT-1 was found to interact with HCV NS3 protein. Binding domains of HCV NS3 and cellular cdN proteins were also determined using the yeast two-hybrid system. Interactions between HCV NS3 and cdN proteins were further demonstrated by co-immunoprecipitation and confocal analysis in cultured cells. The cellular cdN activity was partially repressed by NS3 protein in both the transiently-transfected and the stably-transfected systems. Furthermore, HCV partially repressed the cdN activity while had no effect on its protein expression in the systems of HCV sub-genomic replicons and infectious HCV virions. Deoxyribonucleotidases are present in most mammalian cells and involve in the regulation of intracellular deoxyribonucleotides pools by substrate cycles. Control of DNA precursor concentration is essential for the maintenance of genetic stability. Reduction of cdN activity would result in the imbalance of DNA precursor concentrations. Thus, our results suggested that HCV partially reduced the cdN activity via its NS3 protein and this may in turn cause diseases.

Bioinformatic Analysis of Deleterious Non-Synonymous Single Nucleotide Polymorphisms (nsSNPs in the Coding Regions of Human Prion Protein Gene (PRNP

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Kourosh Bamdad

2016-12-01

Full Text Available Background & Objective: Single nucleotide polymorphisms are the cause of genetic variation to living organisms. Single nucleotide polymorphisms alter residues in the protein sequence. In this investigation, the relationship between prion protein gene polymorphisms and its relevance to pathogenicity was studied. Material & Method: Amino acid sequence of the main isoform from the human prion protein gene (PRNP was extracted from UniProt database and evaluated by FoldAmyloid and AmylPred servers. All non-synonymous single nucleotide polymorphisms (nsSNPs from SNP database (dbSNP were further analyzed by bioinformatics servers including SIFT, PolyPhen-2, I-Mutant-3.0, PANTHER, SNPs & GO, PHD-SNP, Meta-SNP, and MutPred to determine the most damaging nsSNPs. Results: The results of the first structure analyses by FoldAmyloid and AmylPerd servers implied that regions including 5-15, 174-178, 180-184, 211-217, and 240-252 were the most sensitive parts of the protein sequence to amyloidosis. Screening all nsSNPs of the main protein isoform using bioinformatic servers revealed that substitution of Aspartic acid with Valine at position 178 (ID code: rs11538766 was the most deleterious nsSNP in the protein structure. Conclusion:  Substitution of the Aspartic acid with Valine at position 178 (D178V was the most pathogenic mutation in the human prion protein gene. Analyses from the MutPred server also showed that beta-sheets’ increment in the secondary structure was the main reason behind the molecular mechanism of the prion protein aggregation.

Structural features of NS3 of Dengue virus serotypes 2 and 4 in solution and insight into RNA binding and the inhibitory role of quercetin.

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Pan, Ankita; Saw, Wuan Geok; Subramanian Manimekalai, Malathy Sony; Grüber, Ardina; Joon, Shin; Matsui, Tsutomu; Weiss, Thomas M; Grüber, Gerhard

2017-05-01

Dengue virus (DENV), which has four serotypes (DENV-1 to DENV-4), is the causative agent of the viral infection dengue. DENV nonstructural protein 3 (NS3) comprises a serine protease domain and an RNA helicase domain which has nucleotide triphosphatase activities that are essential for RNA replication and viral assembly. Here, solution X-ray scattering was used to provide insight into the overall structure and flexibility of the entire NS3 and its recombinant helicase and protease domains for Dengue virus serotypes 2 and 4 in solution. The DENV-2 and DENV-4 NS3 forms are elongated and flexible in solution. The importance of the linker residues in flexibility and domain-domain arrangement was shown by the compactness of the individual protease and helicase domains. Swapping of the 174 PPAVP 179 linker stretch of the related Hepatitis C virus (HCV) NS3 into DENV-2 NS3 did not alter the elongated shape of the engineered mutant. Conformational alterations owing to RNA binding are described in the protease domain, which undergoes substantial conformational alterations that are required for the optimal catalysis of bound RNA. Finally, the effects of ATPase inhibitors on the enzymatically active DENV-2 and DENV-4 NS3 and the individual helicases are presented, and insight into the allosteric effect of the inhibitor quercetin is provided.

The Future of HCV Therapy: NS4B as an Antiviral Target

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Hadas Dvory-Sobol

2010-11-01

Full Text Available Chronic hepatitis C virus (HCV infection is a major worldwide cause of liver disease, including cirrhosis and hepatocellular carcinoma. It is estimated that more than 170 million individuals are infected with HCV, with three to four million new cases each year. The current standard of care, combination treatment with interferon and ribavirin, eradicates the virus in only about 50% of chronically infected patients. Notably, neither of these drugs directly target HCV. Many new antiviral therapies that specifically target hepatitis C (e.g. NS3 protease or NS5B polymerase inhibitors are therefore in development, with a significant number having advanced into clinical trials. The nonstructural 4B (NS4B protein, is among the least characterized of the HCV structural and nonstructural proteins and has been subjected to few pharmacological studies. NS4B is an integral membrane protein with at least four predicted transmembrane (TM domains. A variety of functions have been postulated for NS4B, such as the ability to induce the membranous web replication platform, RNA binding and NTPase activity. This review summarizes potential targets within the nonstructural protein NS4B, with a focus on novel classes of NS4B inhibitors.

Supersymmetric non-singular fractional D2-branes and NS-NS 2-branes

International Nuclear Information System (INIS)

Cvetic, M.; Gibbons, G.W.; Lue, H.; Pope, C.N.

2001-01-01

We obtain regular deformed D2-brane solutions with fractional D2-branes arising as wrapped D4-branes. The space transverse to the D2-brane is a complete Ricci-flat 7-manifold of G 2 holonomy, which is asymptotically conical with principal orbits that are topologically CP 3 or the flag manifold SU(3)/(U(1)xU(1)). We obtain the solution by first constructing an L 2 normalisable harmonic 3-form. We also review a previously-obtained regular deformed D2-brane whose transverse space is a different 7-manifold of G 2 holonomy, with principal orbits that are topologically S 3 xS 3 . This describes D2-branes with fractional NS-NS 2-branes coming from the wrapping of 5-branes, which is supported by a non-normalisable harmonic 3-form on the 7-manifold. We prove that both types of solutions are supersymmetric, preserving 1/16 of the maximal supersymmetry and hence that they are dual to N=1 three-dimensional gauge theories. In each case, the spectrum for minimally-coupled scalars is discrete, indicating confinement in the infrared region of the dual gauge theories. We examine resolutions of other branes, and obtain necessary conditions for their regularity. The resolution of many of these seems to lie beyond supergravity. In the process of studying these questions, we construct new explicit examples of complete Ricci-flat metrics

Ebselen inhibits hepatitis C virus NS3 helicase binding to nucleic acid and prevents viral replication.

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Mukherjee, Sourav; Weiner, Warren S; Schroeder, Chad E; Simpson, Denise S; Hanson, Alicia M; Sweeney, Noreena L; Marvin, Rachel K; Ndjomou, Jean; Kolli, Rajesh; Isailovic, Dragan; Schoenen, Frank J; Frick, David N

2014-10-17

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 μM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 μM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure-activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines.

Correlation of random urine protein creatinine (P-C ratio with 24-hour urine protein and P-C ratio, based on physical activity: a pilot study

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Seyed-Ali Sadjadi

2010-07-01

Full Text Available Seyed-Ali Sadjadi1,2, Navin Jaipaul1,21Jerry L Pettis Memorial VA Medical Center, 2Loma Linda University School of Medicine, Loma Linda, CA, USAAbstract: Quantification of proteinuria is usually predicated upon 24-hour urine collection. Multiple factors influence urine collection and the rate of protein and creatinine excretion. Urine collection is often incomplete, and therefore creatinine and protein excretion rates are underestimated. A random urine protein-creatinine (P-C ratio has been shown over the years to be a reliable alternative to the 24-hour collection for detection and follow up of proteinuria. However, urine protein excretion may be influenced by physical activity. We studied 48 patients with proteinuria and varying levels of physical activity to determine the correlation between the measures of urine protein excretion. The correlation coefficient (r between 24-hour urine total protein and random urine P-C ratio was 0.75 (P < 0.01 in the overall study population, but varied according to the level of proteinuria and physical activity in a stratified analysis: r = 0.99 (P < 0.001 and r = 0.95 (P < 0.01 in bedridden patients; r = 0.44 (P = not significant [NS] and r = 0.54 (P = NS in semiactive patients; and r = 0.44 (P = NS and r = 0.58 (P < 0.05 in active patients with nephrotic- (>3500 mg/day and non-nephrotic (<3500 mg/day range proteinuria, respectively. The correlation appeared to be stronger between random urine and 24-hour urine P-C ratio for the overall study population (r = 0.84; P < 0.001, and when stratified according to the level of proteinuria and physical activity: r = 0.99 (P < 0.001 and r = 0.92 (P < 0.01 in bedridden patients; r = 0.61 (P = NS and r = 0.54 (P = NS in semiactive patients; and r = 0.64 (P < 0.02 and r = 0.52 (P < 0.05 in active patients with nephrotic and non-nephrotic range proteinuria, respectively. We conclude that the random urine P-C ratio is a reliable and practical way of estimating and

Discovery of a novel compound with anti-venezuelan equine encephalitis virus activity that targets the nonstructural protein 2.

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Dong-Hoon Chung

2014-06-01

Full Text Available Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV, a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM, for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.

Comparative Investigation of Normal Modes and Molecular Dynamics of Hepatitis C NS5B Protein

International Nuclear Information System (INIS)

Asafi, M S; Tekpinar, M; Yildirim, A

2016-01-01

Understanding dynamics of proteins has many practical implications in terms of finding a cure for many protein related diseases. Normal mode analysis and molecular dynamics methods are widely used physics-based computational methods for investigating dynamics of proteins. In this work, we studied dynamics of Hepatitis C NS5B protein with molecular dynamics and normal mode analysis. Principal components obtained from a 100 nanoseconds molecular dynamics simulation show good overlaps with normal modes calculated with a coarse-grained elastic network model. Coarse-grained normal mode analysis takes at least an order of magnitude shorter time. Encouraged by this good overlaps and short computation times, we analyzed further low frequency normal modes of Hepatitis C NS5B. Motion directions and average spatial fluctuations have been analyzed in detail. Finally, biological implications of these motions in drug design efforts against Hepatitis C infections have been elaborated. (paper)

In-Silico Computing of the Most Deleterious nsSNPs in HBA1 Gene.

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Sayed AbdulAzeez

Full Text Available α-Thalassemia (α-thal is a genetic disorder caused by the substitution of single amino acid or large deletions in the HBA1 and/or HBA2 genes.Using modern bioinformatics tools as a systematic in-silico approach to predict the deleterious SNPs in the HBA1 gene and its significant pathogenic impact on the functions and structure of HBA1 protein was predicted.A total of 389 SNPs in HBA1 were retrieved from dbSNP database, which includes: 201 non-coding synonymous (nsSNPs, 43 human active SNPs, 16 intronic SNPs, 11 mRNA 3' UTR SNPs, 9 coding synonymous SNPs, 9 5' UTR SNPs and other types. Structural homology-based method (PolyPhen and sequence homology-based tool (SIFT, SNPs&Go, PROVEAN and PANTHER revealed that 2.4% of the nsSNPs are pathogenic.A total of 5 nsSNPs (G60V, K17M, K17T, L92F and W15R were predicted to be responsible for the structural and functional modifications of HBA1 protein. It is evident from the deep comprehensive in-silico analysis that, two nsSNPs such as G60V and W15R in HBA1 are highly deleterious. These "2 pathogenic nsSNPs" can be considered for wet-lab confirmatory analysis.

New binding site conformations of the dengue virus NS3 protease accessed by molecular dynamics simulation.

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Hugo de Almeida

Full Text Available Dengue fever is caused by four distinct serotypes of the dengue virus (DENV1-4, and is estimated to affect over 500 million people every year. Presently, there are no vaccines or antiviral treatments for this disease. Among the possible targets to fight dengue fever is the viral NS3 protease (NS3PRO, which is in part responsible for viral processing and replication. It is now widely recognized that virtual screening campaigns should consider the flexibility of target protein by using multiple active conformational states. The flexibility of the DENV NS3PRO could explain the relatively low success of previous virtual screening studies. In this first work, we explore the DENV NS3PRO conformational states obtained from molecular dynamics (MD simulations to take into account protease flexibility during the virtual screening/docking process. To do so, we built a full NS3PRO model by multiple template homology modeling. The model comprised the NS2B cofactor (essential to the NS3PRO activation, a glycine flexible link and the proteolytic domain. MD simulations had the purpose to sample, as closely as possible, the ligand binding site conformational landscape prior to inhibitor binding. The obtained conformational MD sample was clustered into four families that, together with principal component analysis of the trajectory, demonstrated protein flexibility. These results allowed the description of multiple binding modes for the Bz-Nle-Lys-Arg-Arg-H inhibitor, as verified by binding plots and pair interaction analysis. This study allowed us to tackle protein flexibility in our virtual screening campaign against the dengue virus NS3 protease.

Suppression of Rac1 Signaling by Influenza A Virus NS1 Facilitates Viral Replication

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Jiang, Wei; Sheng, Chunjie; Gu, Xiuling; Liu, Dong; Yao, Chen; Gao, Shijuan; Chen, Shuai; Huang, Yinghui; Huang, Wenlin; Fang, Min

2016-01-01

Influenza A virus (IAV) is a major human pathogen with the potential to become pandemic. IAV contains only eight RNA segments; thus, the virus must fully exploit the host cellular machinery to facilitate its own replication. In an effort to comprehensively characterize the host machinery taken over by IAV in mammalian cells, we generated stable A549 cell lines with over-expression of the viral non-structural protein (NS1) to investigate the potential host factors that might be modulated by the NS1 protein. We found that the viral NS1 protein directly interacted with cellular Rac1 and facilitated viral replication. Further research revealed that NS1 down-regulated Rac1 activity via post-translational modifications. Therefore, our results demonstrated that IAV blocked Rac1-mediated host cell signal transduction through the NS1 protein to facilitate its own replication. Our findings provide a novel insight into the mechanism of IAV replication and indicate new avenues for the development of potential therapeutic targets. PMID:27869202

Efficacy of NS-018, a potent and selective JAK2/Src inhibitor, in primary cells and mouse models of myeloproliferative neoplasms

International Nuclear Information System (INIS)

Nakaya, Y; Shide, K; Niwa, T; Homan, J; Sugahara, S; Horio, T; Kuramoto, K; Kotera, T; Shibayama, H; Hori, K; Naito, H; Shimoda, K

2011-01-01

Aberrant activation of Janus kinase 2 (JAK2) caused by somatic mutation of JAK2 (JAK2V617F) or the thrombopoietin receptor (MPLW515L) plays an essential role in the pathogenesis of myeloproliferative neoplasms (MPNs), suggesting that inhibition of aberrant JAK2 activation would have a therapeutic benefit. Our novel JAK2 inhibitor, NS-018, was highly active against JAK2 with a 50% inhibition (IC 50 ) of <1 n, and had 30–50-fold greater selectivity for JAK2 over other JAK-family kinases, such as JAK1, JAK3 and tyrosine kinase 2. In addition to JAK2, NS-018 inhibited Src-family kinases. NS-018 showed potent antiproliferative activity against cell lines expressing a constitutively activated JAK2 (the JAK2V617F or MPLW515L mutations or the TEL–JAK2 fusion gene; IC 50 =11–120 n), but showed only minimal cytotoxicity against most other hematopoietic cell lines without a constitutively activated JAK2. Furthermore, NS-018 preferentially suppressed in vitro erythropoietin-independent endogenous colony formation from polycythemia vera patients. NS-018 also markedly reduced splenomegaly and prolonged the survival of mice inoculated with Ba/F3 cells harboring JAK2V617F. In addition, NS-018 significantly reduced leukocytosis, hepatosplenomegaly and extramedullary hematopoiesis, improved nutritional status, and prolonged survival in JAK2V617F transgenic mice. These results suggest that NS-018 will be a promising candidate for the treatment of MPNs

The reliability of DIVA test based on M2e peptide exceed those based on HA2 or NS1 peptides

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Simson Tarigan

2015-06-01

Full Text Available One of the most important disadvantage of vaccination against avian influenza is that it cannot protect vaccinated birds against infection. When vaccinated poultry are heavily exposed to the virus, prolonged, unrecognised, subclinical infection may persist on the farm. The condition can only be serologically monitored by a DIVA (differentiation of infected from vaccinated animals test, whereas conventional diagnostic tests cannot be used. The DIVA tests based on an antibody response following virus replication is the most appropriate approach. For H5N1 influenza such antibodies includes those to the M2e and NS1 proteins and an epitope on the HA2 subunit (HA_488-516. The purpose of this study was to compare the magnitude of the antibody response in chickens vaccinated and infected with an H5N1 virus strain. For that purpose, sera collected from naïve, vaccinated and infected birds, at 1, 2-3, ≥4 weeks post challenge were used. Antibodies were measured by ELISA using biotinylated synthetic peptides as coating antigens. The peptides used include four NS1 peptides corresponding to different regions of the NS1 protein and HA_488-516and M2e peptides. Peptides were coated onto microtitre plates either directly or via a streptavidin bridge. The results showed that vaccination did not cause antibody conversion to any of the peptides, where as challenged birds developed a high antibody response to M2e but, low response to the NS1 and HA2 peptides. Antibodies to the later peptides were detected only by the streptavidin-peptide ELISA. The ELISA based on NS1 or HA_488-516 peptides, therefore, are not reliable for use as DIVA test in H5N1 avian influenza virus infection

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

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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

Correlation of random urine protein creatinine (P-C) ratio with 24-hour urine protein and P-C ratio, based on physical activity: a pilot study.

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Sadjadi, Seyed-Ali; Jaipaul, Navin

2010-09-07

Quantification of proteinuria is usually predicated upon 24-hour urine collection. Multiple factors influence urine collection and the rate of protein and creatinine excretion. Urine collection is often incomplete, and therefore creatinine and protein excretion rates are underestimated. A random urine protein-creatinine (P-C) ratio has been shown over the years to be a reliable alternative to the 24-hour collection for detection and follow up of proteinuria. However, urine protein excretion may be influenced by physical activity. We studied 48 patients with proteinuria and varying levels of physical activity to determine the correlation between the measures of urine protein excretion. The correlation coefficient (r) between 24-hour urine total protein and random urine P-C ratio was 0.75 (P r = 0.99 (P r = 0.95 (P bedridden patients; r = 0.44 (P = not significant [NS]) and r = 0.54 (P = NS) in semiactive patients; and r = 0.44 (P = NS) and r = 0.58 (P 3500 mg/day) and non-nephrotic (r = 0.84; P r = 0.99 (P r = 0.92 (P bedridden patients; r = 0.61 (P = NS) and r = 0.54 (P = NS) in semiactive patients; and r = 0.64 (P r = 0.52 (P < 0.05) in active patients with nephrotic and non-nephrotic range proteinuria, respectively. We conclude that the random urine P-C ratio is a reliable and practical way of estimating and following proteinuria, but its precision and accuracy may be affected by the level of patient physical activity.

Antiviral Activity and Resistance Analysis of NS3/4A Protease Inhibitor Grazoprevir and NS5A Inhibitor Elbasvir in Hepatitis C Virus GT4 Replicons.

Science.gov (United States)

Asante-Appiah, Ernest; Curry, Stephanie; McMonagle, Patricia; Ingravallo, Paul; Chase, Robert; Nickle, David; Qiu, Ping; Howe, Anita; Lahser, Frederick C

2017-07-01

Although genotype 4 (GT4)-infected patients represent a minor overall percentage of the global hepatitis C virus (HCV)-infected population, the high prevalence of the genotype in specific geographic regions coupled with substantial sequence diversity makes it an important genotype to study for antiviral drug discovery and development. We evaluated two direct-acting antiviral agents-grazoprevir, an HCV NS3/4A protease inhibitor, and elbasvir, an HCV NS5A inhibitor-in GT4 replicons prior to clinical studies in this genotype. Following a bioinformatics analysis of available GT4 sequences, a set of replicons bearing representative GT4 clinical isolates was generated. For grazoprevir, the 50% effective concentration (EC 50 ) against the replicon bearing the reference GT4a (ED43) NS3 protease and NS4A was 0.7 nM. The median EC 50 for grazoprevir against chimeric replicons encoding NS3/4A sequences from GT4 clinical isolates was 0.2 nM (range, 0.11 to 0.33 nM; n = 5). The difficulty in establishing replicons bearing NS3/4A resistance-associated substitutions was substantially overcome with the identification of a G162R adaptive substitution in NS3. Single NS3 substitutions D168A/V identified from de novo resistance selection studies reduced grazoprevir antiviral activity by 137- and 47-fold, respectively, in the background of the G162R replicon. For elbasvir, the EC 50 against the replicon bearing the reference full-length GT4a (ED43) NS5A gene was 0.0002 nM. The median EC 50 for elbasvir against chimeric replicons bearing clinical isolates from GT4 was 0.0007 nM (range, 0.0002 to 34 nM; n = 14). De novo resistance selection studies in GT4 demonstrated a high propensity to suppress the emergence of amino acid substitutions that confer high-potency reductions to elbasvir. Phenotypic characterization of the NS5A amino acid substitutions identified (L30F, L30S, M31V, and Y93H) indicated that they conferred 15-, 4-, 2.5-, and 7.5-fold potency losses, respectively, to elbasvir

The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication.

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Morais, Ana Ts; Terzian, Ana Cb; Duarte, Danilo Vb; Bronzoni, Roberta Vm; Madrid, Maria Cfs; Gavioli, Arieli F; Gil, Laura Hvg; Oliveira, Amanda G; Zanelli, Cleslei F; Valentini, Sandro R; Rahal, Paula; Nogueira, Mauricio L

2013-06-22

Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role

H-NS represses transcription of the flagellin gene lafA of lateral flagella in Vibrio parahaemolyticus.

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Wang, Yan; Zhang, Yiquan; Yin, Zhe; Wang, Jie; Zhu, Yongzhe; Peng, Haoran; Zhou, Dongsheng; Qi, Zhongtian; Yang, Wenhui

2018-01-01

Swarming motility is ultimately mediated by the proton-powered lateral flagellar (laf) system in Vibrio parahaemolyticus. Expression of laf genes is tightly regulated by a number of environmental conditions and regulatory factors. The nucleoid-associated DNA-binding protein H-NS is a small and abundant protein that is widely distributed in bacteria, and H-NS-like protein-dependent expression of laf genes has been identified in Vibrio cholerae and V. parahaemolyticus. The data presented here show that H-NS acts as a repressor of the swarming motility in V. parahaemolyticus. A single σ 28 -dependent promoter was detected for lafA encoding the flagellin of the lateral flagella, and its activity was directly repressed by H-NS. Thus, H-NS represses swarming motility by directly acting on lafA. Briefly, this work revealed a novel function for H-NS as a repressor of the expression of lafA and swarming motility in V. parahaemolyticus.

Differences in kinetics of xanomeline binding and selectivity of activation of G proteins at M(1) and M(2) muscarinic acetylcholine receptors

Czech Academy of Sciences Publication Activity Database

Jakubík, Jan; El-Fakahany, E. E.; Doležal, Vladimír

2006-01-01

Roč. 70, č. 2 (2006), s. 656-666 ISSN 0026-895X R&D Projects: GA ČR(CZ) GA305/05/0452; GA MŠk(CZ) LC554 Grant - others:NIH(US) NS25743 Institutional research plan: CEZ:AV0Z5011922 Keywords : xanomeline * muscarinic receptors * G-protein activation Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 4.469, year: 2006

Efficacy of NS-018, a potent and selective JAK2/Src inhibitor, in primary cells and mouse models of myeloproliferative neoplasms.

Science.gov (United States)

Nakaya, Y; Shide, K; Niwa, T; Homan, J; Sugahara, S; Horio, T; Kuramoto, K; Kotera, T; Shibayama, H; Hori, K; Naito, H; Shimoda, K

2011-07-01

Aberrant activation of Janus kinase 2 (JAK2) caused by somatic mutation of JAK2 (JAK2V617F) or the thrombopoietin receptor (MPLW515L) plays an essential role in the pathogenesis of myeloproliferative neoplasms (MPNs), suggesting that inhibition of aberrant JAK2 activation would have a therapeutic benefit. Our novel JAK2 inhibitor, NS-018, was highly active against JAK2 with a 50% inhibition (IC(50)) of MPLW515L mutations or the TEL-JAK2 fusion gene; IC(50)=11-120 n), but showed only minimal cytotoxicity against most other hematopoietic cell lines without a constitutively activated JAK2. Furthermore, NS-018 preferentially suppressed in vitro erythropoietin-independent endogenous colony formation from polycythemia vera patients. NS-018 also markedly reduced splenomegaly and prolonged the survival of mice inoculated with Ba/F3 cells harboring JAK2V617F. In addition, NS-018 significantly reduced leukocytosis, hepatosplenomegaly and extramedullary hematopoiesis, improved nutritional status, and prolonged survival in JAK2V617F transgenic mice. These results suggest that NS-018 will be a promising candidate for the treatment of MPNs.

