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  1. The Vpr protein from HIV-1: distinct roles along the viral life cycle

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    Benichou Serge

    2005-02-01

    Full Text Available Abstract The genomes of human and simian immunodeficiency viruses (HIV and SIV encode the gag, pol and env genes and contain at least six supplementary open reading frames termed tat, rev, nef, vif, vpr, vpx and vpu. While the tat and rev genes encode regulatory proteins absolutely required for virus replication, nef, vif, vpr, vpx and vpu encode for small proteins referred to "auxiliary" (or "accessory", since their expression is usually dispensable for virus growth in many in vitro systems. However, these auxiliary proteins are essential for viral replication and pathogenesis in vivo. The two vpr- and vpx-related genes are found only in members of the HIV-2/SIVsm/SIVmac group, whereas primate lentiviruses from other lineages (HIV-1, SIVcpz, SIVagm, SIVmnd and SIVsyk contain a single vpr gene. In this review, we will mainly focus on vpr from HIV-1 and discuss the most recent developments in our understanding of Vpr functions and its role during the virus replication cycle.

  2. Characterization of the molecular determinants of primary HIV-1 Vpr proteins: impact of the Q65R and R77Q substitutions on Vpr functions.

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    Guillaume Jacquot

    Full Text Available Although HIV-1 Vpr displays several functions in vitro, limited information exists concerning their relevance during infection. Here, we characterized Vpr variants isolated from a rapid and a long-term non-progressor (LTNP. Interestingly, vpr alleles isolated from longitudinal samples of the LTNP revealed a dominant sequence that subsequently led to diversity similar to that observed in the progressor patient. Most of primary Vpr proteins accumulated at the nuclear envelope and interacted with host-cell partners of Vpr. They displayed cytostatic and proapoptotic activities, although a LTNP allele, harboring the Q65R substitution, failed to bind the DCAF1 subunit of the Cul4a/DDB1 E3 ligase and was inactive. This Q65R substitution correlated with impairment of Vpr docking at the nuclear envelope, raising the possibility of a functional link between this property and the Vpr cytostatic activity. In contradiction with published results, the R77Q substitution, found in LTNP alleles, did not influence Vpr proapoptotic activity.

  3. HIV-1 Vpr increases HCV replication through VprBP in cell culture.

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    Yan, Yanling; Huang, Fang; Yuan, Ting; Sun, Binlian; Yang, Rongge

    2016-09-02

    Coinfection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) occurs at a high frequency, in which HIV shows a promotion of HCV-derived liver diseases. However, the mechanism of how this occurs is not well understood. Our previous work has demonstrated that the HIV-1 accessory protein Vpr enhances HCV RNA replication in cell culture. Because Vpr performs most of its functions through host protein VprBP (DCAF1), the role of VprBP in the regulation of HCV by Vpr was investigated in this study. We found that the Vpr mutant Q65R, which is deficient in VprBP binding, could not enhance HCV replication. Furthermore, Vpr-mediated enhancement of HCV replication was severely diminished in VprBP knockdown cells. In addition, an inhibitor of Cullin RING E3 ligases, MLN4924, impaired the function of Vpr during HCV replication. Together, these results suggest that Vpr promotes HCV replication in a VprBP-dependent manner, and that the activity of Cullin RING E3 ligases is essential to this process. In conclusion, our findings demonstrate that HIV-1 Vpr makes the cellular environment more suitable for HCV replication, which might relate with the host ubiquitination system.