In Vitro Antiviral Activity and Resistance Profile of the Next-Generation Hepatitis C Virus NS5A Inhibitor Pibrentasvir.

Science.gov (United States)

Ng, Teresa I; Krishnan, Preethi; Pilot-Matias, Tami; Kati, Warren; Schnell, Gretja; Beyer, Jill; Reisch, Thomas; Lu, Liangjun; Dekhtyar, Tatyana; Irvin, Michelle; Tripathi, Rakesh; Maring, Clarence; Randolph, John T; Wagner, Rolf; Collins, Christine

2017-05-01

Pibrentasvir (ABT-530) is a novel and pan-genotypic hepatitis C virus (HCV) NS5A inhibitor with 50% effective concentration (EC 50 ) values ranging from 1.4 to 5.0 pM against HCV replicons containing NS5A from genotypes 1 to 6. Pibrentasvir demonstrated similar activity against a panel of chimeric replicons containing HCV NS5A of genotypes 1 to 6 from clinical samples. Resistance selection studies were conducted using HCV replicon cells with NS5A from genotype 1a, 1b, 2a, 2b, 3a, 4a, 5a, or 6a at a concentration of pibrentasvir that was 10- or 100-fold over its EC 50 for the respective replicon. With pibrentasvir at 10-fold over the respective EC 50 , only a small number of colonies (0.00015 to 0.0065% of input cells) with resistance-associated amino acid substitutions were selected in replicons containing genotype 1a, 2a, or 3a NS5A, and no viable colonies were selected in replicons containing NS5A from other genotypes. With pibrentasvir at 100-fold over the respective EC 50 , very few colonies (0.0002% of input cells) were selected by pibrentasvir in genotype 1a replicon cells while no colonies were selected in other replicons. Pibrentasvir is active against common resistance-conferring substitutions in HCV genotypes 1 to 6 that were identified for other NS5A inhibitors, including those at key amino acid positions 28, 30, 31, or 93. The combination of pibrentasvir with HCV inhibitors of other classes produced synergistic inhibition of HCV replication. In summary, pibrentasvir is a next-generation HCV NS5A inhibitor with potent and pan-genotypic activity, and it maintains activity against common amino acid substitutions of HCV genotypes 1 to 6 that are known to confer resistance to currently approved NS5A inhibitors. Copyright © 2017 Ng et al.

Mycobacterium tuberculosis nucleoid-associated DNA-binding protein H-NS binds with high-affinity to the Holliday junction and inhibits strand exchange promoted by RecA protein.

Science.gov (United States)

Sharadamma, N; Harshavardhana, Y; Singh, Pawan; Muniyappa, K

2010-06-01

A number of studies have shown that the structure and composition of bacterial nucleoid influences many a processes related to DNA metabolism. The nucleoid-associated proteins modulate not only the DNA conformation but also regulate the DNA metabolic processes such as replication, recombination, repair and transcription. Understanding of how these processes occur in the context of Mycobacterium tuberculosis nucleoid is of considerable medical importance because the nucleoid structure may be constantly remodeled in response to environmental signals and/or growth conditions. Many studies have concluded that Escherichia coli H-NS binds to DNA in a sequence-independent manner, with a preference for A-/T-rich tracts in curved DNA; however, recent studies have identified the existence of medium- and low-affinity binding sites in the vicinity of the curved DNA. Here, we show that the M. tuberculosis H-NS protein binds in a more structure-specific manner to DNA replication and repair intermediates, but displays lower affinity for double-stranded DNA with relatively higher GC content. Notably, M. tuberculosis H-NS was able to bind Holliday junction (HJ), the central recombination intermediate, with substantially higher affinity and inhibited the three-strand exchange promoted by its cognate RecA. Likewise, E. coli H-NS was able to bind the HJ and suppress DNA strand exchange promoted by E. coli RecA, although much less efficiently compared to M. tuberculosis H-NS. Our results provide new insights into a previously unrecognized function of H-NS protein, with implications for blocking the genome integration of horizontally transferred genes by homologous and/or homeologous recombination.

Muscarinic M2 receptors directly activate Gq/11 and Gs G-proteins

Czech Academy of Sciences Publication Activity Database

Michal, Pavel; El-Fakahany, E. E.; Doležal, Vladimír

2007-01-01

Roč. 320, č. 2 (2007), s. 607-614 ISSN 0022-3565 R&D Projects: GA ČR GP305/05/P209; GA ČR GA305/05/0452; GA MŠk(CZ) LC554 Grant - others:NIH(US) NS25743 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic * siRNA * G-proteins Subject RIV: CE - Biochemistry Impact factor: 4.003, year: 2007

Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase

Science.gov (United States)

Tai, Chun-Ling; Pan, Wen-Ching; Liaw, Shwu-Huey; Yang, Ueng-Cheng; Hwang, Lih-Hwa; Chen, Ding-Shinn

2001-01-01

The carboxyl terminus of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole microenvironment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3. PMID:11483774

Different Types of nsP3-Containing Protein Complexes in Sindbis Virus-Infected Cells▿

Science.gov (United States)

Gorchakov, Rodion; Garmashova, Natalia; Frolova, Elena; Frolov, Ilya

2008-01-01

Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions. PMID:18684830

Different types of nsP3-containing protein complexes in Sindbis virus-infected cells.

Science.gov (United States)

Gorchakov, Rodion; Garmashova, Natalia; Frolova, Elena; Frolov, Ilya

2008-10-01

Alphaviruses represent a serious public health threat and cause a wide variety of diseases, ranging from severe encephalitis, which can result in death or neurological sequelae, to mild infection, characterized by fever, skin rashes, and arthritis. In the infected cells, alphaviruses express only four nonstructural proteins, which function in the synthesis of virus-specific RNAs and in modification of the intracellular environment. The results of our study suggest that Sindbis virus (SINV) infection in BHK-21 cells leads to the formation of at least two types of nsP3-containing complexes, one of which was found in association with the plasma membrane and endosome-like vesicles, while the second was coisolated with cell nuclei. The latter complexes could be solubilized only with the cytoskeleton-destabilizing detergent. Besides viral nsPs, in the mammalian cells, both complexes contained G3BP1 and G3BP2 (which were found in different ratios), YBX1, and HSC70. Rasputin, an insect cell-specific homolog of G3BP1, was found in the nsP3-containing complexes isolated from mosquito cells, which was suggestive of a high conservation of the complexes in the cells of both vertebrate and invertebrate origin. The endosome- and plasma membrane-associated complexes contained a high concentration of double-stranded RNAs (dsRNAs), which is indicative of their function in viral-RNA synthesis. The dsRNA synthesis is likely to efficiently proceed on the plasma membrane, and at least some of the protein-RNA complexes would then be transported into the cytosol in association with the endosome-like vesicular organelles. These findings provide new insight into the mechanism of SINV replication and virus-host cell interactions.

The Nucleoid Binding Protein H-NS Biases Genome-Wide Transposon Insertion Landscapes

Directory of Open Access Journals (Sweden)

Satoshi Kimura

2016-08-01

Full Text Available Transposon insertion sequencing (TIS; also known as TnSeq is a potent approach commonly used to comprehensively define the genetic loci that contribute to bacterial fitness in diverse environments. A key presumption underlying analyses of TIS datasets is that loci with a low frequency of transposon insertions contribute to fitness. However, it is not known whether factors such as nucleoid binding proteins can alter the frequency of transposon insertion and thus whether TIS output may systematically reflect factors that are independent of the role of the loci in fitness. Here, we investigated whether the histone-like nucleoid structuring (H-NS protein, which preferentially associates with AT-rich sequences, modulates the frequency of Mariner transposon insertion in the Vibrio cholerae genome, using comparative analysis of TIS results from wild-type (wt and Δhns V. cholerae strains. These analyses were overlaid on gene classification based on GC content as well as on extant genome-wide identification of H-NS binding loci. Our analyses revealed a significant dearth of insertions within AT-rich loci in wt V. cholerae that was not apparent in the Δhns insertion library. Additionally, we observed a striking correlation between genetic loci that are overrepresented in the Δhns insertion library relative to their insertion frequency in wt V. cholerae and loci previously found to physically interact with H-NS. Collectively, our findings reveal that factors other than genetic fitness can systematically modulate the frequency of transposon insertions in TIS studies and add a cautionary note to interpretation of TIS data, particularly for AT-rich sequences.

Hepatitis C Virus NS3 Mediated Microglial Inflammation via TLR2/TLR6 MyD88/NF-κB Pathway and Toll Like Receptor Ligand Treatment Furnished Immune Tolerance.

Directory of Open Access Journals (Sweden)

Ayilam Ramachandran Rajalakshmy

Full Text Available Recent evidence suggests the neurotrophic potential of hepatitis C virus (HCV. HCV NS3 protein is one of the potent antigens of this virus mediating inflammatory response in different cell types. Microglia being the immune surveillance cells in the central nervous system (CNS, the inflammatory potential of NS3 on microglia was studied. Role of toll like receptor (TLR ligands Pam2CSK3 and Pam3CSK4 in controlling the NS3 mediated microglial inflammation was studied using microglial cell line CHME3.IL (Interleukin-8, IL-6, TNF-α (Tumor nicrosis factor alpha and IL-1β gene expressions were measured by semi quantitative RT-PCR (reverse transcription-PCR. ELISA was performed to detect IL-8, IL-6, TNF-α, IL-1β and IL-10 secretion. FACS (Flourescent activated cell sorting was performed to quantify TLR1, TLR2, TLR6, MyD88 (Myeloid differntiation factor 88, IkB-α (I kappaB alpha and pNF-κB (phosphorylated nuclear factor kappaB expression. Immunofluorescence staining was performed for MyD88, TLR6 and NF-κB (Nuclear factor kappaB. Student's t-test or One way analysis of variance with Bonferoni post hoc test was performed and p < 0.05 was considered significant.Microglia responded to NS3 by secreting IL-8, IL-6, TNF-α and IL-1β via TLR2 or TLR6 mediated MyD88/NF-κB pathway. Transcription factor NF-κB was involved in activating the cytokine gene expression and the resultant inflammatory response was controlled by NF-κB inhibitor, Ro106-9920, which is known to down regulate pro-inflammatory cytokine secretion. Activation of the microglia by TLR agonists Pam3CSK4 and Pam2CSK4 induced immune tolerance against NS3. TLR ligand treatment significantly down regulated pro-inflammatory cytokine secretion in the microglia. IL-10 secretion was suggested as the possible mechanism by which TLR agonists induced immune tolerance. NS3 as such was not capable of self-inducing immune tolerance in microglia.In conclusion, NS3 protein was capable of activating

In Vitro Antiviral Activity and Resistance Profile Characterization of the Hepatitis C Virus NS5A Inhibitor Ledipasvir.

Science.gov (United States)

Cheng, Guofeng; Tian, Yang; Doehle, Brian; Peng, Betty; Corsa, Amoreena; Lee, Yu-Jen; Gong, Ruoyu; Yu, Mei; Han, Bin; Xu, Simin; Dvory-Sobol, Hadas; Perron, Michel; Xu, Yili; Mo, Hongmei; Pagratis, Nikos; Link, John O; Delaney, William

2016-01-11

Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, including in vitro potency, in vitro resistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC50) values of 0.031 nM and 0.004 nM, respectively. LDV is also active against HCV genotypes 4a, 4d, 5a, and 6a with EC50 values of 0.11 to 1.1 nM. LDV has relatively less in vitro antiviral activity against genotypes 2a, 2b, 3a, and 6e, with EC50 values of 16 to 530 nM. In vitro resistance selection with LDV identified the single Y93H and Q30E resistance-associated variants (RAVs) in the NS5A gene; these RAVs were also observed in patients after a 3-day monotherapy treatment. In vitro antiviral combination studies indicate that LDV has additive to moderately synergistic antiviral activity when combined with other classes of HCV direct-acting antiviral (DAA) agents, including NS3/4A protease inhibitors and the nucleotide NS5B polymerase inhibitor SOF. Furthermore, LDV is active against known NS3 protease and NS5B polymerase inhibitor RAVs with EC50 values equivalent to those for the wild type. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

NS1 of H7N9 Influenza A Virus Induces NO-Mediated Cellular Senescence in Neuro2a Cells

OpenAIRE

Yinxia Yan; Yongming Du; Huali Zheng; Gefei Wang; Rui Li; Jieling Chen; Kangsheng Li

2017-01-01

Background/Aims: The novel avian H7N9 influenza A virus has been detected in brain tissues and associated with central nervous system (CNS) symptoms in infected human and mice. Roles of its virulence factor, NS1 protein in influenza virus infected neuron has yet to be explored. Methods: Nitric oxide (NO) release and inducible nitric oxide synthase (iNOS) expression in H7N9/NS1-expressed Neuro2a cells were detected by Griess test and western blotting. Cell proliferation rate of H7N9/NS1-expres...

Cleavage preference distinguishes the two-component NS2B-NS3 serine proteinases of Dengue and West Nile viruses.

Science.gov (United States)

Shiryaev, Sergey A; Kozlov, Igor A; Ratnikov, Boris I; Smith, Jeffrey W; Lebl, Michal; Strongin, Alex Y

2007-02-01

Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.

NS3 from Hepatitis C Virus Strain JFH-1 Is an Unusually Robust Helicase That Is Primed To Bind and Unwind Viral RNA

Energy Technology Data Exchange (ETDEWEB)

Zhou, Ting; Ren, Xiaoming; Adams, Rebecca L.; Pyle, Anna Marie; Ou, J. -H. James

2017-10-25

Hepatitis C viruses (HCV) encode a helicase enzyme that is essential for viral replication and assembly (nonstructural protein 3 [NS3]). This helicase has become the focus of extensive basic research on the general helicase mechanism, and it is also of interest as a novel drug target. Despite the importance of this protein, mechanistic work on NS3 has been conducted almost exclusively on variants from HCV genotype 1. Our understanding of NS3 from the highly active HCV strains that are used to study HCV genetics and mechanism in cell culture (such as JFH-1) is lacking. We therefore set out to determine whether NS3 from the replicatively efficient genotype 2a strain JFH-1 displays novel functional or structural properties. Using biochemical assays for RNA binding and duplex unwinding, we show that JFH-1 NS3 binds RNA much more rapidly than the previously studied NS3 variants from genotype 1b. Unlike NS3 variants from other genotypes, JFH-1 NS3 binds RNA with high affinity in a functionally active form that is capable of immediately unwinding RNA duplexes without undergoing rate-limiting conformational changes that precede activation. Unlike other superfamily 2 (SF2) helicases, JFH-1 NS3 does not require long 3' overhangs, and it unwinds duplexes that are flanked by only a few nucleotides, as in the folded HCV genome. To understand the physical basis for this, we solved the crystal structure of JFH-1 NS3, revealing a novel conformation that contains an open, positively charged RNA binding cleft that is primed for productive interaction with RNA targets, potentially explaining robust replication by HCV JFH-1.

IMPORTANCEGenotypes of HCV are as divergent as different types of flavivirus, and yet mechanistic features of HCV variants are presumed to be held in common. One of the most well-studied components of the HCV replication complex is a helicase known as nonstructural protein 3 (NS3). We set out to determine whether this important

Allergenic relevance of nonspecific lipid transfer proteins 2: Identification and characterization of Api g 6 from celery tuber as representative of a novel IgE-binding protein family.

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Vejvar, Eva; Himly, Martin; Briza, Peter; Eichhorn, Stephanie; Ebner, Christof; Hemmer, Wolfgang; Ferreira, Fatima; Gadermaier, Gabriele

2013-11-01

Apium graveolens represents a relevant food allergen source linked with severe systemic reactions. We sought to identify an IgE-binding nonspecific lipid transfer protein (nsLTP) in celery tuber. A low molecular weight protein exclusively present in celery tuber was purified and designated Api g 6. The entire protein sequence was obtained by MS and classified as member of the nsLTP2 family. Api g 6 is monomeric in solution with a molecular mass of 6936 Da. The alpha-helical disulfide bond-stabilized structure confers tremendous thermal stability (Tm > 90°C) and high resistance to gastrointestinal digestion. Endolysosomal degradation demonstrated low susceptibility and the presence of a dominant peptide cluster at the C-terminus. Thirty-eight percent of A. graveolens allergic patients demonstrated IgE reactivity to purified natural Api g 6 in ELISA and heat treatment did only partially reduce its allergenic activity. No correlation in IgE binding and limited cross-reactivity was observed with Api g 2 and Art v 3, nsLTP1 from celery stalks and mugwort pollen. Api g 6, a novel nsLTP2 from celery tuber represents the first well-characterized allergen in this protein family. Despite similar structural and physicochemical features as nsLTP1, immunological properties of Api g 6 are distinct which warrants its inclusion in molecule-based diagnosis of A. graveolens allergy. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hepatitis C virus expressing reporter tagged NS5A protein

DEFF Research Database (Denmark)

2014-01-01

Hepatitis C reporter viruses containing Core through NS2 of prototype isolates of all major HCV genotypes and the remaining genes of isolate JFH1, by insertion of reporter genes in domain III of HCV NS5A were developed. A deletion upstream of the inserted reporter gene sequence conferred favorable...... growth kinetics in Huh7.5 cells to these viruses. These reporter viruses can be used for high throughput analysis of drug and vaccine candidates as well as patient samples. JFH1-based intergenotypic recombinants with genotype specific homotypic 5'UTR, or heterotypic 5'UTR (either of genotype 1a (strain H...

Development and characterization of serotype-specific monoclonal antibodies against the dengue virus-4 (DENV-4) non-structural protein (NS1).

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Gelanew, Tesfaye; Hunsperger, Elizabeth

2018-02-06

Dengue, caused by one of the four serologically distinct dengue viruses (DENV-1 to - 4), is a mosquito-borne disease of serious global health significance. Reliable and cost-effective diagnostic tests, along with effective vaccines and vector-control strategies, are highly required to reduce dengue morbidity and mortality. Evaluation studies revealed that many commercially available NS1 antigen (Ag) tests have limited sensitivity to DENV-4 serotype compared to the other three serotypes. These studies indicated the need for development of new NS1 Ag detection test with improved sensitivity to DENV-4. An NS1 capture enzyme linked immunoassay (ELISA) specific to DENV-4 may improve the detection of DENV-4 cases worldwide. In addition, a serotype-specific NS1 Ag test identifies both DENV and the infecting serotype. In this study, we used a small-ubiquitin-like modifier (SUMO*) cloning vector to express a SUMO*-DENV-4 rNS1 fusion protein to develop NS1 DENV-4 specific monoclonal antibodies (MAbs). These newly developed MAbs were then optimized for use in an anti-NS1 DENV-4 capture ELISA. The serotype specificity and sensitivity of this ELISA was evaluated using (i) supernatants from DENV (1-4)-infected Vero cell cultures, (ii) rNS1s from all the four DENV (1-4) and, (iii) rNS1s of related flaviviruses (yellow fever virus; YFV and West Nile virus; WNV). From the evaluation studies of the newly developed MAbs, we identified three DENV-4 specific anti-NS1 MAbs: 3H7A9, 8A6F2 and 6D4B10. Two of these MAbs were optimal for use in a DENV-4 serotype-specific NS1 capture ELISA: MAb 8A6F2 as the capture antibody and 6D4B10 as a detection antibody. This ELISA was sensitive and specific to DENV-4 with no cross-reactivity to other three DENV (1-3) serotypes and other heterologous flaviviruses. Taken together these data indicated that our MAbs are useful reagents for the development of DENV-4 immunodiagnostic tests.

Adapted J6/JFH1-based Hepatitis C virus recombinants with genotype-specific NS4A show similar efficacies against lead protease inhibitors, alpha interferon, and a putative NS4A inhibitor

DEFF Research Database (Denmark)

Gottwein, Judith M; Jensen, Sanne B; Serre, Stéphanie B N

2013-01-01

To facilitate studies of hepatitis C virus (HCV) NS4A, we aimed at developing J6/JFH1-based recombinants with genotype 1- to 7-specific NS4A proteins. We developed efficient culture systems expressing NS4A proteins of genotypes (isolates) 1a (H77 and TN), 1b (J4), 2a (J6), 4a (ED43), 5a (SA13), 6a...... (HK6a), and 7a (QC69), with peak infectivity titers of ∼3.5 to 4.5 log10 focus-forming units per ml. Except for genotype 2a (J6), growth depended on adaptive mutations identified in long-term culture. Genotype 1a, 1b, and 4a recombinants were adapted by amino acid substitutions F772S (p7) and V1663A...... (NS4A), while 5a, 6a, and 7a recombinants required additional substitutions in the NS3 protease and/or NS4A. We demonstrated applicability of the developed recombinants for study of antivirals. Genotype 1 to 7 NS4A recombinants showed similar responses to the protease inhibitors telaprevir (VX-950...

ns-2 extension to simulate localization system in wireless sensor networks

CSIR Research Space (South Africa)

Abu-Mahfouz, Adnan M

2011-09-01

Full Text Available The ns-2 network simulator is one of the most widely used tools by researchers to investigate the characteristics of wireless sensor networks. Academic papers focus on results and rarely include details of how ns-2 simulations are implemented...

IC-tagged proteins are able to interact with each other and perform complex reactions when integrated into muNS-derived inclusions.

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Brandariz-Nuñez, Alberto; Otero-Romero, Iria; Benavente, Javier; Martinez-Costas, Jose M

2011-09-20

We have recently developed a versatile tagging system (IC-tagging) that causes relocation of the tagged proteins to ARV muNS-derived intracellular globular inclusions. In the present study we demonstrate (i) that the IC-tag can be successfully fused either to the amino or carboxyl terminus of the protein to be tagged and (ii) that IC-tagged proteins are able to interact between them and perform complex reactions that require such interactions while integrated into muNS inclusions, increasing the versatility of the IC-tagging system. Also, our studies with the DsRed protein add some light on the structure/function relationship of the evolution of DsRed chromophore. Copyright © 2011 Elsevier B.V. All rights reserved.

The dopamine D2 receptor can directly recruit and activate GRK2 without G protein activation.

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Pack, Thomas F; Orlen, Margo I; Ray, Caroline; Peterson, Sean M; Caron, Marc G

2018-04-20

The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that is critical for many central nervous system functions. The D2R carries out these functions by signaling through two transducers: G proteins and β-arrestins (βarrs). Selectively engaging either the G protein or βarr pathway may be a way to improve drugs targeting GPCRs. The current model of GPCR signal transduction posits a chain of events where G protein activation ultimately leads to βarr recruitment. GPCR kinases (GRKs), which are regulated by G proteins and whose kinase action facilitates βarr recruitment, bridge these pathways. Therefore, βarr recruitment appears to be intimately tied to G protein activation via GRKs. Here we sought to understand how GRK2 action at the D2R would be disrupted when G protein activation is eliminated and the effect of this on βarr recruitment. We used two recently developed biased D2R mutants that can preferentially interact either with G proteins or βarrs as well as a βarr-biased D2R ligand, UNC9994. With these functionally selective tools, we investigated the mechanism whereby the βarr-preferring D2R achieves βarr pathway activation in the complete absence of G protein activation. We describe how direct, G protein-independent recruitment of GRK2 drives interactions at the βarr-preferring D2R and also contributes to βarr recruitment at the WT D2R. Additionally, we found an additive interaction between the βarr-preferring D2R mutant and UNC9994. These results reveal that the D2R can directly recruit GRK2 without G protein activation and that this mechanism may have relevance to achieving βarr-biased signaling. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

2D non-separable linear canonical transform (2D-NS-LCT) based cryptography

Science.gov (United States)

Zhao, Liang; Muniraj, Inbarasan; Healy, John J.; Malallah, Ra'ed; Cui, Xiao-Guang; Ryle, James P.; Sheridan, John T.

2017-05-01

The 2D non-separable linear canonical transform (2D-NS-LCT) can describe a variety of paraxial optical systems. Digital algorithms to numerically evaluate the 2D-NS-LCTs are not only important in modeling the light field propagations but also of interest in various signal processing based applications, for instance optical encryption. Therefore, in this paper, for the first time, a 2D-NS-LCT based optical Double-random- Phase-Encryption (DRPE) system is proposed which offers encrypting information in multiple degrees of freedom. Compared with the traditional systems, i.e. (i) Fourier transform (FT); (ii) Fresnel transform (FST); (iii) Fractional Fourier transform (FRT); and (iv) Linear Canonical transform (LCT), based DRPE systems, the proposed system is more secure and robust as it encrypts the data with more degrees of freedom with an augmented key-space.