  4. HIV-1 Vpr protein activates the NF-κB pathway to promote G2/M cell cycle arrest

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    Zhibin Liang; Ruikang Liu; Yongquan Lin; Chen Liang; Juan Tan; Wentao Qiao

    2015-01-01

    Viral protein R(Vpr) plays an important role in the replication and pathogenesis of Human immunodeficiency virus type 1(HIV-1). Some of the various functions attributed to Vpr, including the induction of G2/M cell cycle arrest, activating the NF-κB pathway, and promoting viral reverse transcription, might be interrelated. To test this hypothesis, a panel of Vpr mutants were investigated for their ability to induce G2/M arrest and to activate the NF-κB pathway. The results showed that the Vpr mutants that failed to activate NF-κB also lost the activity to induce G2/M arrest, which suggests that inducing G2/M arrest via Vpr depends at least partially on the activation of NF-κB. This latter possibility is supported by data showing that knocking down the key factors in the NF-κB pathway – p65, Rel B, IKKα, or IKKβ– partially rescued the G2/M arrest induced by Vpr.Our results suggest that the NF-κB pathway is probably involved in Vpr-induced G2/M cell cycle arrest.

  5. SLX4-SLX1 Protein-independent Down-regulation of MUS81-EME1 Protein by HIV-1 Viral Protein R (Vpr).

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    Zhou, Xiaohong; DeLucia, Maria; Ahn, Jinwoo

    2016-08-12

    Evolutionarily conserved structure-selective endonuclease MUS81 forms a complex with EME1 and further associates with another endonuclease SLX4-SLX1 to form a four-subunit complex of MUS81-EME1-SLX4-SLX1, coordinating distinctive biochemical activities of both endonucleases in DNA repair. Viral protein R (Vpr), a highly conserved accessory protein in primate lentiviruses, was previously reported to bind SLX4 to mediate down-regulation of MUS81. However, the detailed mechanism underlying MUS81 down-regulation is unclear. Here, we report that HIV-1 Vpr down-regulates both MUS81 and its cofactor EME1 by hijacking the host CRL4-DCAF1 E3 ubiquitin ligase. Multiple Vpr variants, from HIV-1 and SIV, down-regulate both MUS81 and EME1. Furthermore, a C-terminally truncated Vpr mutant and point mutants R80A and Q65R, all of which lack G2 arrest activity, are able to down-regulate MUS81-EME1, suggesting that Vpr-induced G2 arrest is not correlated with MUS81-EME1 down-regulation. We also show that neither the interaction of MUS81-EME1 with Vpr nor their down-regulation is dependent on SLX4-SLX1. Together, these data provide new insight on a conserved function of Vpr in a host endonuclease down-regulation.

  6. Defining the roles for Vpr in HIV-1-associated neuropathogenesis.

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    James, Tony; Nonnemacher, Michael R; Wigdahl, Brian; Krebs, Fred C

    2016-08-01

    It is increasingly evident that the human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has a unique role in neuropathogenesis. Its ability to induce G2/M arrest coupled with its capacity to increase viral gene transcription gives it a unique role in sustaining viral replication and aiding in the establishment and maintenance of a systemic infection. The requirement of Vpr for HIV-1 infection and replication in cells of monocytic origin (a key lineage of cells involved in HIV-1 neuroinvasion) suggests an important role in establishing and sustaining infection in the central nervous system (CNS). Contributions of Vpr to neuropathogenesis can be expanded further through (i) naturally occurring HIV-1 sequence variation that results in functionally divergent Vpr variants; (ii) the dual activities of Vpr as a intracellular protein delivered and expressed during HIV-1 infection and as an extracellular protein that can act on neighboring, uninfected cells; (iii) cell type-dependent consequences of Vpr expression and exposure, including cell cycle arrest, metabolic dysregulation, and cytotoxicity; and (iv) the effects of Vpr on exosome-based intercellular communication in the CNS. Revealing that the effects of this pleiotropic viral protein is an essential part of a greater understanding of HIV-1-associated pathogenesis and potential approaches to treating and preventing disease caused by HIV-1 infection.