A PTPN11 allele encoding a catalytically impaired SHP2 protein in a patient with a Noonan syndrome phenotype.

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Edwards, Jonathan J; Martinelli, Simone; Pannone, Luca; Lo, Ivan Fai-Man; Shi, Lisong; Edelmann, Lisa; Tartaglia, Marco; Luk, Ho-Ming; Gelb, Bruce D

2014-09-01

The RASopathies are a relatively common group of phenotypically similar and genetically related autosomal dominant genetic syndromes caused by missense mutations affecting genes participating in the RAS/mitogen-activated protein kinase (MAPK) pathway that include Noonan syndrome (NS) and Noonan syndrome with multiple lentigines (NSML, formerly LEOPARD syndrome). NS and NSML can be difficult to differentiate during infancy, but the presence of multiple lentigines, café au lait spots, and specific cardiac defects facilitate the diagnosis. Furthermore, individual PTPN11 missense mutations are highly specific to each syndrome and engender opposite biochemical alterations on the function of SHP-2, the protein product of that gene. Here, we report on a 5-year-old male with two de novo PTPN11 mutations in cis, c.1471C>T (p.Pro491Ser), and c.1492C>T (p.Arg498Trp), which are associated with NS and NSML, respectively. This boy's phenotype is intermediate between NS and NSML with facial dysmorphism, short stature, mild global developmental delay, pulmonic stenosis, and deafness but absence of café au lait spots or lentigines. The double-mutant SHP-2 was found to be catalytically impaired. This raises the question of whether clinical differences between NS and NSML can be ascribed solely to the relative SHP-2 catalytic activity. © 2014 Wiley Periodicals, Inc.

Identification of Hydroxyanthraquinones as Novel Inhibitors of Hepatitis C Virus NS3 Helicase

Science.gov (United States)

Furuta, Atsushi; Tsubuki, Masayoshi; Endoh, Miduki; Miyamoto, Tatsuki; Tanaka, Junichi; Abdus Salam, Kazi; Akimitsu, Nobuyoshi; Tani, Hidenori; Yamashita, Atsuya; Moriishi, Kohji; Nakakoshi, Masamichi; Sekiguchi, Yuji; Tsuneda, Satoshi; Noda, Naohiro

2015-01-01

Hepatitis C virus (HCV) is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3) helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure–activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 µM. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 µM, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition. PMID:26262613

Identification of Hydroxyanthraquinones as Novel Inhibitors of Hepatitis C Virus NS3 Helicase

Directory of Open Access Journals (Sweden)

Atsushi Furuta

2015-08-01

Full Text Available Hepatitis C virus (HCV is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3 helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure–activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 µM. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 µM, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition.

Specific interaction of the nonstructural protein NS1 of minute virus of mice (MVM) with [ACCA](2) motifs in the centre of the right-end MVM DNA palindrome induces hairpin-primed viral DNA replication.

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Willwand, Kurt; Moroianu, Adela; Hörlein, Rita; Stremmel, Wolfgang; Rommelaere, Jean

2002-07-01

The linear single-stranded DNA genome of minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate DNA. Amplification of viral RF DNA requires the structural transition of the right-end palindrome from a linear duplex into a double-hairpin structure, which serves for the repriming of unidirectional DNA synthesis. This conformational transition was found previously to be induced by the MVM nonstructural protein NS1. Elimination of the cognate NS1-binding sites, [ACCA](2), from the central region of the right-end palindrome next to the axis of symmetry was shown to markedly reduce the efficiency of hairpin-primed DNA replication, as measured in a reconstituted in vitro replication system. Thus, [ACCA](2) sequence motifs are essential as NS1-binding elements in the context of the structural transition of the right-end MVM palindrome.

Exploration of structural stability in deleterious nsSNPs of the XPA gene: A molecular dynamics approach

Directory of Open Access Journals (Sweden)

N NagaSundaram

2011-01-01

Full Text Available Background: Distinguishing the deleterious from the massive number of non-functional nsSNPs that occur within a single genome is a considerable challenge in mutation research. In this approach, we have used the existing in silico methods to explore the mutation-structure-function relationship in the XPA gene. Materials and Methods: We used the Sorting Intolerant From Tolerant (SIFT, Polymorphism Phenotyping (PolyPhen, I-Mutant 2.0, and the Protein Analysis THrough Evolutionary Relationships methods to predict the effects of deleterious nsSNPs on protein function and evaluated the impact of mutation on protein stability by Molecular Dynamics simulations. Results: By comparing the scores of all the four in silico methods, nsSNP with an ID rs104894131 at position C108F was predicted to be highly deleterious. We extended our Molecular dynamics approach to gain insight into the impact of this non-synonymous polymorphism on structural changes that may affect the activity of the XPA gene. Conclusion: Based on the in silico methods score, potential energy, root-mean-square deviation, and root-mean-square fluctuation, we predict that deleterious nsSNP at position C108F would play a significant role in causing disease by the XPA gene. Our approach would present the application of in silico tools in understanding the functional variation from the perspective of structure, evolution, and phenotype.

Y-box-binding protein 1 interacts with hepatitis C virus NS3/4A and influences the equilibrium between viral RNA replication and infectious particle production.

Science.gov (United States)

Chatel-Chaix, Laurent; Melançon, Pierre; Racine, Marie-Ève; Baril, Martin; Lamarre, Daniel

2011-11-01

The hepatitis C virus (HCV) NS3/4A protein has several essential roles in the virus life cycle, most probably through dynamic interactions with host factors. To discover cellular cofactors that are co-opted by HCV for its replication, we elucidated the NS3/4A interactome using mass spectrometry and identified Y-box-binding protein 1 (YB-1) as an interacting partner of NS3/4A protein and HCV genomic RNA. Importantly, silencing YB-1 expression decreased viral RNA replication and severely impaired the propagation of the infectious HCV molecular clone JFH-1. Immunofluorescence studies further revealed a drastic HCV-dependent redistribution of YB-1 to the surface of the lipid droplets, an important organelle for HCV assembly. Core and NS3 protein-dependent polyprotein maturation were shown to be required for YB-1 relocalization. Unexpectedly, YB-1 knockdown cells showed the increased production of viral infectious particles while HCV RNA replication was impaired. Our data support that HCV hijacks YB-1-containing ribonucleoparticles and that YB-1-NS3/4A-HCV RNA complexes regulate the equilibrium between HCV RNA replication and viral particle production.

A sensor tip based on carbon nanotube-ink printed electrode for the dengue virus NS1 protein.

Science.gov (United States)

Dias, Ana Carolina M S; Gomes-Filho, Sérgio L R; Silva, Mízia M S; Dutra, Rosa F

2013-06-15

An immunosensor for the non-structural protein 1 (NS1) of the dengue virus based on carbon nanotube-screen printed electrodes (CNT-SPE) was successfully developed. A homogeneous mixture containing carboxylated carbon nanotubes was dispersed in carbon ink to prepare a screen printed working electrode. Anti-NS1 antibodies were covalently linked to CNT-SPE by an ethylenediamine film strategy. Amperometrical responses were generated at -0.5 V vs. Ag/AgCl by hydrogen peroxide reaction with peroxidase (HRP) conjugated to the anti-NS1. An excellent detection limit (in the order of 12 ng mL(-1)) and a sensitivity of 85.59 μA mM(-1)cm(-2) were achieved permitting dengue diagnostic according to the clinical range required. The matrix effect, as well as the performance of the assays, was successfully evaluated using spiked blood serum sample obtaining excellent recovery values in the results. Carbon nanotubes incorporated to the carbon ink improved the reproducibility and sensitivity of the CNT-SPE immunosensor. This point-of-care approach represents a great potential value for use in epidemic situations and can facilitate the early screening of patients in acute phase of dengue virus. Copyright © 2012 Elsevier B.V. All rights reserved.

Identification of drug resistance and immune-driven variations in hepatitis C virus (HCV) NS3/4A, NS5A and NS5B regions reveals a new approach toward personalized medicine.

Science.gov (United States)

Ikram, Aqsa; Obaid, Ayesha; Awan, Faryal Mehwish; Hanif, Rumeza; Naz, Anam; Paracha, Rehan Zafar; Ali, Amjad; Janjua, Hussnain Ahmed

2017-01-01

Cellular immune responses (T cell responses) during hepatitis C virus (HCV) infection are significant factors for determining the outcome of infection. HCV adapts to host immune responses by inducing mutations in its genome at specific sites that are important for HLA processing/presentation. Moreover, HCV also adapts to resist potential drugs that are used to restrict its replication, such as direct-acting antivirals (DAAs). Although DAAs have significantly reduced disease burden, resistance to these drugs is still a challenge for the treatment of HCV infection. Recently, drug resistance mutations (DRMs) observed in HCV proteins (NS3/4A, NS5A and NS5B) have heightened concern that the emergence of drug resistance may compromise the effectiveness of DAAs. Therefore, the NS3/4A, NS5A and NS5B drug resistance variations were investigated in this study, and their prevalence was examined in a large number of protein sequences from all HCV genotypes. Furthermore, potential CD4 + and CD8 + T cell epitopes were predicted and their overlap with genetic variations was explored. The findings revealed that many reported DRMs within NS3/4A, NS5A and NS5B are not drug-induced; rather, they are already present in HCV strains, as they were also detected in HCV-naïve patients. This study highlights several hot spots in which HLA and drug selective pressure overlap. Interestingly, these overlapping mutations were frequently observed among many HCV genotypes. This study implicates that knowledge of the host HLA type and HCV subtype/genotype can provide important information in defining personalized therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression of the rice hoja blanca virus (RHBV non-structural protein 3 (NS3 in Escherichia coli and its in situ localization in RHBV-infected rice tissues

Directory of Open Access Journals (Sweden)

Miguel Muñoz

2004-09-01

Full Text Available The non-structural NS3 protein gene from the rice hoja blanca virus (RHBV was fused to the glutathione- S-transferase carboxilic end and expressed in Escherichia coli strain JM83. Large quantities of fusion protein were produced in insoluble form. The fusion protein was fractionated in SDS-PAGE and purified by electroelution, polyclonal antibodies were raised in rabbit and the antiserum was absorbed with bacterial crude extract. A band of similar size as that of NS3 protein was observed in Western blots using extracts from RHBV-infected rice plants. Immunoelectron microscopy with colloidal gold-labeled antibodies against NS3 protein and the viral nucleocapsid protein revealed in situ accumulation of NS3 protein in the cytoplasm but not in the viral inclusion bodies, vacuoles or chloroplasts of RHBV-infected plants, following the same pattern of distribution as the RHBV nucleocapsid protein. Rev. Biol. Trop. 52(3: 765-775. Epub 2004 Dic 15.El gen que codifica por la proteína no estructural NS3 del virus de la hoja blanca de arroz (RHBV se fusionó al extremo carboxilo del gen de la glutationa-S-transferasa y se expresó en la cepa JM83 de Escherichia coli. Se obtuvieron altas concentraciones de la proteína de fusion (GST-NS3 en forma insoluble. La proteína de fusión se fraccionó en geles de SDS-PAGE, se purificó por electroelución, y se utilizó para producir anticuerpos policlonales en conejo . El antisuero producido se absorbió con extractos crudos de E. coli. Extractos crudos de plantas de arroz sanas e infectadas con el RHBV se evaluaron por Western blots detectándose una banda de peso molecular similar al estimado para la proteína NS3 (23KDa en las plantas infectadas con el virus. Los tejidos provenientes de plantas infectadas con el RHBV se analizaron por medio de microscopia inmunoelectrónica con oro colloidal marcado con anticuerpos contra la proteína NS3 y la nucleoproteína viral N. Se observó una acumulación in situ de la

Discovery of Dengue Virus NS4B Inhibitors

Science.gov (United States)

Wang, Qing-Yin; Dong, Hongping; Zou, Bin; Karuna, Ratna; Wan, Kah Fei; Zou, Jing; Susila, Agatha; Yip, Andy; Shan, Chao; Yeo, Kim Long; Xu, Haoying; Ding, Mei; Chan, Wai Ling; Gu, Feng; Seah, Peck Gee; Liu, Wei; Lakshminarayana, Suresh B.; Kang, CongBao; Lescar, Julien; Blasco, Francesca; Smith, Paul W.

2015-01-01

ABSTRACT The four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV both in vitro and in vivo. The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC50], 10 to 80 nM) but not DENV-1 and -4 (EC50, >20 μM). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial “hit” (compound 1), leading to compound 14a, which has good in vivo pharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2- and -3-infected patients. IMPORTANCE Dengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions in the world where it was not previously endemic. While there are several promising vaccine candidates in clinical trials, approved vaccines or antivirals are not yet available. Here we describe the identification and characterization of a spiropyrazolopyridone as a novel inhibitor of DENV by targeting the viral NS4B protein. The compound potently inhibits two of the four serotypes of DENV (DENV-2 and -3) both in vitro and in vivo. Our

Selection of suitable detergents for obtaining an active dengue protease in its natural form from E. coli.

Science.gov (United States)

Liew, Lynette Sin Yee; Lee, Michelle Yueqi; Wong, Ying Lei; Cheng, Jinting; Li, Qingxin; Kang, CongBao

2016-05-01

Dengue protease is a two-component enzyme and is an important drug target against dengue virus. The protease activity and protein stability of dengue nonstructural protein 3 (NS3) require a co-factor region from a four-span membrane protein NS2B. A natural form of dengue protease containing full-length NS2B and NS3 protease domain NS2BFL-NS3pro will be useful for dengue drug discovery. In current study, detergents that can be used for protease purification were tested. Using a water soluble protease construct, 39 detergents were selected for both NS2B and NS2BFL-NS3pro purification. The results showed that 18 detergents were able to sustain the activity of the natural dengue protease and 11 detergents could be used for NS2B purification. The results obtained in this study will be useful for biochemical and biophysical studies on dengue protease. Copyright © 2016 Elsevier Inc. All rights reserved.

Recombinant HCV variants with NS5A from genotypes 1-7 have different sensitivities to an NS5A inhibitor but not interferon-a

DEFF Research Database (Denmark)

Scheel, Troels K H; Gottwein, Judith M; Mikkelsen, Lotte S

2011-01-01

Heterogeneity in the hepatitis C virus (HCV) protein NS5A influences its sensitivity to interferon-based therapy. Furthermore, NS5A is an important target for development of HCV-specific inhibitors. We aimed to develop recombinant infectious cell culture systems that express NS5A from isolates...

A Model of H-NS Mediated Compaction of Bacterial DNA

NARCIS (Netherlands)

Joyeux, M.; Vreede, J.

2013-01-01

The histone-like nucleoid structuring protein (H-NS) is a nucleoid-associated protein, which is involved in both gene regulation and DNA compaction. H-NS can bind to DNA in two different ways: in trans, by binding to two separate DNA duplexes, or in cis, by binding to different sites on the same

Respiratory Syncytial Virus Nonstructural Proteins Upregulate SOCS1 and SOCS3 in the Different Manner from Endogenous IFN Signaling

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Junwen Zheng

2015-01-01

Full Text Available Respiratory syncytial virus (RSV infection upregulates genes of the suppressor of cytokine signaling (SOCS family, which utilize a feedback loop to inhibit type I interferon dependent antiviral signaling pathway. Here, we reconstituted RSV nonstructural (NS protein expression plasmids (pNS1, pNS2, and pNS1/2 and tested whether NS1 or NS2 would trigger SOCS1 and SOCS3 protein expression. These NS proteins inhibited interferon- (IFN- α signaling through a mechanism involving the induction of SOCS1 and SOCS3, which appeared to be different from autocrine IFN dependent. NS1 induced both SOCS1 and SOCS3 upregulation, while NS2 only induced SOCS1 expression. The induced expression of SOCS1 and SOCS3 preceded endogenous IFN-signaling activation and inhibited the IFN-inducible antiviral response as well as chemokine induction. Treatments with INF-α and NS proteins both induced SOCS1 expression; however, they had opposing effects on IFN-α-dependent antiviral gene expression. Our results indicate that NS1 and NS2, which induce the expression of SOCS1 or SOCS3, might represent an independent pathway of stimulating endogenous IFN signaling.

Recombinant Nonstructural 3 Protein, rNS3, of Hepatitis C Virus Along With Recombinant GP96 Induce IL-12, TNFα and α5integrin Expression in Antigen Presenting Cells

Science.gov (United States)

Hajizadeh, Mohammad Reza; Mokarram, Pooneh; Kamali sarvestani, Eskandar; Bolhassani, Azam; Mostafavi Pour, Zohreh

2013-01-01

Background Hepatitis C virus (HCV) infection is the main cause of chronic liver disease and to date there has been no vaccine development to prevent this infection. Among non-structural HCV proteins, NS3 protein is an excellent goal for a therapeutic vaccine, due to its large size and less variation in conserved regions. The immunogenic properties of heat shock proteins (HSPs) for instance GP96 have prompted investigations into their function as strong adjuvant to improve innate and adaptive immunity. Objectives The aim of this study was to examine additive effects of recombinant GP96 (rGP96) fragments accompanied by rNS3 on expression levels of α5integrin and pro-inflammatory cytokines, IL-12 and TNFα, in Antigen Presenting Cells (APCs). Materials and Methods Recombinant viral proteins (rNS3 and rRGD-NS3), N-terminal and C-terminal fragments of GP96 were produced and purified from E. coli in order to treat the cells; mouse spleen Dendritic Cells (DCs) and THP-1 macrophages. Results Our results showed that rNT-GP96 alone significantly increases the expression level of IL-12, TNFα and α5integrin in THP-1 macrophages and DCs, while IL-12 and TNFα expression levels were unaffected by either rNS3 or rRGD-NS3. Interestingly, the co-addition of these recombinant proteins with rNT-GP96 increased IL-12, TNFα and α5integrin expression. Pearson Correlation showed a direct association between α5integrin with IL-12 and TNF-α expression. Conclusions we have highlighted the role of rNS3 plus rNT-GP96 mediated by α5integrin in producing IL-12 and TNFα. It can be suggested that rNT-GP96 could enhance immunity characteristic of rNS3 protein via production of pro-inflammatory cytokines. PMID:24032046

Design, synthesis and identification of silicon-containing HCV NS5A inhibitors with pan-genotype activity.

Science.gov (United States)

Liu, Baomin; Gai, Kuo; Qin, Hui; Liu, Xushi; Cao, Yuan; Lu, Qin; Lu, Dandan; Chen, Deyang; Shen, Hengqiao; Song, Wei; Zhang, Yang; Wang, Xiaojin; Xu, Hongjiang; Zhang, Yinsheng

2018-03-25

Modification of a HCV NS5A inhibitor, ombitasvir, led to the identification of 10d with improved pan-genotype NS5A inhibition and better pharmacokinetic properties. The key structural changes to ombitasvir include bioisosteric replacement of carbon with silicon atom. Compared with ombitasvir, the activity of anti-HCV genotypes (GT 1 to 6) of 10d is increased to some extent, especially the inhibitory activity against genotype 3a and 6a is increased by more than seven times, and the dog's in vivo pharmacokinetics properties were also superior to ombitasvir. Further drug evaluation showed that 10d was similar to ombitasvir on plasma protein binding and liver distribution profiles, with no cytotoxicity and no inhibitory effect on both CYP 450 and hERG ligand binding. However, permeability assay results indicated that 10d was not the substrate of P-gp or BCRP transporter, which is different from that of ombitasvir. The results of a 14-day repeat-dose toxicity study identified no toxicity with 10d. Our findings in preclinical tests suggest that the silicon-containing compound 10d could be worthy of continued study as a potential drug candidate. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

In Vitro and in Vivo Evaluation of Mutations in the NS Region of Lineage 2 West Nile Virus Associated with Neuroinvasiveness in a Mammalian Model

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Katalin Szentpáli-Gavallér

2016-02-01

Full Text Available West Nile virus (WNV strains may differ significantly in neuroinvasiveness in vertebrate hosts. In contrast to genetic lineage 1 WNVs, molecular determinants of pathogenic lineage 2 strains have not been experimentally confirmed so far. A full-length infectious clone of a neurovirulent WNV lineage 2 strain (578/10; Central Europe was generated and amino acid substitutions that have been shown to attenuate lineage 1 WNVs were introduced into the nonstructural proteins (NS1 (P250L, NS2A (A30P, NS3 (P249H NS4B (P38G, C102S, E249G. The mouse neuroinvasive phenotype of each mutant virus was examined following intraperitoneal inoculation of C57BL/6 mice. Only the NS1-P250L mutation was associated with a significant attenuation of virulence in mice compared to the wild-type. Multiplication kinetics in cell culture revealed significantly lower infectious virus titres for the NS1 mutant compared to the wild-type, as well as significantly lower amounts of positive and negative stranded RNA.

The NS1 polypeptide of the murine parvovirus minute virus of mice binds to DNA sequences containing the motif [ACCA]2-3.

Science.gov (United States)

Cotmore, S F; Christensen, J; Nüesch, J P; Tattersall, P

1995-03-01

A DNA fragment containing the minute virus of mice 3' replication origin was specifically coprecipitated in immune complexes containing the virally coded NS1, but not the NS2, polypeptide. Antibodies directed against the amino- or carboxy-terminal regions of NS1 precipitated the NS1-origin complexes, but antibodies directed against NS1 amino acids 284 to 459 blocked complex formation. Using affinity-purified histidine-tagged NS1 preparations, we have shown that the specific protein-DNA interaction is of moderate affinity, being stable in 0.1 M salt but rapidly lost at higher salt concentrations. In contrast, generalized (or nonspecific) DNA binding by NS1 could be demonstrated only in low salt. Addition of ATP or gamma S-ATP enhanced specific DNA binding by wild-type NS1 severalfold, but binding was lost under conditions which favored ATP hydrolysis. NS1 molecules with mutations in a critical lysine residue (amino acid 405) in the consensus ATP-binding site bound to the origin, but this binding could not be enhanced by ATP addition. DNase I protection assays carried out with wild-type NS1 in the presence of gamma S-ATP gave footprints which extended over 43 nucleotides on both DNA strands, from the middle of the origin bubble sequence to a position some 14 bp beyond the nick site. The DNA-binding site for NS1 was mapped to a 22-bp fragment from the middle of the 3' replication origin which contains the sequence ACCAACCA. This conforms to a reiterated motif (ACCA)2-3, which occurs, in more or less degenerate form, at many sites throughout the minute virus of mice genome (J. W. Bodner, Virus Genes 2:167-182, 1989). Insertion of a single copy of the sequence (ACCA)3 was shown to be sufficient to confer NS1 binding on an otherwise unrecognized plasmid fragment. The functions of NS1 in the viral life cycle are reevaluated in the light of this result.

Protein clustering and RNA phylogenetic reconstruction of the influenza A [corrected] virus NS1 protein allow an update in classification and identification of motif conservation.

Science.gov (United States)

Sevilla-Reyes, Edgar E; Chavaro-Pérez, David A; Piten-Isidro, Elvira; Gutiérrez-González, Luis H; Santos-Mendoza, Teresa

2013-01-01

The non-structural protein 1 (NS1) of influenza A virus (IAV), coded by its third most diverse gene, interacts with multiple molecules within infected cells. NS1 is involved in host immune response regulation and is a potential contributor to the virus host range. Early phylogenetic analyses using 50 sequences led to the classification of NS1 gene variants into groups (alleles) A and B. We reanalyzed NS1 diversity using 14,716 complete NS IAV sequences, downloaded from public databases, without host bias. Removal of sequence redundancy and further structured clustering at 96.8% amino acid similarity produced 415 clusters that enhanced our capability to detect distinct subgroups and lineages, which were assigned a numerical nomenclature. Maximum likelihood phylogenetic reconstruction using RNA sequences indicated the previously identified deep branching separating group A from group B, with five distinct subgroups within A as well as two and five lineages within the A4 and A5 subgroups, respectively. Our classification model proposes that sequence patterns in thirteen amino acid positions are sufficient to fit >99.9% of all currently available NS1 sequences into the A subgroups/lineages or the B group. This classification reduces host and virus bias through the prioritization of NS1 RNA phylogenetics over host or virus phenetics. We found significant sequence conservation within the subgroups and lineages with characteristic patterns of functional motifs, such as the differential binding of CPSF30 and crk/crkL or the availability of a C-terminal PDZ-binding motif. To understand selection pressures and evolution acting on NS1, it is necessary to organize the available data. This updated classification may help to clarify and organize the study of NS1 interactions and pathogenic differences and allow the drawing of further functional inferences on sequences in each group, subgroup and lineage rather than on a strain-by-strain basis.

Effects of the novel anti-inflammatory compounds, N-[2-(cyclohexyloxy)-4-nitrophenyl] methanesulphonamide (NS-398) and 5-methanesulphonamido-6-(2,4-difluorothio-phenyl)-1-inda none (L-745,337), on the cyclo-oxygenase activity of human blood prostaglandin endoperoxide synthases.

Science.gov (United States)

Panara, M R; Greco, A; Santini, G; Sciulli, M G; Rotondo, M T; Padovano, R; di Giamberardino, M; Cipollone, F; Cuccurullo, F; Patrono, C

1995-11-01

1. We have evaluated the selectivity of ketoprofen and two novel nonsteroidal anti-inflammatory drugs, N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulphonamide (NS-398) and 5-methanesulphonamido-6-(2,4-difluorothiophenyl)-1-indano ne (L-745,337), in inhibiting the cyclo-oxygenase activity of prostaglandin endoperoxide synthase-2 (PGHS-2) vs PGHS-1 in human blood monocytes and platelets, respectively. 2. Heparinized whole blood samples were drawn from healthy volunteers pretreated with aspirin, 300 mg 48 h before sampling, to suppress the activity of platelet PGHS-1 and incubated at 37 degrees C for 24 h with increasing concentrations of the test compounds in the presence of lipopolysaccharide (LPS, 10 micrograms ml-1). Immunoreactive PGE2 levels were measured in plasma by a specific radioimmunoassay as an index of the cyclo-oxygenase activity of LPS-induced monocyte PGHS-2. 3. The effects of the same inhibitors on platelet PGHS-1 activity were assessed by allowing whole blood samples, drawn from the same subjects in aspirin-free periods, to clot at 37 degrees C for 1 h in the presence of the compounds and measuring immunoreactive thromboxane B2 (TXB2) levels in serum by a specific radioimmunoassay. 4. Under these experimental conditions, ketoprofen enantioselectively inhibited the cyclo-oxygenase activity of both PGHS-1 and PGHS-2 with equal potency (IC50 ratio: approx. 0.5 for both enantiomers), while L-745,337 and NS-398 achieved selective inhibition of monocyte PGHS-2 (IC50 ratio: > 150). L-745,337 and NS-398 did not affect LPS-induced monocyte PGHS-2 biosynthesis to any detectable extent. 5. We conclude that L-745,337 and NS-398 are selective inhibitors of the cyclo-oxygenase activity of human monocyte PGHS-2. These compounds may provide adequate tools to test the contribution of this novel pathway of arachidonate metabolism to human inflammatory disease.

The small molecule NS11021 is a potent and specific activator of Ca2+-activated big-conductance K+ channels

DEFF Research Database (Denmark)

Bentzen, Bo Hjorth; Nardi, Antonio; Calloe, Kirstine

2007-01-01

, a mitochondrial K(+) channel with KCa1.1-resembling properties has been found in the heart, where it may be involved in regulation of energy consumption. In the present study, the effect of a novel NeuroSearch compound, 1-(3,5-bis-trifluoromethyl-phenyl)-3-[4-bromo-2-(1H-tetrazol-5-yl)-phenyl]-thiourea (NS11021...

Pomegranate ( Punica granatum L.) expresses several nsLTP isoforms characterized by different immunoglobulin E-binding properties.

Science.gov (United States)

Bolla, Michela; Zenoni, Sara; Scheurer, Stephan; Vieths, Stefan; San Miguel Moncin, Maria Del Mar; Olivieri, Mario; Antico, Andrea; Ferrer, Marta; Berroa, Felicia; Enrique, Ernesto; Avesani, Linda; Marsano, Francesco; Zoccatelli, Gianni

2014-01-01

Pomegranate allergy is associated with sensitization to non-specific lipid transfer proteins (nsLTPs). Our aim was to identify and characterize the non-specific nsLTPs expressed in pomegranate at the molecular level and to study their allergenic properties in terms of immunoglobulin E (IgE)-binding and cross-reactivity with peach nsLTP (Pru p 3). A non-equilibrium two-dimensional (2-D) electrophoretic approach based on acid-urea PAGE and sodium dodecyl sulfate PAGE was set up to separate pomegranate nsLTPs. Their immunoreactivity was tested by immunoblotting carried out with anti-Pru p 3 polyclonal antibodies and sera from pomegranate-allergic patients. For final identification, pomegranate nsLTPs were purified by chromatography and subjected to trypsin digestion and mass spectrometry (MS) analysis. For this purpose, the sequences obtained by cDNA cloning of three pomegranate nsLTPs were integrated in the database that was subsequently searched for MS data interpretation. Four nsLTPs were identified by 2-D immunoblotting. The detected proteins showed different IgE-binding capacity and partial cross-reactivity with Pru p 3. cDNA cloning and MS analyses led to the identification of three nsLTP isoforms with 66-68% amino acid sequence identity named Pun g 1.0101, Pun g 1.0201 and Pun g 1.0301. By 2-D electrophoresis, we could separate different nsLTP isoforms possessing different IgE-binding properties, which might reflect peculiar allergenic potencies. The contribution of Pru p 3 to prime sensitization is not central as in other plant nsLTPs. © 2014 S. Karger AG, Basel.

Development and Characterization of a High Sensitivity Segmented Fast Neutron Spectrometer (FaNS-2).

Science.gov (United States)

Langford, T J; Beise, E J; Breuer, H; Heimbach, C R; Ji, G; Nico, J S

2016-01-01

We present the development of a segmented fast neutron spectrometer (FaNS-2) based upon plastic scintillator and 3 He proportional counters. It was designed to measure both the flux and spectrum of fast neutrons in the energy range of few MeV to 1 GeV. FaNS-2 utilizes capture-gated spectroscopy to identify neutron events and reject backgrounds. Neutrons deposit energy in the plastic scintillator before capturing on a 3 He nucleus in the proportional counters. Segmentation improves neutron energy reconstruction while the large volume of scintillator increases sensitivity to low neutron fluxes. A main goal of its design is to study comparatively low neutron fluxes, such as cosmogenic neutrons at the Earth's surface, in an underground environment, or from low-activity neutron sources. In this paper, we present details of its design and construction as well as its characterization with a calibrated 252 Cf source and monoenergetic neutron fields of 2.5 MeV and 14 MeV. Detected monoenergetic neutron spectra are unfolded using a Singular Value Decomposition method, demonstrating a 5% energy resolution at 14 MeV. Finally, we discuss plans for measuring the surface and underground cosmogenic neutron spectra with FaNS-2.

Exacerbating effects of human parvovirus B19 NS1 on liver fibrosis in NZB/W F1 mice.

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Tsai-Ching Hsu

Full Text Available Systemic lupus erythematosus (SLE is an autoimmune disorder with unknown etiology that impacts various organs including liver. Recently, human parvovirus B19 (B19 is recognized to exacerbate SLE. However, the effects of B19 on liver in SLE are still unclear. Herein we aimed to investigate the effects of B19 on liver in NZB/W F1 mice by injecting subcutaneously with PBS, recombinant B19 NS1, VP1u or VP2, respectively. Our experimental results revealed that B19 NS1 protein significantly enhanced the TGF-β/Smad fibrotic signaling by increasing the expressions of TGF-β, Smad2/3, phosphorylated Smad2/3, Smad4 and Sp1. The consequent fibrosis-related proteins, PAI-1 and α-SMA, were also significantly induced in livers of NZB/W F1 mice receiving B19 NS1 protein. Accordingly, markedly increased collagen deposition was also observed in livers of NZB/W F1 mice receiving B19 NS1 protein. However, no significant difference was observed in livers of NZB/W F1 mice receiving B19 VP1u or VP2 as compared to the controls. These findings indicate that B19 NS1 plays a crucial role in exacerbating liver fibrosis in NZB/W F1 mice through enhancing the TGF-â/Smad fibrotic signaling.

Exacerbating Effects of Human Parvovirus B19 NS1 on Liver Fibrosis in NZB/W F1 Mice

Science.gov (United States)

Hsu, Tsai-Ching; Tsai, Chun-Chou; Chiu, Chun-Ching; Hsu, Jeng-Dong; Tzang, Bor-Show

2013-01-01

Systemic lupus erythematosus (SLE) is an autoimmune disorder with unknown etiology that impacts various organs including liver. Recently, human parvovirus B19 (B19) is recognized to exacerbate SLE. However, the effects of B19 on liver in SLE are still unclear. Herein we aimed to investigate the effects of B19 on liver in NZB/W F1 mice by injecting subcutaneously with PBS, recombinant B19 NS1, VP1u or VP2, respectively. Our experimental results revealed that B19 NS1 protein significantly enhanced the TGF-β/Smad fibrotic signaling by increasing the expressions of TGF-β, Smad2/3, phosphorylated Smad2/3, Smad4 and Sp1. The consequent fibrosis-related proteins, PAI-1 and α-SMA, were also significantly induced in livers of NZB/W F1 mice receiving B19 NS1 protein. Accordingly, markedly increased collagen deposition was also observed in livers of NZB/W F1 mice receiving B19 NS1 protein. However, no significant difference was observed in livers of NZB/W F1 mice receiving B19 VP1u or VP2 as compared to the controls. These findings indicate that B19 NS1 plays a crucial role in exacerbating liver fibrosis in NZB/W F1 mice through enhancing the TGF-â/Smad fibrotic signaling. PMID:23840852

HCV RNA traffic and association with NS5A in living cells

Energy Technology Data Exchange (ETDEWEB)

Fiches, Guillaume N.; Eyre, Nicholas S.; Aloia, Amanda L.; Van Der Hoek, Kylie [Department of Molecular and Cellular Biology, Research Centre for Infectious Diseases, University of Adelaide, Adelaide and Centre for Cancer Biology, SA Pathology, Adelaide, SA (Australia); Betz-Stablein, Brigit; Luciani, Fabio [Systems Immunology, School of Medical Sciences, University of New South Wales, Sydney, NSW (Australia); Chopra, Abha [Institute for Immunology and infectious diseases (IIID), Murdoch University, Perth, WA (Australia); Beard, Michael R., E-mail: michael.beard@adelaide.edu.au [Department of Molecular and Cellular Biology, Research Centre for Infectious Diseases, University of Adelaide, Adelaide and Centre for Cancer Biology, SA Pathology, Adelaide, SA (Australia)

2016-06-15

The spatiotemporal dynamics of Hepatitis C Virus (HCV) RNA localisation are poorly understood. To address this we engineered HCV genomes harbouring MS2 bacteriophage RNA stem-loops within the 3′-untranslated region to allow tracking of HCV RNA via specific interaction with a MS2-Coat-mCherry fusion protein. Despite the impact of these insertions on viral fitness, live imaging revealed that replication of tagged-HCV genomes induced specific redistribution of the mCherry-tagged-MS2-Coat protein to motile and static foci. Further analysis showed that HCV RNA was associated with NS5A in both static and motile structures while a subset of motile NS5A structures was devoid of HCV RNA. Further investigation of viral RNA traffic with respect to lipid droplets (LDs) revealed HCV RNA-positive structures in close association with LDs. These studies provide new insights into the dynamics of HCV RNA traffic with NS5A and LDs and provide a platform for future investigations of HCV replication and assembly. - Highlights: • HCV can tolerate can bacteriophage MS2 stem-loop insertions within the 3′ UTR. • MS2 stem-loop containing HCV genomes allow for real-time imaging of HCV RNA. • HCV RNA is both static and motile and associates with NS5A and lipid droplets.

HCV RNA traffic and association with NS5A in living cells

International Nuclear Information System (INIS)

Fiches, Guillaume N.; Eyre, Nicholas S.; Aloia, Amanda L.; Van Der Hoek, Kylie; Betz-Stablein, Brigit; Luciani, Fabio; Chopra, Abha; Beard, Michael R.

2016-01-01

The spatiotemporal dynamics of Hepatitis C Virus (HCV) RNA localisation are poorly understood. To address this we engineered HCV genomes harbouring MS2 bacteriophage RNA stem-loops within the 3′-untranslated region to allow tracking of HCV RNA via specific interaction with a MS2-Coat-mCherry fusion protein. Despite the impact of these insertions on viral fitness, live imaging revealed that replication of tagged-HCV genomes induced specific redistribution of the mCherry-tagged-MS2-Coat protein to motile and static foci. Further analysis showed that HCV RNA was associated with NS5A in both static and motile structures while a subset of motile NS5A structures was devoid of HCV RNA. Further investigation of viral RNA traffic with respect to lipid droplets (LDs) revealed HCV RNA-positive structures in close association with LDs. These studies provide new insights into the dynamics of HCV RNA traffic with NS5A and LDs and provide a platform for future investigations of HCV replication and assembly. - Highlights: • HCV can tolerate can bacteriophage MS2 stem-loop insertions within the 3′ UTR. • MS2 stem-loop containing HCV genomes allow for real-time imaging of HCV RNA. • HCV RNA is both static and motile and associates with NS5A and lipid droplets.

The NS3 proteins of global strains of bluetongue virus evolve into regional topotypes through negative (purifying) selection.

Science.gov (United States)

Balasuriya, U B R; Nadler, S A; Wilson, W C; Pritchard, L I; Smythe, A B; Savini, G; Monaco, F; De Santis, P; Zhang, N; Tabachnick, W J; Maclachlan, N J

2008-01-01

Comparison of the deduced amino acid sequences of the genes (S10) encoding the NS3 protein of 137 strains of bluetongue virus (BTV) from Africa, the Americas, Asia, Australia and the Mediterranean Basin showed limited variation. Common to all NS3 sequences were potential glycosylation sites at amino acid residues 63 and 150 and a cysteine at residue 137, whereas a cysteine at residue 181 was not conserved. The PPXY and PS/TAP late-domain motifs were conserved in all but three of the viruses. Phylogenetic analyses of these same sequences yielded two principal clades that grouped the viruses irrespective of their serotype or year of isolation (1900-2003). All viruses from Asia and Australia were grouped in one clade, whereas those from the other regions were present in both clades. Each clade segregated into distinct subclades that included viruses from single or multiple regions, and the S10 genes of some field viruses were identical to those of live-attenuated BTV vaccines. There was no evidence of positive selection on the S10 gene as assessed by reconstruction of ancestral codon states on the phylogeny, rather the functional constraints of the NS3 protein are expressed through substantial negative (purifying) selection.

Insights into the interaction between nucleoid-associated proteins H ha and H-NS by NMR and fluorescence anisotropy

International Nuclear Information System (INIS)

Cordeiro, T.N.; Garcia, J.; Pons, M.

2005-01-01

NMR and fluorescence anisotropy are both valuable tools for studying bio molecular interactions. NMR can provide structural insights at atomic resolution. Still, it can be wisely complemented by lower-resolution biophysical techniques, such as fluorescence anisotropy. In this article we report the combination of NMR and fluorescence anisotropy in establishing novel structure-function insights into the interaction between two bacterial nucleoid-associated proteins, H ha and H-NS. H ha (H-NS) complexes are known to play an important role in modulating the expression of some environmentally regulated genes that confer survival advantage in a particular growth condition. (author)

Insights into the interaction between nucleoid-associated proteins H ha and H-NS by NMR and fluorescence anisotropy

Energy Technology Data Exchange (ETDEWEB)

Cordeiro, T.N.; Garcia, J. [Institut de Recerca Biomedica-Parc Cientific de (Spain). Lab. of Biomolecular NMR; Pons, M. [Universitat de Barcelona (Spain). Dept. de Quimica Organica]. E-mail: mpons@ub.edu

2005-07-01

NMR and fluorescence anisotropy are both valuable tools for studying bio molecular interactions. NMR can provide structural insights at atomic resolution. Still, it can be wisely complemented by lower-resolution biophysical techniques, such as fluorescence anisotropy. In this article we report the combination of NMR and fluorescence anisotropy in establishing novel structure-function insights into the interaction between two bacterial nucleoid-associated proteins, H ha and H-NS. H ha (H-NS) complexes are known to play an important role in modulating the expression of some environmentally regulated genes that confer survival advantage in a particular growth condition. (author)

Performance of commercial dengue NS1 ELISA and molecular analysis of NS1 gene of dengue viruses obtained during surveillance in Indonesia.

Science.gov (United States)

Aryati, Aryati; Trimarsanto, Hidayat; Yohan, Benediktus; Wardhani, Puspa; Fahri, Sukmal; Sasmono, R Tedjo

2013-12-29

Early diagnosis of dengue infection is crucial for better management of the disease. Diagnostic tests based on the detection of dengue virus (DENV) Non Structural Protein 1 (NS1) antigen are commercially available with different sensitivities and specificities observed in various settings. Dengue is endemic in Indonesia and clinicians are increasingly using the NS1 detection for dengue confirmation. This study described the performance of Panbio Dengue Early NS1 and IgM Capture ELISA assays for dengue detection during our surveillance in eight cities in Indonesia as well as the genetic diversity of DENV NS1 genes and its relationship with the NS1 detection. The NS1 and IgM/IgG ELISA assays were used for screening and confirmation of dengue infection during surveillance in 2010-2012. Collected serum samples (n = 440) were subjected to RT-PCR and virus isolation, in which 188 samples were confirmed for dengue infection. The positivity of the ELISA assays were correlated with the RT-PCR results to obtain the sensitivity of the assays. The NS1 genes of 48 Indonesian virus isolates were sequenced and their genetic characteristics were studied. Using molecular data as gold standard, the sensitivity of NS1 ELISA assay for samples from Indonesia was 56.4% while IgM ELISA was 73.7%. When both NS1 and IgM results were combined, the sensitivity increased to 89.4%. The NS1 sensitivity varied when correlated with city/geographical origins and DENV serotype, in which the lowest sensitivity was observed for DENV-4 (19.0%). NS1 sensitivity was higher in primary (67.6%) compared to secondary infection (48.2%). The specificity of NS1 assay for non-dengue samples were 100%. The NS1 gene sequence analysis of 48 isolates revealed the presence of polymorphisms of the NS1 genes which apparently did not influence the NS1 sensitivity. We observed a relatively low sensitivity of NS1 ELISA for dengue detection on RT-PCR-positive dengue samples. The detection rate increased significantly

Optical and EPR spectra of the thionitrosyl complex [Cr(OH2)5(NS)]2+

DEFF Research Database (Denmark)

Døssing, Anders Rørbæk; Dethlefsen, Johannes Wied

2008-01-01

. The optical data indicate that the NS ligand is a weaker p-acceptor than the NO ligand. The EPR parameters of [Cr(OH2)5(NS)]2+ were determined: giso, g¦ and g-: 1.96515, 1.92686(5) and 1.986860(8); Aiso(53Cr), A¦(53Cr) and A-(53Cr): 25.3´10-4, 38´10-4 and 18.5´10-4cm-1; Aiso(14N), A¦(14N) and A-(14N): 6...

Genetic characterization of the non-structural protein-3 gene of bluetongue virus serotype-2 isolate from India

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Raghavendra Sumanth Pudupakam

2017-03-01

Full Text Available Aim: Sequence analysis and phylogenetic studies based on non-structural protein-3 (NS3 gene are important in understanding the evolution and epidemiology of bluetongue virus (BTV. This study was aimed at characterizing the NS3 gene sequence of Indian BTV serotype-2 (BTV2 to elucidate its genetic relationship to global BTV isolates. Materials and Methods: The NS3 gene of BTV2 was amplified from infected BHK-21 cell cultures, cloned and subjected to sequence analysis. The generated NS3 gene sequence was compared with the corresponding sequences of different BTV serotypes across the world, and a phylogenetic relationship was established. Results: The NS3 gene of BTV2 showed moderate levels of variability in comparison to different BTV serotypes, with nucleotide sequence identities ranging from 81% to 98%. The region showed high sequence homology of 93-99% at amino acid level with various BTV serotypes. The PPXY/PTAP late domain motifs, glycosylation sites, hydrophobic domains, and the amino acid residues critical for virus-host interactions were conserved in NS3 protein. Phylogenetic analysis revealed that BTV isolates segregate into four topotypes and that the Indian BTV2 in subclade IA is closely related to Asian and Australian origin strains. Conclusion: Analysis of the NS3 gene indicated that Indian BTV2 isolate is closely related to strains from Asia and Australia, suggesting a common origin of infection. Although the pattern of evolution of BTV2 isolate is different from other global isolates, the deduced amino acid sequence of NS3 protein demonstrated high molecular stability.

Genetic characterization of the non-structural protein-3 gene of bluetongue virus serotype-2 isolate from India.

Science.gov (United States)

Pudupakam, Raghavendra Sumanth; Raghunath, Shobana; Pudupakam, Meghanath; Daggupati, Sreenivasulu

2017-03-01

Sequence analysis and phylogenetic studies based on non-structural protein-3 (NS3) gene are important in understanding the evolution and epidemiology of bluetongue virus (BTV). This study was aimed at characterizing the NS3 gene sequence of Indian BTV serotype-2 (BTV2) to elucidate its genetic relationship to global BTV isolates. The NS3 gene of BTV2 was amplified from infected BHK-21 cell cultures, cloned and subjected to sequence analysis. The generated NS3 gene sequence was compared with the corresponding sequences of different BTV serotypes across the world, and a phylogenetic relationship was established. The NS3 gene of BTV2 showed moderate levels of variability in comparison to different BTV serotypes, with nucleotide sequence identities ranging from 81% to 98%. The region showed high sequence homology of 93-99% at amino acid level with various BTV serotypes. The PPXY/PTAP late domain motifs, glycosylation sites, hydrophobic domains, and the amino acid residues critical for virus-host interactions were conserved in NS3 protein. Phylogenetic analysis revealed that BTV isolates segregate into four topotypes and that the Indian BTV2 in subclade IA is closely related to Asian and Australian origin strains. Analysis of the NS3 gene indicated that Indian BTV2 isolate is closely related to strains from Asia and Australia, suggesting a common origin of infection. Although the pattern of evolution of BTV2 isolate is different from other global isolates, the deduced amino acid sequence of NS3 protein demonstrated high molecular stability.

Ebselen Inhibits Hepatitis C Virus NS3 Helicase Binding to Nucleic Acid and Prevents Viral Replication

OpenAIRE

Mukherjee, Sourav; Weiner, Warren S.; Schroeder, Chad E.; Simpson, Denise S.; Hanson, Alicia M.; Sweeney, Noreena L.; Marvin, Rachel K.; Ndjomou, Jean; Kolli, Rajesh; Isailovic, Dragan; Schoenen, Frank J.; Frick, David N.

2014-01-01

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previousl...

Direct binding of ledipasvir to HCV NS5A: mechanism of resistance to an HCV antiviral agent.

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Hyock Joo Kwon

Full Text Available Ledipasvir, a direct acting antiviral agent (DAA targeting the Hepatitis C Virus NS5A protein, exhibits picomolar activity in replicon cells. While its mechanism of action is unclear, mutations that confer resistance to ledipasvir in HCV replicon cells are located in NS5A, suggesting that NS5A is the direct target of ledipasvir. To date co-precipitation and cross-linking experiments in replicon or NS5A transfected cells have not conclusively shown a direct, specific interaction between NS5A and ledipasvir. Using recombinant, full length NS5A, we show that ledipasvir binds directly, with high affinity and specificity, to NS5A. Ledipasvir binding to recombinant NS5A is saturable with a dissociation constant in the low nanomolar range. A mutant form of NS5A (Y93H that confers resistance to ledipasvir shows diminished binding to ledipasvir. The current study shows that ledipasvir inhibits NS5A through direct binding and that resistance to ledipasvir is the result of a reduction in binding affinity to NS5A mutants.

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

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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.

Purification, crystallization and X-ray diffraction analysis of the C-terminal protease domain of Venezuelan equine encephalitis virus nsP2

International Nuclear Information System (INIS)

Russo, Andrew T.; Watowich, Stanley J.