  7. Human Immunodeficiency Virus Type 1 (HIV-1) Viral Protein R (Vpr)-Mediated Cell Cycle Arrest: An Analysis of Current Mechanistic Models

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    2006-06-08

    with vpr + , but not vpr - , experimental HIV strains became tetraploid . The presence of both multi- and mono-nucleated sub-populations of these 8N...the direct binding of Vpr with human 14-3-3 isoforms. This group performed a series of yeast two- hybrid screens, using wild-type and two mutant Vpr

  8. HIV-1 Vpr induces interferon-stimulated genes in human monocyte-derived macrophages.

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    Muhammad Atif Zahoor

    Full Text Available Macrophages act as reservoirs of human immunodeficiency virus type 1 (HIV-1 and play an important role in its transmission to other cells. HIV-1 Vpr is a multi-functional protein involved in HIV-1 replication and pathogenesis; however, its exact role in HIV-1-infected human macrophages remains poorly understood. In this study, we used a microarray approach to explore the effects of HIV-1 Vpr on the transcriptional profile of human monocyte-derived macrophages (MDMs. More than 500 genes, mainly those involved in the innate immune response, the type I interferon pathway, cytokine production, and signal transduction, were differentially regulated (fold change >2.0 after infection with a recombinant adenovirus expressing HIV-1 Vpr protein. The differential expression profiles of select interferon-stimulated genes (ISGs and genes involved in the innate immune response, including STAT1, IRF7, MX1, MX2, ISG15, ISG20, IFIT1, IFIT2, IFIT3, IFI27, IFI44L, APOBEC3A, DDX58 (RIG-I, TNFSF10 (TRAIL, and RSAD2 (viperin were confirmed by real-time quantitative PCR and were consistent with the microarray data. In addition, at the post-translational level, HIV-1 Vpr induced the phosphorylation of STAT1 at tyrosine 701 in human MDMs. These results demonstrate that HIV-1 Vpr leads to the induction of ISGs and expand the current understanding of the function of Vpr and its role in HIV-1 immune pathogenesis.

  9. Nuclear exportin receptor CAS regulates the NPI-1-mediated nuclear import of HIV-1 Vpr.

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    Eri Takeda

    Full Text Available Vpr, an accessory protein of human immunodeficiency virus type 1, is a multifunctional protein that plays an important role in viral replication. We have previously shown that the region between residues 17 and 74 of Vpr (Vpr(N17C74 contained a bona fide nuclear localization signal and it is targeted Vpr(N17C74 to the nuclear envelope and then imported into the nucleus by importin α (Impα alone. The interaction between Impα and Vpr is important not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages; however, it was unclear whether full-length Vpr enters the nucleus in a manner similar to Vpr(N17C74. This study investigated the nuclear import of full-length Vpr using the three typical Impα isoforms, Rch1, Qip1 and NPI-1, and revealed that full-length Vpr is selectively imported by NPI-1, but not Rch1 and Qip1, after it makes contact with the perinuclear region in digitonin-permeabilized cells. A binding assay using the three Impα isoforms showed that Vpr bound preferentially to the ninth armadillo repeat (ARM region (which is also essential for the binding of CAS, the export receptor for Impα in all three isoforms. Comparison of biochemical binding affinities between Vpr and the Impα isoforms using surface plasmon resonance analysis demonstrated almost identical values for the binding of Vpr to the full-length isoforms and to their C-terminal domains. By contrast, the data showed that, in the presence of CAS, Vpr was released from the Vpr/NPI-1 complex but was not released from Rch1 or Qip1. Finally, the NPI-1-mediated nuclear import of Vpr was greatly reduced in semi-intact CAS knocked-down cells and was recovered by the addition of exogenous CAS. This report is the first to show the requirement for and the regulation of CAS in the functioning of the Vpr-Impα complex.