2006-01-01

The C-terminal protease domain of Venezuelan equine encephalitis virus (VEEV) nsP2 has been overexpressed in E. coli, purified and successfully crystallized. Native crystals diffract to beyond 2.5 Å resolution and isomorphous heavy-atom derivatives suitable for phase analysis have been identified. The C-terminal region of Venezuelan equine encephalitis virus (VEEV) nsP2 is responsible for proteolytic processing of the VEEV polyprotein replication complex. This action regulates the activity of the replication complex and is essential for viral replication, thus making nsP2 a very attractive target for development of VEEV therapeutics. The 338-amino-acid C-terminal region of VEEV nsP2 has been overexpressed in Escherichia coli, purified and crystallized. Crystals diffract to beyond 2.5 Å resolution and belong to the orthorhombic space group P2 1 2 1 2 1 . Isomorphous heavy-atom derivatives suitable for phase analysis have been obtained and work on building a complete structural model is under way

Purification, crystallization and X-ray diffraction analysis of the C-terminal protease domain of Venezuelan equine encephalitis virus nsP2

Energy Technology Data Exchange (ETDEWEB)

Russo, Andrew T.; Watowich, Stanley J., E-mail: watowich@xray.utmb.edu [Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX (United States)

2006-06-01

The C-terminal protease domain of Venezuelan equine encephalitis virus (VEEV) nsP2 has been overexpressed in E. coli, purified and successfully crystallized. Native crystals diffract to beyond 2.5 Å resolution and isomorphous heavy-atom derivatives suitable for phase analysis have been identified. The C-terminal region of Venezuelan equine encephalitis virus (VEEV) nsP2 is responsible for proteolytic processing of the VEEV polyprotein replication complex. This action regulates the activity of the replication complex and is essential for viral replication, thus making nsP2 a very attractive target for development of VEEV therapeutics. The 338-amino-acid C-terminal region of VEEV nsP2 has been overexpressed in Escherichia coli, purified and crystallized. Crystals diffract to beyond 2.5 Å resolution and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}. Isomorphous heavy-atom derivatives suitable for phase analysis have been obtained and work on building a complete structural model is under way.

Host-derived apolipoproteins play comparable roles with viral secretory proteins Erns and NS1 in the infectious particle formation of Flaviviridae.

Directory of Open Access Journals (Sweden)

Takasuke Fukuhara

2017-06-01

Full Text Available Amphipathic α-helices of exchangeable apolipoproteins have shown to play crucial roles in the formation of infectious hepatitis C virus (HCV particles through the interaction with viral particles. Among the Flaviviridae members, pestivirus and flavivirus possess a viral structural protein Erns or a non-structural protein 1 (NS1 as secretory glycoproteins, respectively, while Hepacivirus including HCV has no secretory glycoprotein. In case of pestivirus replication, the C-terminal long amphipathic α-helices of Erns are important for anchoring to viral membrane. Here we show that host-derived apolipoproteins play functional roles similar to those of virally encoded Erns and NS1 in the formation of infectious particles. We examined whether Erns and NS1 could compensate for the role of apolipoproteins in particle formation of HCV in apolipoprotein B (ApoB and ApoE double-knockout Huh7 (BE-KO, and non-hepatic 293T cells. We found that exogenous expression of either Erns or NS1 rescued infectious particle formation of HCV in the BE-KO and 293T cells. In addition, expression of apolipoproteins or NS1 partially rescued the production of infectious pestivirus particles in cells upon electroporation with an Erns-deleted non-infectious RNA. As with exchangeable apolipoproteins, the C-terminal amphipathic α-helices of Erns play the functional roles in the formation of infectious HCV or pestivirus particles. These results strongly suggest that the host- and virus-derived secretory glycoproteins have overlapping roles in the viral life cycle of Flaviviridae, especially in the maturation of infectious particles, while Erns and NS1 also participate in replication complex formation and viral entry, respectively. Considering the abundant hepatic expression and liver-specific propagation of these apolipoproteins, HCV might have evolved to utilize them in the formation of infectious particles through deletion of a secretory viral glycoprotein gene.

New NS varieties of six-rowed winter barley

Directory of Open Access Journals (Sweden)

Pržulj Novo

2009-01-01

Full Text Available The paper describes the characteristics of several new NS varieties of winter six-rowed barley released in Serbia between 2004 and 2007. These are Somborac, Ozren, Javor, Novosadski 773, Sremac and Leotar. In the official variety trials in the country, all six of these varieties outyielded the check variety, and the margins were as follows: Somborac - 3.4%, Ozren - 5.0%, Javor - 7.3%, Novosadski 773 - 3.4%, Sremac - 7.4%, and Leotar - 7.2%. Yield levels in absolute terms depended on the variety as well as year. All six-rowed NS varieties headed earlier than the check and had better resistance to lodging than the check has. The test weight of the new varieties was 70.2-73.8 kg/hl and the 1000-grain weight 33.4-50.2 g. The cellulose content was 4.4-4.8%, the fat content 1.4%, and the protein content 13.3-14.6%. The high variability of the new NS varieties of winter six-rowed barley makes it possible to choose the most suitable genotype for each barley-growing area in the country. .

Silencing by H-NS potentiated the evolution of Salmonella.

Directory of Open Access Journals (Sweden)

Sabrina S Ali

2014-11-01

Full Text Available The bacterial H-NS protein silences expression from sequences with higher AT-content than the host genome and is believed to buffer the fitness consequences associated with foreign gene acquisition. Loss of H-NS results in severe growth defects in Salmonella, but the underlying reasons were unclear. An experimental evolution approach was employed to determine which secondary mutations could compensate for the loss of H-NS in Salmonella. Six independently derived S. Typhimurium hns mutant strains were serially passaged for 300 generations prior to whole genome sequencing. Growth rates of all lineages dramatically improved during the course of the experiment. Each of the hns mutant lineages acquired missense mutations in the gene encoding the H-NS paralog StpA encoding a poorly understood H-NS paralog, while 5 of the mutant lineages acquired deletions in the genes encoding the Salmonella Pathogenicity Island-1 (SPI-1 Type 3 secretion system critical to invoke inflammation. We further demonstrate that SPI-1 misregulation is a primary contributor to the decreased fitness in Salmonella hns mutants. Three of the lineages acquired additional loss of function mutations in the PhoPQ virulence regulatory system. Similarly passaged wild type Salmonella lineages did not acquire these mutations. The stpA missense mutations arose in the oligomerization domain and generated proteins that could compensate for the loss of H-NS to varying degrees. StpA variants most able to functionally substitute for H-NS displayed altered DNA binding and oligomerization properties that resembled those of H-NS. These findings indicate that H-NS was central to the evolution of the Salmonellae by buffering the negative fitness consequences caused by the secretion system that is the defining characteristic of the species.

Molecular Dynamics of the ZIKA Virus NS3 Helicase

Science.gov (United States)

Raubenolt, Bryan; Rick, Steven; The Rick Group Team

The recent outbreaks of the ZIKA virus (ZIKV) and its connection to microcephaly in newborns has raised its awareness as a global threat and many scientific research efforts are currently underway in attempt to create a vaccine. Molecular Dynamics is a powerful method of investigating the physical behavior of protein complexes. ZIKV is comprised of 3 structural and 7 nonstructural proteins. The NS3 helicase protein appears to play a significant role in the replication complex and its inhibition could be a crucial source of antiviral drug design. This research primarily focuses on studying the structural dynamics, over the course of few hundred nanoseconds, of NS3 helicase in the free state, as well as in complex form with human ssRNA, ATP, and an analogue of GTP. RMSD and RMSF plots of each simulation will provide details on the forces involved in the overall stability of the active and inactive states. Furthermore, free energy calculations on a per residue level will reveal the most interactive residues between states and ultimately the primary driving force behind these interactions. Together these analyses will provide highly relevant information on the binding surface chemistry and thus serve as the basis for potential drug design.

NS simulator for beginners

CERN Document Server

Altman, Eitan

2012-01-01

NS-2 is an open-source discrete event network simulator which is widely used by both the research community as well as by the people involved in the standardization protocols of IETF. The goal of this book is twofold: on one hand to learn how to use the NS-2 simulator, and on the other hand, to become acquainted with and to understand the operation of some of the simulated objects using NS-2 simulations. The book is intended to help students, engineers or researchers who need not have much background in programming or who want to learn through simple examples how to analyse some simulated obje

A novel hepacivirus with an unusually long and intrinsically disordered NS5A protein in a wild Old World primate.

Science.gov (United States)

Lauck, Michael; Sibley, Samuel D; Lara, James; Purdy, Michael A; Khudyakov, Yury; Hyeroba, David; Tumukunde, Alex; Weny, Geoffrey; Switzer, William M; Chapman, Colin A; Hughes, Austin L; Friedrich, Thomas C; O'Connor, David H; Goldberg, Tony L

2013-08-01

GB virus B (GBV-B; family Flaviviridae, genus Hepacivirus) has been studied in New World primates as a model for human hepatitis C virus infection, but the distribution of GBV-B and its relatives in nature has remained obscure. Here, we report the discovery of a novel and highly divergent GBV-B-like virus in an Old World monkey, the black-and-white colobus (Colobus guereza), in Uganda. The new virus, guereza hepacivirus (GHV), clusters phylogenetically with GBV-B and recently described hepaciviruses infecting African bats and North American rodents, and it shows evidence of ancient recombination with these other hepaciviruses. Direct sequencing of reverse-transcribed RNA from blood plasma from three of nine colobus monkeys yielded near-complete GHV genomes, comprising two distinct viral variants. The viruses contain an exceptionally long nonstructural 5A (NS5A) gene, approximately half of which codes for a protein with no discernible homology to known proteins. Computational structure-based analyses indicate that the amino terminus of the GHV NS5A protein may serve a zinc-binding function, similar to the NS5A of other viruses within the family Flaviviridae. However, the 521-amino-acid carboxy terminus is intrinsically disordered, reflecting an unusual degree of structural plasticity and polyfunctionality. These findings shed new light on the natural history and evolution of the hepaciviruses and on the extent of structural variation within the Flaviviridae.

Serotype-specific Differences in Dengue Virus Non-structural Protein 5 Nuclear Localization*

Science.gov (United States)

Hannemann, Holger; Sung, Po-Yu; Chiu, Han-Chen; Yousuf, Amjad; Bird, Jim; Lim, Siew Pheng; Davidson, Andrew D.

2013-01-01

The four serotypes of dengue virus (DENV-1 to -4) cause the most important arthropod-borne viral disease of humans. DENV non-structural protein 5 (NS5) contains enzymatic activities required for capping and replication of the viral RNA genome that occurs in the host cytoplasm. However, previous studies have shown that DENV-2 NS5 accumulates in the nucleus during infection. In this study, we examined the nuclear localization of NS5 for all four DENV serotypes. We demonstrate for the first time that there are serotypic differences in NS5 nuclear localization. Whereas the DENV-2 and -3 proteins accumulate in the nucleus, DENV-1 and -4 NS5 are predominantly if not exclusively localized to the cytoplasm. Comparative studies on the DENV-2 and -4 NS5 proteins revealed that the difference in DENV-4 NS5 nuclear localization was not due to rapid nuclear export but rather the lack of a functional nuclear localization sequence. Interaction studies using DENV-2 and -4 NS5 and human importin-α isoforms failed to identify an interaction that supported the differential nuclear localization of NS5. siRNA knockdown of the human importin-α isoform KPNA2, corresponding to the murine importin-α isoform previously shown to bind to DENV-2 NS5, did not substantially affect DENV-2 NS5 nuclear localization, whereas knockdown of importin-β did. The serotypic differences in NS5 nuclear localization did not correlate with differences in IL-8 gene expression. The results show that NS5 nuclear localization is not strictly required for virus replication but is more likely to have an auxiliary function in the life cycle of specific DENV serotypes. PMID:23770669

Serotype-specific differences in dengue virus non-structural protein 5 nuclear localization.

Science.gov (United States)

Hannemann, Holger; Sung, Po-Yu; Chiu, Han-Chen; Yousuf, Amjad; Bird, Jim; Lim, Siew Pheng; Davidson, Andrew D

2013-08-02

The four serotypes of dengue virus (DENV-1 to -4) cause the most important arthropod-borne viral disease of humans. DENV non-structural protein 5 (NS5) contains enzymatic activities required for capping and replication of the viral RNA genome that occurs in the host cytoplasm. However, previous studies have shown that DENV-2 NS5 accumulates in the nucleus during infection. In this study, we examined the nuclear localization of NS5 for all four DENV serotypes. We demonstrate for the first time that there are serotypic differences in NS5 nuclear localization. Whereas the DENV-2 and -3 proteins accumulate in the nucleus, DENV-1 and -4 NS5 are predominantly if not exclusively localized to the cytoplasm. Comparative studies on the DENV-2 and -4 NS5 proteins revealed that the difference in DENV-4 NS5 nuclear localization was not due to rapid nuclear export but rather the lack of a functional nuclear localization sequence. Interaction studies using DENV-2 and -4 NS5 and human importin-α isoforms failed to identify an interaction that supported the differential nuclear localization of NS5. siRNA knockdown of the human importin-α isoform KPNA2, corresponding to the murine importin-α isoform previously shown to bind to DENV-2 NS5, did not substantially affect DENV-2 NS5 nuclear localization, whereas knockdown of importin-β did. The serotypic differences in NS5 nuclear localization did not correlate with differences in IL-8 gene expression. The results show that NS5 nuclear localization is not strictly required for virus replication but is more likely to have an auxiliary function in the life cycle of specific DENV serotypes.

Mechanistic Characterization of GS-9190 (Tegobuvir), a Novel Nonnucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase▿

Science.gov (United States)

Shih, I-hung; Vliegen, Inge; Peng, Betty; Yang, Huiling; Hebner, Christy; Paeshuyse, Jan; Pürstinger, Gerhard; Fenaux, Martijn; Tian, Yang; Mabery, Eric; Qi, Xiaoping; Bahador, Gina; Paulson, Matthew; Lehman, Laura S.; Bondy, Steven; Tse, Winston; Reiser, Hans; Lee, William A.; Schmitz, Uli; Neyts, Johan; Zhong, Weidong

2011-01-01

GS-9190 (Tegobuvir) is a novel imidazopyridine inhibitor of hepatitis C virus (HCV) RNA replication in vitro and has demonstrated potent antiviral activity in patients chronically infected with genotype 1 (GT1) HCV. GS-9190 exhibits reduced activity against GT2a (JFH1) subgenomic replicons and GT2a (J6/JFH1) infectious virus, suggesting that the compound's mechanism of action involves a genotype-specific viral component. To further investigate the GS-9190 mechanism of action, we utilized the susceptibility differences between GT1b and GT2a by constructing a series of replicon chimeras where combinations of 1b and 2a nonstructural proteins were encoded within the same replicon. The antiviral activities of GS-9190 against the chimeric replicons were reduced to levels comparable to that of the wild-type GT2a replicon in chimeras expressing GT2a NS5B. GT1b replicons in which the β-hairpin region (amino acids 435 to 455) was replaced by the corresponding sequence of GT2a were markedly less susceptible to GS-9190, indicating the importance of the thumb subdomain of the polymerase in this effect. Resistance selection in GT1b replicon cells identified several mutations in NS5B (C316Y, Y448H, Y452H, and C445F) that contributed to the drug resistance phenotype. Reintroduction of these mutations into wild-type replicons conferred resistance to GS-9190, with the number of NS5B mutations correlating with the degree of resistance. Analysis of GS-9190 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of GS-9190 is different from other nonnucleoside inhibitors. Collectively, these data demonstrate that GS-9190 represents a novel class of nonnucleoside polymerase inhibitors that interact with NS5B likely through involvement of the β-hairpin in the thumb subdomain. PMID:21746939

Giant magnons under NS-NS and Melvin fields

International Nuclear Information System (INIS)

Huang, W.-H.

2006-01-01

The giant magnon is a rotating spiky string configuration which has the same dispersion relation between the energy and angular momentum as that of a spin magnon. In this paper we investigate the effects of the NS-NS and Melvin fields on the giant magnon. We first analyze the energy and angular momenta of the two-spin spiky D-string moving on the AdS 3 x S 1 with the NS-NS field. Due to the infinite boundary of the AdS spacetime the D-string solution will extend to infinity and it appears the divergences. After adding the counter terms we obtain the dispersion relation of the corresponding giant magnon. The result shows that there will appear a prefactor before the angular momentum, in addition to some corrections in the sine function. We also see that the spiky profile of a rotating D-string plays an important role in mapping it to a spin magnon. We next investigate the energy and angular momentum of the one-spin spiky fundamental string moving on the R x S 2 with the electric or magnetic Melvin field. The dispersion relation of the corresponding deformed giant magnon is also obtained. We discuss some properties of the correction terms and their relations to the spin chain with deformations

Process Performances of 2 ns Pulsed Discharge Plasma

Science.gov (United States)

Matsumoto, Takao; Wang, Douyan; Namihira, Takao; Akiyama, Hidenori

2011-08-01

Pulsed discharge plasmas have been used to treat exhaust gases. Since pulse duration and the rise time of applied voltage to the discharge electrode has a strong influence on the energy efficiency of pollutant removal, the development of a short-pulse generator is of paramount importance for practical applications. In this work, it is demonstrated that the non thermal plasma produced by the 2 ns pulsed discharge has a higher energy efficiency than the 5 ns pulsed discharge plasma for NO removal and ozone generation. Typically, the NO removal efficiency was 1.0 mol kW-1 h-1 for 70% NO removal (initial NO concentration = 200 ppm, gas flow = 10 L/min). Meanwhile, the ozone yield was 500 g kW-1 h-1 for 20 g/m3 ozone concentration in the case of oxygen feeding. These energy efficiencies are the highest in the literature.

Positioning system in wireless sensor networks using NS-2

CSIR Research Space (South Africa)

Abu-Mahfouz, Adnan M

2012-10-01

Full Text Available The practical difficulties of setting up a wireless sensor network (WSN) and analysing its performance have made simulation essential for the study of WSNs. The ns-2 network simulator is one of the most widely used tools by researchers...

Hepatitis C virus NS4B carboxy terminal domain is a membrane binding domain

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Spaan Willy JM

2009-05-01

Full Text Available Abstract Background Hepatitis C virus (HCV induces membrane rearrangements during replication. All HCV proteins are associated to membranes, pointing out the importance of membranes for HCV. Non structural protein 4B (NS4B has been reported to induce cellular membrane alterations like the membranous web. Four transmembrane segments in the middle of the protein anchor NS4B to membranes. An amphipatic helix at the amino-terminus attaches to membranes as well. The carboxy-terminal domain (CTD of NS4B is highly conserved in Hepaciviruses, though its function remains unknown. Results A cytosolic localization is predicted for the NS4B-CTD. However, using membrane floatation assays and immunofluorescence, we now show targeting of the NS4B-CTD to membranes. Furthermore, a profile-profile search, with an HCV NS4B-CTD multiple sequence alignment, indicates sequence similarity to the membrane binding domain of prokaryotic D-lactate dehydrogenase (d-LDH. The crystal structure of E. coli d-LDH suggests that the region similar to NS4B-CTD is located in the membrane binding domain (MBD of d-LDH, implying analogy in membrane association. Targeting of d-LDH to membranes occurs via electrostatic interactions of positive residues on the outside of the protein with negative head groups of lipids. To verify that anchorage of d-LDH MBD and NS4B-CTD is analogous, NS4B-CTD mutants were designed to disrupt these electrostatic interactions. Membrane association was confirmed by swopping the membrane contacting helix of d-LDH with the corresponding domain of the 4B-CTD. Furthermore, the functionality of these residues was tested in the HCV replicon system. Conclusion Together these data show that NS4B-CTD is associated to membranes, similar to the prokaryotic d-LDH MBD, and is important for replication.

Determination of the catalytic activity of LEOPARD syndrome-associated SHP2 mutants toward parafibromin, a bona fide SHP2 substrate involved in Wnt signaling

International Nuclear Information System (INIS)

Noda, Saori; Takahashi, Atsushi; Hayashi, Takeru; Tanuma, Sei-ichi; Hatakeyama, Masanori

2016-01-01

SHP2, encoded by the PTPN11 gene, is a protein tyrosine phosphatase that plays a key role in the proliferation of cells via RAS-ERK activation. SHP2 also promotes Wnt signaling by dephosphorylating parafibromin. Germline missense mutations of PTPN11 are found in more than half of patients with Noonan syndrome (NS) and LEOPARD syndrome (LS), both of which are congenital developmental disorders with multiple common symptoms. However, whereas NS-associated PTPN11 mutations give rise to gain-of-function SHP2 mutants, LS-associated SHP2 mutants are reportedly loss-of-function mutants. To determine the phosphatase activity of LS-associated SHP2 more appropriately, we performed an in vitro phosphatase assay using tyrosine-phosphorylated parafibromin, a biologically relevant substrate of SHP2 and the positive regulator of Wnt signaling that is activated through SHP2-mediated dephosphorylation. We found that LS-associated SHP2 mutants (Y279C, T468M, Q506P, and Q510E) exhibited a substantially reduced phosphatase activity toward parafibromin when compared with wild-type SHP2. Furthermore, each of the LS-associated mutants displayed a differential degree of decrease in phosphatase activity. Deviation of the SHP2 catalytic activity from a certain range, either too strong or too weak, may therefore lead to similar clinical outcomes in NS and LS, possibly through an imbalanced Wnt signal caused by inadequate dephosphorylation of parafibromin. - Highlights: • LS-associated SHP2 mutants dephosphorylate parafibromin on Y290, Y293, and Y315. • LS-associated SHP2 mutants display a reduced tyrosine phosphatase activity. • LS-specific SHP2-Y279C is catalytically less active than LS-specific SHP2-T468M. • NS/LS-associated SHP2-Q506P has both hyper- and hypomorphic enzymatic properties.

Determination of the catalytic activity of LEOPARD syndrome-associated SHP2 mutants toward parafibromin, a bona fide SHP2 substrate involved in Wnt signaling

Energy Technology Data Exchange (ETDEWEB)

Noda, Saori [Division of Microbiology, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan); Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba (Japan); Takahashi, Atsushi; Hayashi, Takeru [Division of Microbiology, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan); Tanuma, Sei-ichi [Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba (Japan); Hatakeyama, Masanori, E-mail: mhata@m.u-tokyo.ac.jp [Division of Microbiology, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan)

2016-01-22

SHP2, encoded by the PTPN11 gene, is a protein tyrosine phosphatase that plays a key role in the proliferation of cells via RAS-ERK activation. SHP2 also promotes Wnt signaling by dephosphorylating parafibromin. Germline missense mutations of PTPN11 are found in more than half of patients with Noonan syndrome (NS) and LEOPARD syndrome (LS), both of which are congenital developmental disorders with multiple common symptoms. However, whereas NS-associated PTPN11 mutations give rise to gain-of-function SHP2 mutants, LS-associated SHP2 mutants are reportedly loss-of-function mutants. To determine the phosphatase activity of LS-associated SHP2 more appropriately, we performed an in vitro phosphatase assay using tyrosine-phosphorylated parafibromin, a biologically relevant substrate of SHP2 and the positive regulator of Wnt signaling that is activated through SHP2-mediated dephosphorylation. We found that LS-associated SHP2 mutants (Y279C, T468M, Q506P, and Q510E) exhibited a substantially reduced phosphatase activity toward parafibromin when compared with wild-type SHP2. Furthermore, each of the LS-associated mutants displayed a differential degree of decrease in phosphatase activity. Deviation of the SHP2 catalytic activity from a certain range, either too strong or too weak, may therefore lead to similar clinical outcomes in NS and LS, possibly through an imbalanced Wnt signal caused by inadequate dephosphorylation of parafibromin. - Highlights: • LS-associated SHP2 mutants dephosphorylate parafibromin on Y290, Y293, and Y315. • LS-associated SHP2 mutants display a reduced tyrosine phosphatase activity. • LS-specific SHP2-Y279C is catalytically less active than LS-specific SHP2-T468M. • NS/LS-associated SHP2-Q506P has both hyper- and hypomorphic enzymatic properties.

Innate Immune Evasion Mediated by Flaviviridae Non-Structural Proteins.

Science.gov (United States)

Chen, Shun; Wu, Zhen; Wang, Mingshu; Cheng, Anchun

2017-10-07

Flaviviridae-caused diseases are a critical, emerging public health problem worldwide. Flaviviridae infections usually cause severe, acute or chronic diseases, such as liver damage and liver cancer resulting from a hepatitis C virus (HCV) infection and high fever and shock caused by yellow fever. Many researchers worldwide are investigating the mechanisms by which Flaviviridae cause severe diseases. Flaviviridae can interfere with the host's innate immunity to achieve their purpose of proliferation. For instance, dengue virus (DENV) NS2A, NS2B3, NS4A, NS4B and NS5; HCV NS2, NS3, NS3/4A, NS4B and NS5A; and West Nile virus (WNV) NS1 and NS4B proteins are involved in immune evasion. This review discusses the interplay between viral non-structural Flaviviridae proteins and relevant host proteins, which leads to the suppression of the host's innate antiviral immunity.