  10. Roles of p53 and caspases in the induction of cell cycle arrest and apoptosis by HIV-1 vpr.

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    Shostak, L D; Ludlow, J; Fisk, J; Pursell, S; Rimel, B J; Nguyen, D; Rosenblatt, J D; Planelles, V

    1999-08-25

    The vpr gene from the human immunodeficiency virus type-1 (HIV-1) encodes a 14-kDa protein that prevents cell proliferation by causing a block in the G(2) phase of the cell cycle. This cellular function of vpr is conserved in evolution because other primate lentiviruses, including HIV-2, SIV(mac), and SIV(agm) encode related genes that also induce G(2) arrest. After G(2) arrest, cells expressing vpr undergo apoptosis. The signaling pathways that result in vpr-induced cell cycle arrest and apoptosis have yet to be determined. The p53 tumor suppressor protein is involved in signaling pathways leading to cell cycle arrest and apoptosis in a variety of cell types. In this work, we examine the potential role of p53 in mediating cell cycle block and/or apoptosis by HIV-1 vpr and demonstrate that both phenomena occur independently of the presence and function of p53. Caspases are common mediators of apoptosis. We examined the potential role of caspases in mediating vpr-induced apoptosis by treating vpr-expressing cells with Boc-D-FMK, a broad spectrum, irreversible inhibitor of the caspase family. Boc-D-FMK significantly reduced the numbers of apoptotic cells induced by vpr. Therefore, we conclude that vpr-induced apoptosis is effected via the activation of caspases. Copyright 1999 Academic Press.

  11. HIV-1 replication through hHR23A-mediated interaction of Vpr with 26S proteasome.

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    Ge Li

    Full Text Available HIV-1 Vpr is a virion-associated protein. Its activities link to viral pathogenesis and disease progression of HIV-infected patients. In vitro, Vpr moderately activates HIV-1 replication in proliferating T cells, but it is required for efficient viral infection and replication in vivo in non-dividing cells such as macrophages. How exactly Vpr contributes to viral replication remains elusive. We show here that Vpr stimulates HIV-1 replication at least in part through its interaction with hHR23A, a protein that binds to 19S subunit of the 26S proteasome and shuttles ubiquitinated proteins to the proteasome for degradation. The Vpr-proteasome interaction was initially discovered in fission yeast, where Vpr was shown to associate with Mts4 and Mts2, two 19S-associated proteins. The interaction of Vpr with the 19S subunit of the proteasome was further confirmed in mammalian cells where Vpr associates with the mammalian orthologues of fission yeast Mts4 and S5a. Consistently, depletion of hHR23A interrupts interaction of Vpr with proteasome in mammalian cells. Furthermore, Vpr promotes hHR23A-mediated protein-ubiquitination, and down-regulation of hHR23A using RNAi significantly reduced viral replication in non-proliferating MAGI-CCR5 cells and primary macrophages. These findings suggest that Vpr-proteasome interaction might counteract certain host restriction factor(s to stimulate viral replication in non-dividing cells.

  12. Delineating HIV-associated neurocognitive disorders using transgenic models: the neuropathogenic actions of Vpr.

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    Power, Christopher; Hui, Elizabeth; Vivithanaporn, Pornpun; Acharjee, Shaona; Polyak, Maria

    2012-06-01

    HIV-associated neurocognitive disorders (HAND) represent a constellation of neurological disabilities defined by neuropsychological impairments, neurobehavioral abnormalities and motor deficits. To gain insights into the mechanisms underlying the development of these disabilities, several transgenic models have been developed over the past two decades, which have provided important information regarding the cellular and molecular factors contributing to the neuropathogenesis of HAND. Herein, we concentrate on the neuropathogenic effects of HIV-1 Vpr expressed under the control of c-fms, resulting transgene expression in myeloid cells in both the central and peripheral nervous systems. Vpr's actions, possibly through its impact on cell cycle machinery, in brain culminate in neuronal and astrocyte injury and death through apoptosis involving activation of caspases-3, -6 and -9 depending on the individual target cell type. Indeed, these outcomes are also induced by soluble Vpr implying Vpr's effects stem from direct interaction with target cells. Remarkably, in vivo transgenic Vpr expression induces a neurodegenerative phenotype defined by neurobehavioral deficits and neuronal loss in the absence of frank inflammation. Implantation of another viral protein, hepatitis C virus (HCV) core, into Vpr transgenic animals' brains stimulated neuroinflammation and amplified the neurodegenerative disease phenotype, thereby recapitulating HCV's putative neuropathogenic actions. The availability of different transgenic models to study HIV neuropathogenesis represents exciting and innovative approaches to understanding disease mechanisms and perhaps developing new therapeutic strategies in the future.

  13. Vpr Promotes Macrophage-Dependent HIV-1 Infection of CD4+ T Lymphocytes.

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    David R Collins

    2015-07-01

    Full Text Available Vpr is a conserved primate lentiviral protein that promotes infection of T lymphocytes in vivo by an unknown mechanism. Here we demonstrate that Vpr and its cellular co-factor, DCAF1, are necessary for efficient cell-to-cell spread of HIV-1 from macrophages to CD4+ T lymphocytes when there is inadequate cell-free virus to support direct T lymphocyte infection. Remarkably, Vpr functioned to counteract a macrophage-specific intrinsic antiviral pathway that targeted Env-containing virions to LAMP1+ lysosomal compartments. This restriction of Env also impaired virological synapses formed through interactions between HIV-1 Env on infected macrophages and CD4 on T lymphocytes. Treatment of infected macrophages with exogenous interferon-alpha induced virion degradation and blocked synapse formation, overcoming the effects of Vpr. These results provide a mechanism that helps explain the in vivo requirement for Vpr and suggests that a macrophage-dependent stage of HIV-1 infection drives the evolutionary conservation of Vpr.

  14. Vpr14-88-Apobec3G fusion protein is efficiently incorporated into Vif-positive HIV-1 particles and inhibits viral infection.

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    Zhujun Ao

    Full Text Available BACKGROUND: APOBEC3G (A3G, a deoxycytidine deaminase, is a potent host antiviral factor that can restrict HIV-1 infection. During Vif-negative HIV-1 replication, A3G is incorporated into HIV-1 particles, induces mutations in reverse transcribed viral DNA and inhibits reverse transcription. However, HIV-1 Vif counteracts A3G's activities by inducing its degradation and by blocking its incorporation into HIV-1 particles. Thus, it is interesting to elucidate a mechanism that would allow A3G to escape the effects of Vif in order to rescue its potent antiviral activity and to provide a possible novel therapeutic strategy for treating HIV-1 infection. METHODS AND FINDINGS: In this study, we generated an R88-A3G fusion protein by fusing A3G to a virion-targeting polypeptide (R14-88 derived from HIV-1 Vpr protein and compared its antiviral effects relative to those of HA-tagged native A3G (HA-A3G. Our study showed that transient expression of the R88-A3G fusion protein in both Vif(- and Vif(+ HIV-1 producing cells drastically inhibited viral infection in HeLa-CD4-CCR5-cells, CD4(+ C8166 T cells and human primary PBMCs. Moreover, we established CD4(+ C8166 T cell lines that stably express either R88-A3G or HA-A3G by transduction with VSV-G-pseudotyped lentiviral vector that harbor expression cassettes for R88-A3G or HA-A3G, respectively, and tested their susceptibility to Vif(+ HIV-1 infection. Our results clearly reveal that expression of R88-A3G in transduced CD4(+ C8166 cells significantly blocked Vif(+ HIV-1 infection. In an attempt to understand the mechanism underlying the antiviral activity of R88-A3G, we demonstrated that R88-A3G was efficiently incorporated into viral particles in the presence of Vif. Moreover, PCR analysis revealed that R88-A3G significantly inhibited viral cDNA synthesis during the early stage of Vif(+ virus infection. CONCLUSIONS: Our results clearly indicate that R88 delivers A3G into Vif(+ HIV-1 particles and inhibits

  15. HIV-1 Vpr N-terminal tagging affects alternative splicing of the viral genome

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    Baeyens, Ann; Naessens, Evelien; Van Nuffel, Anouk; Weening, Karin E.; Reilly, Anne-Marie; Claeys, Eva; Trypsteen, Wim; Vandekerckhove, Linos; Eyckerman, Sven; Gevaert, Kris; Verhasselt, Bruno