Illustrating and homology modeling the proteins of the Zika virus [version 2; referees: 2 approved

Directory of Open Access Journals (Sweden)

Sean Ekins

2016-09-01

Full Text Available The Zika virus (ZIKV is a flavivirus of the family Flaviviridae, which is similar to dengue virus, yellow fever and West Nile virus. Recent outbreaks in South America, Latin America, the Caribbean and in particular Brazil have led to concern for the spread of the disease and potential to cause Guillain-Barré syndrome and microcephaly. Although ZIKV has been known of for over 60 years there is very little in the way of knowledge of the virus with few publications and no crystal structures. No antivirals have been tested against it either in vitro or in vivo. ZIKV therefore epitomizes a neglected disease. Several suggested steps have been proposed which could be taken to initiate ZIKV antiviral drug discovery using both high throughput screens as well as structure-based design based on homology models for the key proteins. We now describe preliminary homology models created for NS5, FtsJ, NS4B, NS4A, HELICc, DEXDc, peptidase S7, NS2B, NS2A, NS1, E stem, glycoprotein M, propeptide, capsid and glycoprotein E using SWISS-MODEL. Eleven out of 15 models pass our model quality criteria for their further use. While a ZIKV glycoprotein E homology model was initially described in the immature conformation as a trimer, we now describe the mature dimer conformer which allowed the construction of an illustration of the complete virion. By comparing illustrations of ZIKV based on this new homology model and the dengue virus crystal structure we propose potential differences that could be exploited for antiviral and vaccine design. The prediction of sites for glycosylation on this protein may also be useful in this regard. While we await a cryo-EM structure of ZIKV and eventual crystal structures of the individual proteins, these homology models provide the community with a starting point for structure-based design of drugs and vaccines as well as a for computational virtual screening.

Membrane alterations induced by nonstructural proteins of human norovirus.

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Sylvie Y Doerflinger

2017-10-01

Full Text Available Human noroviruses (huNoV are the most frequent cause of non-bacterial acute gastroenteritis worldwide, particularly genogroup II genotype 4 (GII.4 variants. The viral nonstructural (NS proteins encoded by the ORF1 polyprotein induce vesical clusters harboring the viral replication sites. Little is known so far about the ultrastructure of these replication organelles or the contribution of individual NS proteins to their biogenesis. We compared the ultrastructural changes induced by expression of norovirus ORF1 polyproteins with those induced upon infection with murine norovirus (MNV. Characteristic membrane alterations induced by ORF1 expression resembled those found in MNV infected cells, consisting of vesicle accumulations likely built from the endoplasmic reticulum (ER which included single membrane vesicles (SMVs, double membrane vesicles (DMVs and multi membrane vesicles (MMVs. In-depth analysis using electron tomography suggested that MMVs originate through the enwrapping of SMVs with tubular structures similar to mechanisms reported for picornaviruses. Expression of GII.4 NS1-2, NS3 and NS4 fused to GFP revealed distinct membrane alterations when analyzed by correlative light and electron microscopy. Expression of NS1-2 induced proliferation of smooth ER membranes forming long tubular structures that were affected by mutations in the active center of the putative NS1-2 hydrolase domain. NS3 was associated with ER membranes around lipid droplets (LDs and induced the formation of convoluted membranes, which were even more pronounced in case of NS4. Interestingly, NS4 was the only GII.4 protein capable of inducing SMV and DMV formation when expressed individually. Our work provides the first ultrastructural analysis of norovirus GII.4 induced vesicle clusters and suggests that their morphology and biogenesis is most similar to picornaviruses. We further identified NS4 as a key factor in the formation of membrane alterations of huNoV and

Transactivation of a cellular promoter by the NS1 protein of the parvovirus minute virus of mice through a putative hormone-responsive element.

OpenAIRE

Vanacker, J M; Corbau, R; Adelmant, G; Perros, M; Laudet, V; Rommelaere, J

1996-01-01

The promoter of the thyroid hormone receptor alpha gene (c-erbA-1) is activated by the nonstructural protein 1 (NS1) of parvovirus minute virus of mice (prototype strain [MVMp]) in ras-transformed FREJ4 cells that are permissive for lytic MVMp replication. This stimulation may be related to the sensitivity of host cells to MVMp, as it does not take place in parental FR3T3 cells, which are resistant to the parvovirus killing effect. The analysis of a series of deletion and point mutants of the...

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

Science.gov (United States)

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. © 2014 John Wiley & Sons Ltd.

Utility of dengue NS1 antigen rapid diagnostic test for use in difficult to reach areas and its comparison with dengue NS1 ELISA and qRT-PCR.

Science.gov (United States)

Shukla, Mohan K; Singh, Neeru; Sharma, Ravendra K; Barde, Pradip V

2017-07-01

The objective of this study was to demonstrate the utility of dengue virus (DENV) non structural protein 1 (NS1) based rapid diagnostic test (RDT) for use in tribal and difficult to reach areas for early dengue (DEN) diagnosis in acute phase patients and evaluate its sensitivity and specificity against DENV NS1 enzyme linked immune sorbent assay (ELISA) and real time reverse transcriptase polymerase chain reaction (qRT-PCR). The DENV NS1 RDT was used for preliminary diagnosis during outbreaks in difficult to reach rural and tribal areas. The diagnosis was confirmed by DENV NS1 ELISA in the laboratory. The samples were also tested and serotyped by qRT-PCR. The results were evaluated using statistical tests. The DENV NS1 RDT showed 99.2% sensitivity and 96.0% specificity when analyzed using DENV NS1 ELISA as standard. The specificity and sensitivity of the RDT when compared with qRT-PCR was 93.6% and 91.1%, respectively. The serotype specific evaluation showed more than 90% sensitivity and specificity for DENV-1, 2, and 3. The RDT proved a good diagnostic tool in difficult to reach rural and tribal areas. Further evaluation studies with different commercially available RDTs in different field conditions are essential, that will help clinicians and patients for treatment and programme managers for timely intervention. © 2017 Wiley Periodicals, Inc.

The eukaryotic translation initiation factor 3 subunit E binds to classical swine fever virus NS5A and facilitates viral replication.

Science.gov (United States)

Liu, Xiaofeng; Wang, Xiaoyu; Wang, Qian; Luo, Mingyang; Guo, Huancheng; Gong, Wenjie; Tu, Changchun; Sun, Jinfu

2018-02-01

Classical swine fever virus (CSFV) NS5A protein is a multifunctional protein, playing critical roles in viral RNA replication, translation and assembly. To further explore its functions in viral replication, interaction of NS5A with host factors was assayed using a his-tag "pull down" assay coupled with shotgun LC-MS/MS. Host protein translation initiation factor 3 subunit E was identified as a binding partner of NS5A, and confirmed by co-immunoprecipitation and co-localization analysis. Overexpression of eIF3E markedly enhanced CSFV genomic replication, viral protein expression and production of progeny virus, and downregulation of eIF3E by siRNA significantly decreased viral proliferation in PK-15 cells. Luciferase reporter assay showed an enhancement of translational activity of the internal ribosome entry site of CSFV by eIF3E and a decrease in cellular translation by NS5A. These data indicate that eIF3E plays an important role in CSFV replication, thereby identifying it as a potential target for inhibition of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

Therapeutic Effects of Monoclonal Antibody against Dengue Virus NS1 in a STAT1 Knockout Mouse Model of Dengue Infection.

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Wan, Shu-Wen; Chen, Pei-Wei; Chen, Chin-Yu; Lai, Yen-Chung; Chu, Ya-Ting; Hung, Chia-Yi; Lee, Han; Wu, Hsuan Franziska; Chuang, Yung-Chun; Lin, Jessica; Chang, Chih-Peng; Wang, Shuying; Liu, Ching-Chuan; Ho, Tzong-Shiann; Lin, Chiou-Feng; Lee, Chien-Kuo; Wu-Hsieh, Betty A; Anderson, Robert; Yeh, Trai-Ming; Lin, Yee-Shin

2017-10-15

Dengue virus (DENV) is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome and is endemic to tropical and subtropical regions of the world. Our previous studies showed the existence of epitopes in the C-terminal region of DENV nonstructural protein 1 (NS1) which are cross-reactive with host Ags and trigger anti-DENV NS1 Ab-mediated endothelial cell damage and platelet dysfunction. To circumvent these potentially harmful events, we replaced the C-terminal region of DENV NS1 with the corresponding region from Japanese encephalitis virus NS1 to create chimeric DJ NS1 protein. Passive immunization of DENV-infected mice with polyclonal anti-DJ NS1 Abs reduced viral Ag expression at skin inoculation sites and shortened DENV-induced prolonged bleeding time. We also investigated the therapeutic effects of anti-NS1 mAb. One mAb designated 2E8 does not recognize the C-terminal region of DENV NS1 in which host-cross-reactive epitopes reside. Moreover, mAb 2E8 recognizes NS1 of all four DENV serotypes. We also found that mAb 2E8 caused complement-mediated lysis in DENV-infected cells. In mouse model studies, treatment with mAb 2E8 shortened DENV-induced prolonged bleeding time and reduced viral Ag expression in the skin. Importantly, mAb 2E8 provided therapeutic effects against all four serotypes of DENV. We further found that mAb administration to mice as late as 1 d prior to severe bleeding still reduced prolonged bleeding time and hemorrhage. Therefore, administration with a single dose of mAb 2E8 can protect mice against DENV infection and pathological effects, suggesting that NS1-specific mAb may be a therapeutic option against dengue disease. Copyright © 2017 by The American Association of Immunologists, Inc.

Processing quality of NS soybean varieties

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Đorđević Vuk

2012-01-01

Full Text Available Current NS soybean varieties are of satisfactory technological quality, and also significant technological diversity. Varieties Triumf and Venera possess higher oil content. Variety Sava has a balanced oil and protein content, and can be used for obtaining different soy products. Variety Rubin has the highest protein content and is suitable for new high protein products. Estimated processing value is a good parameter to describe the processing quality of soybeans. Based on several years and spatial analysis, it is possible to separate the geographic regions with prevailing favourable conditions for obtaining higher protein or oil content.

The modulation of radiosensitivity by combined treatment of selective COX-2 inhibitor, NS 398 and EGF receptor blocker AG 1478 in HeLa cell line

International Nuclear Information System (INIS)

Youn, Seon Min; Oh, Young Kee; Kim, Joo Heon; Park, Mi Ja; Seong, In Ock; Kang, Ki Mun; Chai, Gyu Yong

2005-01-01

Selective inhibition of multiple molecular targets may improve the antitumor activity of radiation. Two specific inhibitors of selective cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) were combined with radiation on the HeLa cell line. To investigate cooperative mechanism with selective COX-2 inhibitor and EGFR blocker, in vitro experiments were done. Antitumor effect was obtained by growth inhibition and apoptosis analysis by annexin V-Flous method. Radiation modulation effects were determined by the clonogenic cell survival assay. Surviving fractions at 2 Gy (SF 2 ) and dose enhancement radio at a surviving fraction of 0.25 were evaluated. To investigate the mechanism of the modulation of radiosensitivity, the cell cycle analyses were done by flow cytometry. The bcl-2 and bax expressions were analyzed by western blot. A cooperative effect were observed on the apoptosis of the HeLa cell line when combination of the two drugs, AG 1478 and NS 398 with radiation at the lowest doses, apoptosis of 22.70% compare with combination of the one drug with radiation, apoptosis of 8.49%. In cell cycle analysis, accumulation of cell on G 0 /G 1 phase and decrement of S phase fraction was observed from 24 hours to 72 hours after treatment with radiation, AG 1478 and NS 398. The combination of NS 398 and AG 1478 enhanced radiosensitivity in a concentration-dependent manner in HeLa cells with dose enhancement ratios of 3.00 and SF 2 of 0.12 but the combination of one drug with radiation was not enhanced radiosensitivity with dose enhancement ratios of 1.12 and SF2 of 0.68 (ρ = 0.005). The expression levels of bcl-2 and bax were reduced when combined with AG 1478 and NS 398. Our results indicate that the selective COX-2 inhibitor and EGFR blocker combined with radiation have potential additive or cooperative effects on radiation treatment and may act through various mechanisms including direct inhibition of tumor cell proliferation, suppression of tumor cell

Regulation of oxidative enzyme activity and eukaryotic elongation factor 2 in human skeletal muscle: influence of gender and exercise

DEFF Research Database (Denmark)

Roepstorff, Carsten; Schjerling, P.; Vistisen, Bodil

2005-01-01

AIM: To investigate gender-related differences in the responses of oxidative enzymes and eukaryotic elongation factor-2 (eEF2) to exercise. METHODS: The influence of exercise (90 min, 60%VO(2peak)) on citrate synthase (CS) and beta-hydroxyacyl-CoA dehydrogenase (HAD) activity and mRNA content...... expression and phosphorylation were unaffected by training status (NS). CONCLUSION: Basal transcriptional, translational, and/or post-translational control of CS and HAD seems to be gender-dependent. Also, gender differences in translation and/or post-translational protein modification of CS occur during...... not differ between females and males (NS). In females only, CS activity was enhanced (P differ between UT and ET but, nevertheless, CS activity was 56% higher in ET than in UT volunteers (P

Downregulation of viral RNA translation by hepatitis C virus non-structural protein NS5A requires the poly(U/UC) sequence in the 3' UTR.

Science.gov (United States)

Hoffman, Brett; Li, Zhubing; Liu, Qiang

2015-08-01

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is essential for viral replication; however, its effect on HCV RNA translation remains controversial partially due to the use of reporters lacking the 3' UTR, where NS5A binds to the poly(U/UC) sequence. We investigated the role of NS5A in HCV translation using a monocistronic RNA containing a Renilla luciferase gene flanked by the HCV UTRs. We found that NS5A downregulated viral RNA translation in a dose-dependent manner. This downregulation required both the 5' and 3' UTRs of HCV because substitution of either sequence with the 5' and 3' UTRs of enterovirus 71 or a cap structure at the 5' end eliminated the effects of NS5A on translation. Translation of the HCV genomic RNA was also downregulated by NS5A. The inhibition of HCV translation by NS5A required the poly(U/UC) sequence in the 3' UTR as NS5A did not affect translation when it was deleted. In addition, we showed that, whilst the amphipathic α-helix of NS5A has no effect on viral translation, the three domains of NS5A can inhibit translation independently, also dependent on the presence of the poly(U/UC) sequence in the 3' UTR. These results suggested that NS5A downregulated HCV RNA translation through a mechanism involving the poly(U/UC) sequence in the 3' UTR.

Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

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Tong, X; Kono, T; Evans-Molina, C

2015-06-18

The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b

Location of the binding domains for the RNA polymerase L and the ribonucleocapsid template within different halves of the NS phosphoprotein of vesicular stomatitis virus

International Nuclear Information System (INIS)

Emerson, S.U.; Schubert, M.

1987-01-01

Recombinant DNA techniques were used to delete regions of a cDNA clone of the phosphoprotein NS gene of vesicular stomatitis virus. The complete NS gene and four mutant genes containing internal or terminal deletions were inserted into a modified pGem4 vector under the transcriptional control of the page T7 promoter. Run-off transcripts were synthesized and translated in vitro to provide [ 35 S]methionine-labeled complete NS or deletion mutant NS proteins. Immune coprecipitation assays involving these proteins were developed to map the regions of the NS protein responsible for binding to the structural viral nucleocapsid protein N and the catalytic RNA polymerase protein L. The data indicate the NS protein is a bivalent protein consisting of two discrete functional domains. Contrary to previous suggestions, the negatively charged amino-terminal half of NS protein binds to L protein, while the carboxyl-terminal half of NS protein binds to both soluble recombinant nucleocapsid protein N and viral ribonucleocapsid template

Murine Efficacy and Pharmacokinetic Evaluation of the Flaviviral NS5 Capping Enzyme 2-Thioxothiazolidin-4-One Inhibitor BG-323.

Science.gov (United States)

Bullard, Kristen M; Gullberg, Rebekah C; Soltani, Elnaz; Steel, J Jordan; Geiss, Brian J; Keenan, Susan M

2015-01-01

Arthropod-borne flavivirus infection continues to cause significant morbidity and mortality worldwide. Identification of drug targets and novel antiflaviviral compounds to treat these diseases has become a global health imperative. A previous screen of 235,456 commercially available small molecules identified the 2-thioxothiazolidin-4-one family of compounds as inhibitors of the flaviviral NS5 capping enzyme, a promising target for antiviral drug development. Rational drug design methodologies enabled identification of lead compound BG-323 from this series. We have shown previously that BG-323 potently inhibits NS5 capping enzyme activity, displays antiviral effects in dengue virus replicon assays and inhibits growth of West Nile and yellow fever viruses with low cytotoxicity in vitro. In this study we further characterized BG-323's antiviral activity in vitro and in vivo. We found that BG-323 was able to reduce replication of WNV (NY99) and Powassan viruses in culture, and we were unable to force resistance into WNV (Kunjin) in long-term culture experiments. We then evaluated the antiviral activity of BG-323 in a murine model. Mice were challenged with WNV NY99 and administered BG-323 or mock by IP inoculation immediately post challenge and twice daily thereafter. Mice were bled and viremia was quantified on day three. No significant differences in viremia were observed between BG-323-treated and control groups and clinical scores indicated both BG-323-treated and control mice developed signs of illness on approximately the same day post challenge. To determine whether differences in in vitro and in vivo efficacy were due to unfavorable pharmacokinetic properties of BG-323, we conducted a pharmacokinetic evaluation of this small molecule. Insights from pharmacokinetic studies indicate that BG-323 is cell permeable, has a low efflux ratio and does not significantly inhibit two common cytochrome P450 (CYP P450) isoforms thus suggesting this molecule may be less

Murine Efficacy and Pharmacokinetic Evaluation of the Flaviviral NS5 Capping Enzyme 2-Thioxothiazolidin-4-One Inhibitor BG-323.

Directory of Open Access Journals (Sweden)

Kristen M Bullard

Full Text Available Arthropod-borne flavivirus infection continues to cause significant morbidity and mortality worldwide. Identification of drug targets and novel antiflaviviral compounds to treat these diseases has become a global health imperative. A previous screen of 235,456 commercially available small molecules identified the 2-thioxothiazolidin-4-one family of compounds as inhibitors of the flaviviral NS5 capping enzyme, a promising target for antiviral drug development. Rational drug design methodologies enabled identification of lead compound BG-323 from this series. We have shown previously that BG-323 potently inhibits NS5 capping enzyme activity, displays antiviral effects in dengue virus replicon assays and inhibits growth of West Nile and yellow fever viruses with low cytotoxicity in vitro. In this study we further characterized BG-323's antiviral activity in vitro and in vivo. We found that BG-323 was able to reduce replication of WNV (NY99 and Powassan viruses in culture, and we were unable to force resistance into WNV (Kunjin in long-term culture experiments. We then evaluated the antiviral activity of BG-323 in a murine model. Mice were challenged with WNV NY99 and administered BG-323 or mock by IP inoculation immediately post challenge and twice daily thereafter. Mice were bled and viremia was quantified on day three. No significant differences in viremia were observed between BG-323-treated and control groups and clinical scores indicated both BG-323-treated and control mice developed signs of illness on approximately the same day post challenge. To determine whether differences in in vitro and in vivo efficacy were due to unfavorable pharmacokinetic properties of BG-323, we conducted a pharmacokinetic evaluation of this small molecule. Insights from pharmacokinetic studies indicate that BG-323 is cell permeable, has a low efflux ratio and does not significantly inhibit two common cytochrome P450 (CYP P450 isoforms thus suggesting this molecule

Hepatitis C Virus NS3/4A Protease Inhibitors: A Light at the End of the Tunnel

Science.gov (United States)

Chatel-Chaix, Laurent; Baril, Martin; Lamarre, Daniel

2010-01-01

Hepatitis C virus (HCV) infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease) that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection. PMID:21994705

Hepatitis C Virus NS3/4A Protease Inhibitors: A Light at the End of the Tunnel

Directory of Open Access Journals (Sweden)

Laurent Chatel-Chaix

2010-08-01

Full Text Available Hepatitis C virus (HCV infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH interaction with 3' ends of Japanese encephalitis virus RNA and colocalization with the viral NS5 protein

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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.

Cellular Hsp27 interacts with classical swine fever virus NS5A protein and negatively regulates viral replication by the NF-κB signaling pathway.

Science.gov (United States)

Ling, Shifeng; Luo, Mingyang; Jiang, Shengnan; Liu, Jiayu; Ding, Chunying; Zhang, Qinghuan; Guo, Huancheng; Gong, Wenjie; Tu, Changchun; Sun, Jinfu

2018-05-01

Classical swine fever virus (CSFV) nonstructural protein NS5A is a multifunctional protein functioning in regulation of viral genome replication, protein translation and assembly by interaction with viral or host proteins. Here, heat shock protein 27 (Hsp27) has been identified as a novel binding partner of NS5A by using His tag "pull down" coupled with shotgun LC-MS/MS, with interaction of both proteins further confirmed by co-immunoprecipitation and laser confocal assays. In PK-15 cells, silencing of Hsp27 expression by siRNA enhanced CSFV replication, and upregulation of Hsp27 inhibited viral proliferation. Additionally, we have shown that overexpression of Hsp27 increased NF-κB signaling induced by TNFα. Blocking NF-κB signaling in PK-15 cells overexpressing Hsp27 by ammonium pyrrolidinedithiocarbamate (PDTC) eliminated the inhibition of CSFV replication by Hsp27. These findings clearly demonstrate that the inhibition of CSFV replication by Hsp27 is mediated via the NF-κB signaling pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

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)

Dubrau, Danilo; Tortorici, M Alejandra; Rey, Félix A; Tautz, Norbert

2017-02-01

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.

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

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

Directory of Open Access Journals (Sweden)

Danilo Dubrau

2017-02-01

Full Text Available 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.

Targeting Dengue Virus NS-3 Helicase by Ligand based Pharmacophore Modeling and Structure based Virtual Screening

Science.gov (United States)

Halim, Sobia A.; Khan, Shanza; Khan, Ajmal; Wadood, Abdul; Mabood, Fazal; Hussain, Javid; Al-Harrasi, Ahmed

2017-10-01

Dengue fever is an emerging public health concern, with several million viral infections occur annually, for which no effective therapy currently exist. Non-structural protein 3 (NS-3) Helicase encoded by the dengue virus (DENV) is considered as a potential drug target to design new and effective drugs against dengue. Helicase is involved in unwinding of dengue RNA. This study was conducted to design new NS-3 Helicase inhibitor by in silico ligand- and structure based approaches. Initially ligand-based pharmacophore model was generated that was used to screen a set of 1201474 compounds collected from ZINC Database. The compounds matched with the pharmacophore model were docked into the active site of NS-3 helicase. Based on docking scores and binding interactions, twenty five compounds are suggested to be potential inhibitors of NS3 Helicase. The pharmacokinetic properties of these hits were predicted. The selected hits revealed acceptable ADMET properties. This study identified potential inhibitors of NS-3 Helicase in silico, and can be helpful in the treatment of Dengue.

Efficacy of Live-Attenuated H9N2 Influenza Vaccine Candidates Containing NS1 Truncations against H9N2 Avian Influenza Viruses

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Sujuan Chen

2017-06-01

Full Text Available H9N2 avian influenza virus is a zoonotic agent with a broad host range that can contribute genetic information to H5 or H7N9 subtype viruses, which are significant threats to both humans and birds. Thus, there is a great need for a vaccine to control H9N2 avian influenza. Three mutant viruses of an H9N2 virus A/chicken/Taixing/10/2010 (rTX-NS1-73, rTX-NS1-100, and rTX-NS1-128 were constructed with different NS1 gene truncations and confirmed by western blot analysis. The genetic stability, pathogenicity, transmissibility, and host immune responses toward these mutants were evaluated. The mutant virus rTX-NS1-128 exhibited the most attenuated phenotype and lost transmissibility. The expression levels of interleukin 12 in the nasal and tracheal tissues from chickens immunized with rTX-NS1-128 were significantly upregulated on day 3 post-immunization and the IgA and IgG antibody levels were significantly increased on days 7, 14, and 21 post-immunization when compared to chickens that received an inactivated vaccine. rTX-NS1-128 also protected chickens from challenge by homologous and heterologous H9N2 avian influenza viruses. The results indicate that rTX-NS1-128 can be used as a potential live-attenuated vaccine against H9N2 avian influenza.

Stress-induced activation of protein kinase CK2 by direct interaction with p38 mitogen-activated protein kinase

DEFF Research Database (Denmark)

Sayed, M; Kim, S O; Salh, B S

2000-01-01

Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2...... in the human cervical carcinoma HeLa cells by up to 8-fold, and this could be blocked by the p38 MAP kinase inhibitor SB203580. We show that p38alpha MAP kinase, in a phosphorylation-dependent manner, can directly interact with the alpha and beta subunits of CK2 to activate the holoenzyme through what appears...

Design of New Competitive Dengue Ns2b/Ns3 Protease Inhibitors—A Computational Approach

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Noorsaadah Abd. Rahman

2011-02-01

Full Text Available Dengue is a serious disease which has become a global health burden in the last decade. Currently, there are no approved vaccines or antiviral therapies to combat the disease. The increasing spread and severity of the dengue virus infection emphasizes the importance of drug discovery strategies that could efficiently and cost-effectively identify antiviral drug leads for development into potent drugs. To this effect, several computational approaches were applied in this work. Initially molecular docking studies of reference ligands to the DEN2 NS2B/NS3 serine protease were carried out. These reference ligands consist of reported competitive inhibitors extracted from Boesenbergia rotunda (i.e., 4-hydroxypanduratin A and panduratin A and three other synthesized panduratin A derivative compounds (i.e., 246DA, 2446DA and 20H46DA. The design of new lead inhibitors was carried out in two stages. In the first stage, the enzyme complexed to the reference ligands was minimized and their complexation energies (i.e., sum of interaction energy and binding energy were computed. New compounds as potential dengue inhibitors were then designed by putting various substituents successively on the benzyl ring A of the reference molecule. These substituted benzyl compounds were then computed for their enzyme-ligand complexation energies. New enzyme-ligand complexes, exhibiting the lowest complexation energies and closest to the computed energy for the reference compounds, were then chosen for the next stage manipulation and design, which involved substituting positions 4 and 5 of the benzyl ring A (positions 3 and 4 for 2446DA with various substituents.

Characterization of monoclonal antibodies that specifically recognize the palm subdomain of hepatitis C virus nonstructural protein 5B polymerase.

Science.gov (United States)

Ingravallo, P; Lahser, F; Xia, E; Sodowich, B; Lai, V C; Hong, Z; Zhong, W

2001-06-01

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) which plays an essential role in viral RNA replication. Antibodies that specifically recognize NS5B will have utilities in monitoring NS5B production and subcellular localization, as well as in structure-function studies. In this report, three mouse monoclonal antibodies (mAbs), 16A9C9, 16D9A4 and 20A12C7, against a recombinant NS5B protein (genotype 1a, H-77 strain) were produced. These mAbs specifically recognize HCV NS5B, but not RdRps of polivirus (PV), bovine viral diarrhea virus (BVDV) or GB virus B (GBV-B). The mAbs can readily detect NS5B in cellular lysates of human osteosarcoma Saos2 cells constitutively expressing the nonstructural region of HCV (NS3-NS4A-NS4B-NS5A-NS5B). NS5B proteins of different HCV genotypes/subtypes (1a, 1b, 2a, 2c, 5a) showed varied affinity for these mAbs. Interestingly, the epitopes for the mAbs were mapped to the palm subdomain (amino acid 188-370) of the HCV RdRp as determined by immunoblotting analysis of a panel of HCV/GBV-B chimeric NS5B proteins. The binding site was mapped between amino acid 231 and 267 of NS5B for 16A9C9, and between 282 and 372 for 16D9A4 and 20A12C7. Furthermore, these mAbs showed no inhibitory effect on the NS5B polymerase activity in vitro.

Hepatitis C Virus NS3 Inhibitors: Current and Future Perspectives

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Kazi Abdus Salam

2013-01-01

Full Text Available Currently, hepatitis C virus (HCV infection is considered a serious health-care problem all over the world. A good number of direct-acting antivirals (DAAs against HCV infection are in clinical progress including NS3-4A protease inhibitors, RNA-dependent RNA polymerase inhibitors, and NS5A inhibitors as well as host targeted inhibitors. Two NS3-4A protease inhibitors (telaprevir and boceprevir have been recently approved for the treatment of hepatitis C in combination with standard of care (pegylated interferon plus ribavirin. The new therapy has significantly improved sustained virologic response (SVR; however, the adverse effects associated with this therapy are still the main concern. In addition to the emergence of viral resistance, other targets must be continually developed. One such underdeveloped target is the helicase portion of the HCV NS3 protein. This review article summarizes our current understanding of HCV treatment, particularly with those of NS3 inhibitors.

The modulation of radiosensitivity by combined treatment of selective COX-2 inhibitor, NS 398 and EGF receptor blocker AG 1478 in HeLa cell line

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Youn, Seon Min; Oh, Young Kee; Kim, Joo Heon; Park, Mi Ja; Seong, In Ock [Eulji University School of Medicine, Daejeon (Korea, Republic of); Kang, Ki Mun; Chai, Gyu Yong [Gyeongsang National University College of Medicine, Jinju (Korea, Republic of)

2005-03-15

Selective inhibition of multiple molecular targets may improve the antitumor activity of radiation. Two specific inhibitors of selective cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) were combined with radiation on the HeLa cell line. To investigate cooperative mechanism with selective COX-2 inhibitor and EGFR blocker, in vitro experiments were done. Antitumor effect was obtained by growth inhibition and apoptosis analysis by annexin V-Flous method. Radiation modulation effects were determined by the clonogenic cell survival assay. Surviving fractions at 2 Gy (SF{sub 2}) and dose enhancement radio at a surviving fraction of 0.25 were evaluated. To investigate the mechanism of the modulation of radiosensitivity, the cell cycle analyses were done by flow cytometry. The bcl-2 and bax expressions were analyzed by western blot. A cooperative effect were observed on the apoptosis of the HeLa cell line when combination of the two drugs, AG 1478 and NS 398 with radiation at the lowest doses, apoptosis of 22.70% compare with combination of the one drug with radiation, apoptosis of 8.49%. In cell cycle analysis, accumulation of cell on G{sub 0}/G{sub 1} phase and decrement of S phase fraction was observed from 24 hours to 72 hours after treatment with radiation, AG 1478 and NS 398. The combination of NS 398 and AG 1478 enhanced radiosensitivity in a concentration-dependent manner in HeLa cells with dose enhancement ratios of 3.00 and SF{sub 2} of 0.12 but the combination of one drug with radiation was not enhanced radiosensitivity with dose enhancement ratios of 1.12 and SF2 of 0.68 ({rho} = 0.005). The expression levels of bcl-2 and bax were reduced when combined with AG 1478 and NS 398. Our results indicate that the selective COX-2 inhibitor and EGFR blocker combined with radiation have potential additive or cooperative effects on radiation treatment and may act through various mechanisms including direct inhibition of tumor cell proliferation

Activation of multiple G-proteins by muscarinic M1 and M2 receptors

Czech Academy of Sciences Publication Activity Database

Michal, Pavel; El-Fakahany, E. E.; Doležal, Vladimír

2006-01-01

Roč. 27, č. S1 (2006), s. 404-404 ISSN 1671-4083. [World Congress of Pharmacology /15./. 02.07.2006-07.07.2006, Beijing] R&D Projects: GA ČR(CZ) GP305/05/P209; GA ČR(CZ) GA305/05/0452; GA MŠk(CZ) LC554 Grant - others:NIH(US) NS25743 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic receptors * multiple G-protein coupling Subject RIV: ED - Physiology

Synthesis and characterisation of NS13558: a new important tool for addressing KCa1.1 channel function ex vivo

DEFF Research Database (Denmark)

Bentzen, Bo Hjorth; Andersen, Rune Wederkinck; Olesen, Søren-Peter

2009-01-01

Pharmacological activation of the large-conductance Ca(2+)-activated K(+) channel (KCa1.1) in the cardiac inner mitochondrial membrane has been found to protect the heart against ischemia reperfusion injuries. However, there are concerns about the selectivity of the pharmacological tools used...... to modulate the channel. Here, we address this issue by synthesising a methylated analogue of the tool KCa1.1 channel activator NS11021. The compound (NS13558) is designed as a structurally closely related and biologically inactive analogue of NS11021. NS13558 did not elicit any significant opening of cloned...... human KCa1.1 channels, but maintained comparable biological activity towards other cardiac ion channels as compared to NS11021. In isolated perfused rat hearts subjected to ischemia-reperfusion, infarct size was reduced from 29% in control to 7% in NS11021 treated hearts. In comparison, the inactive...

Mapping of Leaf Rust Resistance Genes and Molecular Characterization of the 2NS/2AS Translocation in the Wheat Cultivar Jagger.

Science.gov (United States)

Xue, Shulin; Kolmer, James A; Wang, Shuwen; Yan, Liuling

2018-04-19

Winter wheat cultivar 'Jagger' was recently found to have an alien chromosomal segment 2NS that has Lr37 , a gene conferring resistance against leaf rust caused by Puccinia triticina The objective of this study was to map and characterize the gene(s) for seedling leaf rust resistance in Jagger. The recombinant inbred line (RIL) population of Jagger × '2174' was inoculated with leaf rust pathogen THBJG and BBBDB, and evaluated for infection type (IT) response. A major quantitative trait locus (QTL) for THBJG and BBBDB was coincidently mapped to chromosome arm 2AS, and the QTL accounted for 56.6% - 66.2% of total phenotypic variation in infection type (IT) response to THBJG, and 72.1% - 86.9% to BBBDB. The causal gene for resistance to these rust races was mapped to the 2NS segment in Jagger. The 2NS segment was located in a region of approximately 27.8 Mb starting from the telomere of chromosome arm 2AS, based on the sequences of the A genome in tetraploid wheat. The Lr17a gene on chromosome arm 2AS was delimited to 3.1 Mb in the genomic region, which was orthologous to the 2NS segment. Therefore, the Lr37 gene in the 2NS segment can be pyramided with other effective resistance genes, rather than Lr17a in wheat, to improve resistance to rust diseases. Copyright © 2018, G3: Genes, Genomes, Genetics.

Mosquito Rasputin interacts with chikungunya virus nsP3 and determines the infection rate in Aedes albopictus.

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Fros, Jelke J; Geertsema, Corinne; Zouache, Karima; Baggen, Jim; Domeradzka, Natalia; van Leeuwen, Daniël M; Flipse, Jacky; Vlak, Just M; Failloux, Anna-Bella; Pijlman, Gorben P

2015-09-17

Chikungunya virus (CHIKV) is an arthritogenic alphavirus (family Togaviridae), transmitted by Aedes species mosquitoes. CHIKV re-emerged in 2004 with multiple outbreaks worldwide and recently reached the Americas where it has infected over a million individuals in a rapidly expanding epidemic. While alphavirus replication is well understood in general, the specific function (s) of non-structural protein nsP3 remain elusive. CHIKV nsP3 modulates the mammalian stress response by preventing stress granule formation through sequestration of G3BP. In mosquitoes, nsP3 is a determinant of vector specificity, but its functional interaction with mosquito proteins is unclear. In this research we studied the domains required for localization of CHIKV nsP3 in insect cells and demonstrated its molecular interaction with Rasputin (Rin), the mosquito homologue of G3BP. The biological involvement of Rin in CHIKV infection was investigated in live Ae. albopictus mosquitoes. In insect cells, nsP3 localized as cytoplasmic granules, which was dependent on the central domain and the C-terminal variable region but independent of the N-terminal macrodomain. Ae. albopictus Rin displayed a diffuse, cytoplasmic localization, but was effectively sequestered into nsP3-granules upon nsP3 co-expression. Site-directed mutagenesis showed that the Rin-nsP3 interaction involved the NTF2-like domain of Rin and two conserved TFGD repeats in the C-terminal variable domain of nsP3. Although in vitro silencing of Rin did not impact nsP3 localization or CHIKV replication in cell culture, Rin depletion in vivo significantly decreased the CHIKV infection rate and transmissibility in Ae.albopictus. We identified the nsP3 hypervariable C-terminal domain as a critical factor for granular localization and sequestration of mosquito Rin. Our study offers novel insight into a conserved virus-mosquito interaction at the molecular level, and reveals a strong proviral role for G3BP homologue Rin in live mosquitoes

Pmr, a histone-like protein H1 (H-NS) family protein encoded by the IncP-7 plasmid pCAR1, is a key global regulator that alters host function.

Science.gov (United States)

Yun, Choong-Soo; Suzuki, Chiho; Naito, Kunihiko; Takeda, Toshiharu; Takahashi, Yurika; Sai, Fumiya; Terabayashi, Tsuguno; Miyakoshi, Masatoshi; Shintani, Masaki; Nishida, Hiromi; Yamane, Hisakazu; Nojiri, Hideaki

2010-09-01

Histone-like protein H1 (H-NS) family proteins are nucleoid-associated proteins (NAPs) conserved among many bacterial species. The IncP-7 plasmid pCAR1 is transmissible among various Pseudomonas strains and carries a gene encoding the H-NS family protein, Pmr. Pseudomonas putida KT2440 is a host of pCAR1, which harbors five genes encoding the H-NS family proteins PP_1366 (TurA), PP_3765 (TurB), PP_0017 (TurC), PP_3693 (TurD), and PP_2947 (TurE). Quantitative reverse transcription-PCR (qRT-PCR) demonstrated that the presence of pCAR1 does not affect the transcription of these five genes and that only pmr, turA, and turB were primarily transcribed in KT2440(pCAR1). In vitro pull-down assays revealed that Pmr strongly interacted with itself and with TurA, TurB, and TurE. Transcriptome comparisons of the pmr disruptant, KT2440, and KT2440(pCAR1) strains indicated that pmr disruption had greater effects on the host transcriptome than did pCAR1 carriage. The transcriptional levels of some genes that increased with pCAR1 carriage, such as the mexEF-oprN efflux pump genes and parI, reverted with pmr disruption to levels in pCAR1-free KT2440. Transcriptional levels of putative horizontally acquired host genes were not altered by pCAR1 carriage but were altered by pmr disruption. Identification of genome-wide Pmr binding sites by ChAP-chip (chromatin affinity purification coupled with high-density tiling chip) analysis demonstrated that Pmr preferentially binds to horizontally acquired DNA regions. The Pmr binding sites overlapped well with the location of the genes differentially transcribed following pmr disruption on both the plasmid and the chromosome. Our findings indicate that Pmr is a key factor in optimizing gene transcription on pCAR1 and the host chromosome.

Identification and Biochemical Characterization of Halisulfate 3 and Suvanine as Novel Inhibitors of Hepatitis C Virus NS3 Helicase from a Marine Sponge

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Atsushi Furuta

2014-01-01

Full Text Available Hepatitis C virus (HCV is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3 helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3 and suvanine as novel NS3 helicase inhibitors, with IC50 values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC50 values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.

Broadband Fan Noise Prediction System for Turbofan Engines. Volume 2; BFaNS User's Manual and Developer's Guide

Science.gov (United States)

Morin, Bruce L.

2010-01-01

Pratt & Whitney has developed a Broadband Fan Noise Prediction System (BFaNS) for turbofan engines. This system computes the noise generated by turbulence impinging on the leading edges of the fan and fan exit guide vane, and noise generated by boundary-layer turbulence passing over the fan trailing edge. BFaNS has been validated on three fan rigs that were tested during the NASA Advanced Subsonic Technology Program (AST). The predicted noise spectra agreed well with measured data. The predicted effects of fan speed, vane count, and vane sweep also agreed well with measurements. The noise prediction system consists of two computer programs: Setup_BFaNS and BFaNS. Setup_BFaNS converts user-specified geometry and flow-field information into a BFaNS input file. From this input file, BFaNS computes the inlet and aft broadband sound power spectra generated by the fan and FEGV. The output file from BFaNS contains the inlet, aft and total sound power spectra from each noise source. This report is the second volume of a three-volume set documenting the Broadband Fan Noise Prediction System: Volume 1: Setup_BFaNS User s Manual and Developer s Guide; Volume 2: BFaNS User s Manual and Developer s Guide; and Volume 3: Validation and Test Cases. The present volume begins with an overview of the Broadband Fan Noise Prediction System, followed by step-by-step instructions for installing and running BFaNS. It concludes with technical documentation of the BFaNS computer program.

Phospholipid transfer protein activity and incident type 2 diabetes mellitus

NARCIS (Netherlands)

Abbasi, Ali; Dallinga-Thie, Geesje M.; Dullaart, Robin P. F.

2015-01-01

The plasma activity of phospholipid transfer protein (PLTP), which has multifaceted functions in lipoprotein metabolism and in inflammatory responses, is elevated in insulin resistant conditions. We determined the association of plasma PLTP activity with incident type 2 diabetes mellitus (T2DM).

Dynamic Nucleolar Targeting of Dengue Virus Polymerase NS5 in Response to Extracellular pH

Science.gov (United States)

Fraser, Johanna E.; Rawlinson, Stephen M.; Heaton, Steven M.

2016-01-01

ABSTRACT The nucleolar subcompartment of the nucleus is increasingly recognized as an important target of RNA viruses. Here we document for the first time the ability of dengue virus (DENV) polymerase, nonstructural protein 5 (NS5), to accumulate within the nucleolus of infected cells and to target green fluorescent protein (GFP) to the nucleolus of live transfected cells. Intriguingly, NS5 exchange between the nucleus and nucleolus is dynamically modulated by extracellular pH, responding rapidly and reversibly to pH change, in contrast to GFP alone or other nucleolar and non-nucleolar targeted protein controls. The minimal pH-sensitive nucleolar targeting region (pHNTR), sufficient to target GFP to the nucleolus in a pH-sensitive fashion, was mapped to NS5 residues 1 to 244, with mutation of key hydrophobic residues, Leu-165, Leu-167, and Val-168, abolishing pHNTR function in NS5-transfected cells, and severely attenuating DENV growth in infected cells. This is the first report of a viral protein whose nucleolar targeting ability is rapidly modulated by extracellular stimuli, suggesting that DENV has the ability to detect and respond dynamically to the extracellular environment. IMPORTANCE Infections by dengue virus (DENV) threaten 40% of the world's population yet there is no approved vaccine or antiviral therapeutic to treat infections. Understanding the molecular details that govern effective viral replication is key for the development of novel antiviral strategies. Here, we describe for the first time dynamic trafficking of DENV nonstructural protein 5 (NS5) to the subnuclear compartment, the nucleolus. We demonstrate that NS5's targeting to the nucleolus occurs in response to acidic pH, identify the key amino acid residues within NS5 that are responsible, and demonstrate that their mutation severely impairs production of infectious DENV. Overall, this study identifies a unique subcellular trafficking event and suggests that DENV is able to detect and respond

Evaluating vacquinol-1 in rats carrying glioblastoma models RG2 and NS1.

Science.gov (United States)

Ahlstedt, Jonatan; Förnvik, Karolina; Zolfaghari, Shaian; Kwak, Dongoh; Hammarström, Lars G J; Ernfors, Patrik; Salford, Leif G; Redebrandt, Henrietta Nittby

2018-02-02

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor, and available experimental and routine therapies result in limited survival benefits. A vulnerability of GBM cells to catastrophic vacuolization and cell death, a process termed methuosis, induced by Vacquinol-1 (VQ-1) has been described earlier. In the present study, we investigate the efficacy of VQ-1 treatment in two syngeneic rat GBM models, RG2 and NS1. VQ-1 treatment affected growth of both RG2 and NS1 cells in vitro . Intracranially, significant reduction in RG2 tumor size was observed, although no effect was seen on overall survival. No survival advantage or effect on tumor size was seen in animals carrying the NS1 models compared to untreated controls. Furthermore, immunological staining of FOXP3, CD4 and CD8 showed no marked difference in immune cell infiltrate in tumor environment following treatment. Taken together, a survival advantage of VQ-1 treatment alone could not be demonstrated here, even though some effect upon tumor size was seen. Staining for immune cell markers did not indicate that VQ-1 either reduced or increased host anti-tumor immune response.

High affinity human antibody fragments to dengue virus non-structural protein 3.

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Nicole J Moreland

Full Text Available BACKGROUND: The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3 are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5' triphosphatase domain which forms the remainder of the 618-aa long protein. METHODOLOGY/PRINCIPAL FINDINGS: In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531 within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells. CONCLUSIONS/SIGNIFICANCE: Antibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.

Effects of nanosecond pulsed electric fields (nsPEFs) on the human fungal pathogen Candida albicans: an in vitro study

Science.gov (United States)

Guo, Jinsong; Dang, Jie; Wang, Kaile; Zhang, Jue; Fang, Jing

2018-05-01

Candida albicans is the leading human fungal pathogen that causes many life-threatening infections. Notably, the current clinical trial data indicate that Candida species shows the emerging resistance to anti-fungal drugs. The aim of this study was to evaluate the antifungal effects of nanosecond pulsed electric fields (nsPEFs) as a novel drug-free strategy in vitro. In this study, we investigated the inactivation and permeabilization effects of C. albicans under different nsPEFs exposure conditions (100 pulses, 100 ns in duration, intensities of 20, 40 kV cm‑1). Cell death was studied by annexin-V and propidium iodide staining. The changes of intracellular Ca2+ concentration after nsPEFs treatment were observed using Fluo-4 AM. Results show that C. albicans cells and biofilms were both obviously inhibited and destroyed after nsPEFs treatment. Furthermore, C. albicans cells were significantly permeabilized after nsPEFs treatment. Additionally, nsPEFs exposure led to a large amount of DNA and protein leakage. Importantly, nsPEFs induced a field strength-dependent apoptosis in C. albicans cells. Further experiments revealed that Ca2+ involved in nsPEFs induced C. albicans apoptosis. In conclusion, this proof-of-concept study provides a potential alternative drug-free strategy for killing pathogenic Candida species.

Kushenin induces the apoptosis of HCV-infected cells by blocking the PI3K-Akt-mTOR pathway via inhibiting NS5A

Energy Technology Data Exchange (ETDEWEB)

Zhou, Yi; Chen, Na; Liu, Xiaojing; Lin, Shumei [Department of Infectious Diseases, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061 (China); Luo, Wenjuan, E-mail: wenjuanluoxa@163.com [School of Pharmacy, Xi’an Jiaotong University, Xi’an, Shaanxi 710061 (China); Liu, Min, E-mail: minliusx@163.com [Department of Infectious Diseases, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061 (China)

2016-07-01

With the increased burden induced by HCV, there is an urgent need to develop better-tolerated agents with good safety. In this study, we evaluated the anti-HCV capability of kushenin, as well as the possible mechanism to Huh7.5-HCV cells. The results demonstrated that kushenin significantly inhibited the HCV-RNA level. Similarly, the expression of HCV-specific protein NS5A was also decreased. Molecular docking results displayed that kushenin bonded well to the active pockets of HCV NS5A, further confirming the effects of kushenin on HCV replication. Coimmunoprecipitation assay determined that kushenin suppressed the interaction between PI3K and NS5A in HCV-replicon cells. Furthermore, kushenin exerted an obviously induced function on HCV-replicon cells apoptosis by inhibiting PI3K-Akt-mTOR pathway, which could be ameliorated by the specific activator IGF-1 addition. Taken together, kushenin possesses the ability to inhibit HCV replication, and contributes to the increased apoptosis of HCV-infected cells by blocking the PI3K-Akt-mTOR pathway via inhibiting NS5A. Our results provide important evidence for a better understanding of the pathogenesis of HCV infection, and suggest that kushenin has the potential to treat HCV disease. - Highlights: • Kushenin inhibits HCV replication. • Kushenin bonds directly to NS5A protein. • Kushenin induces the apoptosis of HCV-infected cells. • kushenin suppresses the interaction between PI3K and NS5A. • Kushenin inhibits PI3K-Akt-mTOR pathway.

Kushenin induces the apoptosis of HCV-infected cells by blocking the PI3K-Akt-mTOR pathway via inhibiting NS5A

International Nuclear Information System (INIS)

Zhou, Yi; Chen, Na; Liu, Xiaojing; Lin, Shumei; Luo, Wenjuan; Liu, Min

2016-01-01

With the increased burden induced by HCV, there is an urgent need to develop better-tolerated agents with good safety. In this study, we evaluated the anti-HCV capability of kushenin, as well as the possible mechanism to Huh7.5-HCV cells. The results demonstrated that kushenin significantly inhibited the HCV-RNA level. Similarly, the expression of HCV-specific protein NS5A was also decreased. Molecular docking results displayed that kushenin bonded well to the active pockets of HCV NS5A, further confirming the effects of kushenin on HCV replication. Coimmunoprecipitation assay determined that kushenin suppressed the interaction between PI3K and NS5A in HCV-replicon cells. Furthermore, kushenin exerted an obviously induced function on HCV-replicon cells apoptosis by inhibiting PI3K-Akt-mTOR pathway, which could be ameliorated by the specific activator IGF-1 addition. Taken together, kushenin possesses the ability to inhibit HCV replication, and contributes to the increased apoptosis of HCV-infected cells by blocking the PI3K-Akt-mTOR pathway via inhibiting NS5A. Our results provide important evidence for a better understanding of the pathogenesis of HCV infection, and suggest that kushenin has the potential to treat HCV disease. - Highlights: • Kushenin inhibits HCV replication. • Kushenin bonds directly to NS5A protein. • Kushenin induces the apoptosis of HCV-infected cells. • kushenin suppresses the interaction between PI3K and NS5A. • Kushenin inhibits PI3K-Akt-mTOR pathway.

Boron tolerance in NS wheat lines

Directory of Open Access Journals (Sweden)

Brdar Milka

2006-01-01

Full Text Available Boron is an essential micronutrient for higher plants. Present in excessive amounts boron becomes toxic and can limit plant growth and yield. Suppression of root growth is one of the symptoms of boron toxicity in wheat. This study was undertaken to investigate the response of 10 perspective NS lines of wheat to high concentrations of boron. Analysis of root growth was done on young plants, germinated and grown in the presence of different concentrations of boric acid (0, 50,100 and 150 mg/1. Significant differences occurred between analyzed genotypes and treatments regarding root length. Average suppression of root growth was between 11,6 and 34,2%, for line NS 252/02 are even noted 61,4% longer roots at treatments in relation to the control. Lines with mean suppression of root growth less than 20% (NS 101/02, NS 138/01, NS 53/03 and NS 73/02 may be considered as boron tolerant. Spearmans coefficients showed high level of agreement regarding rang of root length for genotypes treated with 100 and 150 mg H3BO3/l.

50 ns Backup Solution

CERN Document Server

Kain, V; Bartosik, H; Goddard, B; Höfle, W; Iadarola, G; Meddahi, M; Pieloni, T; Rumolo, G; Salvant, B; Wenninger, J

2014-01-01

The baseline bunch spacing for LHC high luminosity proton-proton operation after LS3 is 25 ns to maximize the integrated luminosity while keeping the pile-up low. The success of this mode of operation is not guaranteed. Electron cloud, UFOs, long-range beambeam, heating and other effects might make 25 ns operation in the LHC and/or the injectors difficult. This talk will review possible showstoppers in the LHC and injectors for 25 ns operation and discuss possible remedies. An alternative would be re-considering 50 ns operation. An estimate of the 50 ns performance will be given. The question of whether a different upgrade path would have to be chosen in case of 50 ns operation will also be addressed.

50 ns Backup Solution

International Nuclear Information System (INIS)

Kain, V; Arduini, G; Bartosik, H; Goddard, B; Höfle, W; Iadarola, G; Meddahi, M; Pieloni, T; Rumolo, G; Salvant, B; Wenninger, J

2014-01-01

The baseline bunch spacing for LHC high luminosity proton-proton operation after LS3 is 25 ns to maximize the integrated luminosity while keeping the pile-up low. The success of this mode of operation is not guaranteed. Electron cloud, UFOs, long-range beambeam, heating and other effects might make 25 ns operation in the LHC and/or the injectors difficult. This talk will review possible showstoppers in the LHC and injectors for 25 ns operation and discuss possible remedies. An alternative would be re-considering 50 ns operation. An estimate of the 50 ns performance will be given. The question of whether a different upgrade path would have to be chosen in case of 50 ns operation will also be addressed

Evolutionary dynamics of hepatitis C virus NS3 protease domain during and following treatment with narlaprevir, a potent NS3 protease inhibitor

NARCIS (Netherlands)

de Bruijne, J.; Thomas, X. V.; Rebers, S. P.; Weegink, C. J.; Treitel, M. A.; Hughes, E.; Bergmann, J. F.; de Knegt, R. J.; Janssen, H. L. A.; Reesink, H. W.; Molenkamp, R.; Schinkel, J.

2013-01-01

Narlaprevir, a hepatitis C virus (HCV) NS3/4A serine protease inhibitor, has demonstrated robust antiviral activity in a placebo-controlled phase 1 study. To study evolutionary dynamics of resistant variants, the NS3 protease sequence was clonally analysed in thirty-two HCV genotype 1-infected

Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

Science.gov (United States)

Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

2014-01-17

The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

Molecular characterization of Api g 2, a novel allergenic member of the lipid-transfer protein 1 family from celery stalks.

Science.gov (United States)

Gadermaier, Gabriele; Egger, Matthias; Girbl, Tamara; Erler, Anja; Harrer, Andrea; Vejvar, Eva; Liso, Marina; Richter, Klaus; Zuidmeer, Laurian; Mari, Adriano; Ferreira, Fatima

2011-04-01

Celery represents a relevant cross-reactive food allergen source in the adult population. As the currently known allergens are not typical elicitors of severe symptoms, we aimed to identify and characterize a non-specific lipid transfer protein (nsLTP). MS and cDNA cloning were applied to obtain the full-length sequence of a novel allergenic nsLTP from celery stalks. The purified natural molecule consisted of a single isoallergen designated as Api g 2.0101, which was recombinantly produced in Escherichia coli Rosetta-gami. The natural and recombinant molecules displayed equivalent physicochemical and immunological properties. Circular dichroism revealed a typical α-helical fold and high thermal stability. Moreover, Api g 2 was highly resistant to simulated gastrointestinal digestion. As assessed by ELISA, thermal denaturation did not affect the IgE binding of Api g 2. Natural and recombinant Api g 2 showed similar allergenic activity in mediator release assays. Api g 2-specific IgE antibodies cross-reacted with peach and mugwort pollen nsLTPs. Based on our results, it can be anticipated that inclusion of recombinant Api g 2 in the current panel of allergens for molecule-based diagnosis will facilitate the evaluation of the clinical relevance of nsLTP sensitization in celery allergy and help clinicians in the management of food allergic patients. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multiple Functional Domains and Complexes of the Two Nonstructural Proteins of Human Respiratory Syncytial Virus Contribute to Interferon Suppression and Cellular Location▿

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Swedan, Samer; Andrews, Joel; Majumdar, Tanmay; Musiyenko, Alla; Barik, Sailen

2011-01-01

Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKKε, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo- as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKKε reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression. PMID:21795342

A search for RNA insertions and NS3 gene duplication in the genome of cytopathic isolates of bovine viral diarrhea virus

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V.L. Quadros

2006-07-01

Full Text Available Calves born persistently infected with non-cytopathic bovine viral diarrhea virus (ncpBVDV frequently develop a fatal gastroenteric illness called mucosal disease. Both the original virus (ncpBVDV and an antigenically identical but cytopathic virus (cpBVDV can be isolated from animals affected by mucosal disease. Cytopathic BVDVs originate from their ncp counterparts by diverse genetic mechanisms, all leading to the expression of the non-structural polypeptide NS3 as a discrete protein. In contrast, ncpBVDVs express only the large precursor polypeptide, NS2-3, which contains the NS3 sequence within its carboxy-terminal half. We report here the investigation of the mechanism leading to NS3 expression in 41 cpBVDV isolates. An RT-PCR strategy was employed to detect RNA insertions within the NS2-3 gene and/or duplication of the NS3 gene, two common mechanisms of NS3 expression. RT-PCR amplification revealed insertions in the NS2-3 gene of three cp isolates, with the inserts being similar in size to that present in the cpBVDV NADL strain. Sequencing of one such insert revealed a 296-nucleotide sequence with a central core of 270 nucleotides coding for an amino acid sequence highly homologous (98% to the NADL insert, a sequence corresponding to part of the cellular J-Domain gene. One cpBVDV isolate contained a duplication of the NS3 gene downstream from the original locus. In contrast, no detectable NS2-3 insertions or NS3 gene duplications were observed in the genome of 37 cp isolates. These results demonstrate that processing of NS2-3 without bulk mRNA insertions or NS3 gene duplications seems to be a frequent mechanism leading to NS3 expression and BVDV cytopathology.

Oncolytic effects of a novel influenza A virus expressing interleukin-15 from the NS reading frame.

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Marijke van Rikxoort

Full Text Available Oncolytic influenza A viruses with deleted NS1 gene (delNS1 replicate selectively in tumour cells with defective interferon response and/or activated Ras/Raf/MEK/ERK signalling pathway. To develop a delNS1 virus with specific immunostimulatory properties, we used an optimised technology to insert the interleukin-15 (IL-15 coding sequence into the viral NS gene segment (delNS1-IL-15. DelNS1 and delNS1-IL-15 exerted similar oncolytic effects. Both viruses replicated and caused caspase-dependent apoptosis in interferon-defective melanoma cells. Virus replication was required for their oncolytic activity. Cisplatin enhanced the oncolytic activity of delNS1 viruses. The cytotoxic drug increased delNS1 replication and delNS1-induced caspase-dependent apoptosis. Interference with MEK/ERK signalling by RNAi-mediated depletion or the MEK inhibitor U0126 did not affect the oncolytic effects of the delNS1 viruses. In oncolysis sensitive melanoma cells, delNS1-IL-15 (but not delNS1 infection resulted in the production of IL-15 levels ranging from 70 to 1140 pg/mL in the cell culture supernatants. The supernatants of delNS1-IL-15-infected (but not of delNS1-infected melanoma cells induced primary human natural killer cell-mediated lysis of non-infected tumour cells. In conclusion, we constructed a novel oncolytic influenza virus that combines the oncolytic activity of delNS1 viruses with immunostimulatory properties through production of functional IL-15. Moreover, we showed that the oncolytic activity of delNS1 viruses can be enhanced in combination with cytotoxic anti-cancer drugs.

Hepatitis C virus induces E6AP-dependent degradation of the retinoblastoma protein.

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Tsubasa Munakata

2007-09-01

Full Text Available Hepatitis C virus (HCV is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B, forms a complex with the retinoblastoma tumor suppressor protein (pRb, targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP, as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer.

[Clinical significance of NS1-BP expression in esophageal squamous cell carcinoma].

Science.gov (United States)

Ren, K; Qian, D; Wang, Y W; Pang, Q S; Zhang, W C; Yuan, Z Y; Wang, P

2018-01-23

Objective: To investigate the clinical significance of NS1-BP expression in patients with esophageal squamous cell carcinoma (ESCC), and to study the roles of NS1-BP in proliferation and apoptosis of ESCC cells. Methods: A total of 98 tumor tissues and 30 adjacent normal tissues from 98 ESCC patients were used as study group and control group, and these samples were collected in Sun Yat-Sen University Cancer Center between 2002 and 2008. In addition, 46 ESCC tissues which were collected in Cancer Institute and Hospital of Tianjin Medical University were used as validation group. Expression of mucosal NS1-BP was detected by immunohistochemistry. Kaplan-Meier curve and log-rank test were used to analyze the survival rate. Multivariate Cox proportional hazard model was used to analyze the prognostic factors. Furthermore, NS1-BP was over expressed or knocked down in ESCC cells by transient transfection. Protein levels of c-Myc were detected by western blot. Cell viability and apoptosis was analyzed by MTT assay and flow cytometry. Results: Among all of tested samples, NS1-BP were down-regulated in 9 out of 30 non-tumorous normal esophageal tissues (30.0%) and 85 out of 144 ESCC tissues (59.0%), respectively, showing a statistically significant difference ( P =0.012). In the study group, three-year disease-free survival rate of NS1-BP high expression group (53.2%) was significantly higher than that of NS1-BP low expression group (27.6%; P =0.009). In the validation group, the three-year disease-free survival rates were 57.8% and 25.5% in NS1-BP high and low levels groups, respectively, showing a similar results ( P =0.016). Importantly, multivariate analyses showed that low expression of NS1-BP was an independent predictor for chemoradiotherapy sensitivity and shorter disease-free survival time in ESCC patients( P <0.05 for all). Furthermore, overexpressed NS1-BP in TE-1 cells repressed c-Myc expression, inhibited cell proliferation and promoted apoptosis. In contrast

Cell culture-adaptive mutations of NS5A affect replication of hepatitis C virus differentially depending on the viral genotypes.

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Chung, Aeri; Jin, Bora; Han, Kwang-Hyub; Ahn, Sang Hoon; Kim, Seungtaek

2017-01-01

Most of HCV RNAs require cell culture-adaptive mutations for efficient replication in cell culture and a number of such mutations have been described including a well-known S2204I substitution mutation in NS5A protein. In contrast, the replication of genotype 2a JFH1 RNA in cell culture does not require any cell culture-adaptive mutation. Rather, the presence of S2204I mutation impaired the JFH1 RNA replication. In this study, we examined the effect of reversions and substitutions of NS5A cell culture-adaptive mutations on virus replication in different genotypic backgrounds after either placing genotype 1a NS5A in the genotype 2a JFH1 or vice versa. The results from this investigation suggest that the S2204I mutation affects HCV RNA replication differentially depending on the viral genotypes but that the effect was not simply explained by the genotypic background. Perhaps, the effect of the S2204I mutation on HCV replication reflects both intra- and intergenic interactions of NS5A protein. J. Med. Virol. 89:146-152, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Phospholipid transfer protein activity and incident type 2 diabetes mellitus

NARCIS (Netherlands)

Abbasi, Ali; Dallinga-Thie, Geesje M.; Dullaart, Robin P. F.

2015-01-01

Background: The plasma activity of phospholipid transfer protein (PLTP), which has multifaceted functions in lipoprotein metabolism and in inflammatory responses, is elevated in insulin resistant conditions. We determined the association of plasma PLTP activity with incident type 2 diabetes mellitus

Activation of histamine H4 receptor inhibits TNFα/IMD-0354-induced apoptosis in human salivary NS-SV-AC cells.

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Stegajev, Vasili; Kouri, Vesa-Petteri; Salem, Abdelhakim; Rozov, Stanislav; Stark, Holger; Nordström, Dan C E; Konttinen, Yrjö T

2014-12-01

Apoptosis is involved in the pathogenesis of Sjögren's syndrome (SS), an autoimmune disease affecting exocrine glands. Our recent studies revealed diminished histamine H4 receptor (H₄R) expression and impaired histamine transport in the salivary gland epithelial cells in SS. The aim was now to test if nanomolar histamine and high-affinity H₄R signaling affect apoptosis of human salivary gland epithelial cell. Simian virus 40-immortalized acinar NS-SV-AC cells were cultured in serum-free keratinocyte medium ± histamine H₄R agonist HST-10. Expression and internalization of H₄R were studied by immunofluorescence staining ± clathrin inhibitor methyl-β-cyclodextrin (MβCD). Apoptosis induced using tumor necrosis factor-α with nuclear factor-κB inhibitor IMD-0354 was studied using phase contrast microscopy, Western blot, flow cytometry and polymerase chain reaction (qRT-PCR). HST-10-stimulated H₄R internalization was inhibited by MβCD. Western blotting revealed diminished phosphorylated c-Jun N-terminal kinase JNK, but unchanged levels of phosphorylated extracellular signal regulated kinase pERK1/2 in H₄R-stimulated samples compared to controls. qRT-PCR showed up-regulated expression of anti-apoptotic B cell lymphoma-extra large/Bcl-xL mRNAs and proteins, whereas pro-apoptotic Bcl-2-associated X protein/BAX remained unchanged in H4R-stimulated samples. H₄R stimulation diminished cleavage of PARP and flow cytometry showed significant dose-dependent inhibitory effect of H₄R stimulation on apoptosis. As far as we know this is the first study showing inhibitory effect of H₄R activation on apoptosis of human salivary gland cells. Diminished H₄R-mediated activation may contribute to loss of immune tolerance in autoimmune diseases and in SS in particular.

Exploring the molecular basis of dsRNA recognition by NS1 protein of influenza A virus using molecular dynamics simulation and free energy calculation.

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Pan, Dabo; Sun, Huijun; Shen, Yulin; Liu, Huanxiang; Yao, Xiaojun

2011-12-01

The frequent outbreak of influenza pandemic and the limited available anti-influenza drugs highlight the urgent need for the development of new antiviral drugs. The dsRNA-binding surface of nonstructural protein 1 of influenza A virus (NS1A) is a promising target. The detailed understanding of NS1A-dsRNA interaction will be valuable for structure-based anti-influenza drug discovery. To characterize and explore the key interaction features between dsRNA and NS1A, molecular dynamics simulation combined with MM-GBSA calculations were performed. Based on the MM-GBSA calculations, we find that the intermolecular van der Waals interaction and the nonpolar solvation term provide the main driving force for the binding process. Meanwhile, 17 key residues from NS1A were identified to be responsible for the dsRNA binding. Compared with the wild type NS1A, all the studied mutants S42A, T49A, R38A, R35AR46A have obvious reduced binding free energies with dsRNA reflecting in the reduction of the polar and/or nonpolar interactions. In addition, the structural and energy analysis indicate the mutations have a small effect to the backbone structures but the loss of side chain interactions is responsible for the decrease of the binding affinity. The uncovering of NS1A-dsRNA recognition mechanism will provide some useful insights and new chances for the development of anti-influenza drugs. Copyright © 2011 Elsevier B.V. All rights reserved.

Genetic Defects Underlie the Non-syndromic Autosomal Recessive Intellectual Disability (NS-ARID

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Saleha Shamim

2017-05-01

Full Text Available Intellectual disability (ID is a neurodevelopmental disorder which appears frequently as the result of genetic mutations and may be syndromic (S-ID or non-syndromic (NS-ID. ID causes an important economic burden, for patient's family, health systems, and society. Identifying genes that cause S-ID can easily be evaluated due to the clinical symptoms or physical anomalies. However, in the case of NS-ID due to the absence of co-morbid features, the latest molecular genetic techniques can be used to understand the genetic defects that underlie it. Recent studies have shown that non-syndromic autosomal recessive (NS-ARID is extremely heterogeneous and contributes much more than X-linked ID. However, very little is known about the genes and loci involved in NS-ARID relative to X-linked ID, and whose complete genetic etiology remains obscure. In this review article, the known genetic etiology of NS-ARID and possible relationships between genes and the associated molecular pathways of their encoded proteins has been reviewed which will enhance our understanding about the underlying genes and mechanisms in NS-ARID.

StpA and Hha stimulate pausing by RNA polymerase by promoting DNA-DNA bridging of H-NS filaments.

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Boudreau, Beth A; Hron, Daniel R; Qin, Liang; van der Valk, Ramon A; Kotlajich, Matthew V; Dame, Remus T; Landick, Robert

2018-06-20

In enterobacteria, AT-rich horizontally acquired genes, including virulence genes, are silenced through the actions of at least three nucleoid-associated proteins (NAPs): H-NS, StpA and Hha. These proteins form gene-silencing nucleoprotein filaments through direct DNA binding by H-NS and StpA homodimers or heterodimers. Both linear and bridged filaments, in which NAPs bind one or two DNA segments, respectively, have been observed. Hha can interact with H-NS or StpA filaments, but itself lacks a DNA-binding domain. Filaments composed of H-NS alone can inhibit transcription initiation and, in the bridged conformation, slow elongating RNA polymerase (RNAP) by promoting backtracking at pause sites. How the other NAPs modulate these effects of H-NS is unknown, despite evidence that they help regulate subsets of silenced genes in vivo (e.g. in pathogenicity islands). Here we report that Hha and StpA greatly enhance H-NS-stimulated pausing by RNAP at 20°C. StpA:H-NS or StpA-only filaments also stimulate pausing at 37°C, a temperature at which Hha:H-NS or H-NS-only filaments have much less effect. In addition, we report that both Hha and StpA greatly stimulate DNA-DNA bridging by H-NS filaments. Together, these observations indicate that Hha and StpA can affect H-NS-mediated gene regulation by stimulating bridging of H-NS/DNA filaments.

Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

DEFF Research Database (Denmark)

Köpper, Frederik; Bierwirth, Cathrin; Schön, Margarete

2013-01-01

knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation...

Cytotoxic Activity of Coagulase-Negative Staphylococci in Bovine Mastitis

Science.gov (United States)

Zhang, Songlin; Maddox, Carol W.

2000-01-01

Secreted toxins play important roles in the pathogenesis of bacterial infections. In this study, we examined the presence of secreted cytotoxic factors of coagulase-negative staphylococci (CoNS) from bovine clinical and subclinical mastitis. A 34- to 36-kDa protein with cell-rounding cytotoxic activity was found in many CoNS strains, especially in Staphylococcus chromogenes strains. The protein caused cell detachment and cell rounding in several cell lines, including HEp-2, Int 407, CHO-K1, and Y-1 cells. Native protein recovered from nondenatured polyacrylamide gel electrophoresis showed both cytotoxic activity and casein hydrolysis activity. The purified protein had a pH optimal at 7.2 to 7.5 and a pI of 5.1 and was heat labile. The proteolytic activity could be inhibited by zinc and metal specific inhibitors such as 1,10-phenanthroline and EDTA, indicating that it is a metalloprotease. Protein mass analysis and peptide sequencing indicated that the protein is a novel metalloprotease. Different bacterial strains expressed variable levels of 34- to 36-kDa protease, which may provide an indication of strain virulence. PMID:10678913

The protein C omega-loop substitution Asn2Ile is associated with reduced protein C anticoagulant activity.

LENUS (Irish Health Repository)

Preston, Roger J S

2012-02-01

We report a kindred with heritable protein C (PC) deficiency in which two siblings with severe thrombosis showed a composite type I and IIb PC deficiency phenotype, identified using commercial PC assays (proband: PC antigen 42 u\\/dl, amidolytic activity 40 u\\/dl, anticoagulant activity 9 u\\/dl). The independent PROC nucleotide variations c.669C>A (predictive of Ser181Arg) and c.131C>T (predictive of Asn2Ile) segregated with the type I and type IIb PC deficiency phenotypes respectively, but co-segregated in the siblings with severe thrombosis. Soluble thrombomodulin (sTM)-mediated inhibition of plasma thrombin generation from an individual with PC-Asn2Ile was lower (endogenous thrombin potential (ETP) 56 +\\/- 1% that of ETP determined without sTM) than control plasma (ETP 15 +\\/- 2%) indicating reduced PC anticoagulant activity. Recombinant APC-Asn2Ile exhibited normal amidolytic activity but impaired anticoagulant activity. Protein S (PS)-dependent anticoagulant activity of recombinant APC-Asn2Ile and binding of recombinant APC-Asn2Ile to endothelial protein C receptor (EPCR) were reduced compared to recombinant wild-type APC. Asn2 lies within the omega-loop of the PC\\/APC Gla domain and this region is critical for calcium-induced folding and subsequent interactions with anionic phospholipids, EPCR and PS. The disruption of these interactions in this naturally-occurring PC variant highlights their collective importance in mediating APC anticoagulant activity in vivo.

Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering--A Comparison between Wild-Type Protein and a Hinge Mutant.

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Saara Laulumaa

Full Text Available Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS. The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.

Miracle: The Multi-Interface Cross-Layer Extension of ns2

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Guerra Federico

2010-01-01

Full Text Available We present Miracle, a novel framework which extends ns2 to facilitate the simulation and the design of beyond 4G networks. Miracle enhances ns2 by providing an efficient and embedded engine for handling cross-layer messages and, at the same time, enabling the coexistence of multiple modules within each layer of the protocol stack. We also present a novel framework developed as an extension of Miracle called Miracle PHY and MAC. This framework facilitates the development of more realistic Channel, PHY and MAC modules, considering features currently lacking in most state-of-the-art simulators, while at the same time giving a strong emphasis on code modularity, interoperability and reusability. Finally, we provide an overview of the wireless technologies implemented in Miracle, discussing in particular the models for the IEEE 802.11, UMTS and WiMAX standards and for Underwater Acoustic Networks. We observe that, thanks to Miracle and its extensions, it is possible to carefully simulate complex network architectures at all the OSI layers, from the physical reception model to standard applications and system management schemes. This allows to have a comprehensive view of all the interactions among network components, which play an important role in many research areas, such as cognitive networking and cross-layer design.

HTLV-1 Tax-mediated TAK1 activation involves TAB2 adapter protein

International Nuclear Information System (INIS)

Yu Qingsheng; Minoda, Yasumasa; Yoshida, Ryoko; Yoshida, Hideyuki; Iha, Hidekatsu; Kobayashi, Takashi; Yoshimura, Akihiko; Takaesu, Giichi

2008-01-01

Human T cell leukemia virus type 1 (HTLV-1) Tax is an oncoprotein that plays a crucial role in the proliferation and transformation of HTLV-1-infected T lymphocytes. It has recently been reported that Tax activates a MAPKKK family, TAK1. However, the molecular mechanism of Tax-mediated TAK1 activation is not well understood. In this report, we investigated the role of TAK1-binding protein 2 (TAB2) in Tax-mediated TAK1 activation. We found that TAB2 physically interacts with Tax and augments Tax-induced NF-κB activity. Tax and TAB2 cooperatively activate TAK1 when they are coexpressed. Furthermore, TAK1 activation by Tax requires TAB2 binding as well as ubiquitination of Tax. We also found that the overexpression of TRAF2, 5, or 6 strongly induces Tax ubiquitination. These results suggest that TAB2 may be critically involved in Tax-mediated activation of TAK1 and that NF-κB-activating TRAF family proteins are potential cellular E3 ubiquitin ligases toward Tax