    2016-01-01

    To facilitate studies on Vpr function in replicating HIV-1, we aimed to tag the protein in an infectious virus. First we showed that N-, but not C-terminal HA/FLAG tagging of Vpr protein preserves Vpr cytopathicity. Cloning the tags into proviral DNA however ablated viral production and replication. By construction of additional viral variants we could show this defect was not protein- but RNA-dependent and sequence specific, and characterized by oversplicing of the genomic RNA. Simulation of genomic RNA folding suggested that introduction of the tag sequence induced an alternative folding structure in a region enriched in splice sites and splicing regulatory sequences. In silico predictions identified the HA/His6-Vpr tagging in HIV-1 to affect mRNA folding less than HA/FLAG-Vpr tagging. In vitro infectivity and mRNA splice pattern improved but did not reach wild-type values. Thus, sequence-specific insertions may interfere with mRNA splicing, possibly due to altered RNA folding. Our results point to the complexity of viral RNA genome sequence interactions. This should be taken into consideration when designing viral manipulation strategies, for both research as for biological interventions. PMID:27721439

  16. Fast track, dynein-dependent nuclear targeting of human immunodeficiency virus Vpr protein; impaired trafficking in a clinical isolate

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    Caly, Leon [Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic. 3800 (Australia); Kassouf, Vicki T. [Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW 2145 (Australia); Moseley, Gregory W. [Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic. 3800 (Australia); Diefenbach, Russell J.; Cunningham, Anthony L. [Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Westmead, NSW 2145 (Australia); Jans, David A., E-mail: david.jans@monash.edu [Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic. 3800 (Australia)

    2016-02-12

    Nuclear import of the accessory protein Vpr is central to infection by human immunodeficiency virus (HIV). We previously identified the Vpr F72L mutation in a HIV-infected, long-term non-progressor, showing that it resulted in reduced Vpr nuclear accumulation and altered cytoplasmic localisation. Here we demonstrate for the first time that the effects of nuclear accumulation of the F72L mutation are due to impairment of microtubule-dependent-enhancement of Vpr nuclear import. We use high resolution imaging approaches including fluorescence recovery after photobleaching and other approaches to document interaction between Vpr and the dynein light chain protein, DYNLT1, and impaired interaction of the F72L mutant with DYNLT1. The results implicate MTs/DYNLT1 as drivers of Vpr nuclear import and HIV infection, with important therapeutic implications. - Highlights: • HIV-1 Vpr utilizes the microtubule network to traffic towards the nucleus. • Mechanism relies on interaction between Vpr and dynein light chain protein DYNLT1. • Long-term non-progressor derived mutation (F72L) impairs this interaction. • Key residues in the vicinity of F72 contribute to interaction with DYNLT1.

  17. Activation of the DNA Damage Response Is a Conserved Function of HIV-1 and HIV-2 Vpr That Is Independent of SLX4 Recruitment

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    Oliver I. Fregoso

    2016-09-01

    Full Text Available There has been extraordinary progress in understanding the roles of lentiviral accessory proteins in antagonizing host antiviral defense proteins. However, the precise primary function of the accessory gene Vpr remains elusive. Here we suggest that engagement with the DNA damage response is an important function of primate lentiviral Vpr proteins because of its conserved function among diverse lentiviral lineages. In contrast, we show that, for HIV-1, HIV-2, and related Vpr isolates and orthologs, there is a lack of correlation between DNA damage response activation and interaction with the host SLX4 protein complex of structure specific endonucleases; some Vpr proteins are able to interact with SLX4, but the majority are not. Using the clustered regularly interspaced short palindromic repeat (CRISPR/Cas9 method to knock out SLX4, we formally showed that HIV-1 and HIV-2 Vpr orthologs can still activate the DNA damage response and cell cycle arrest in the absence of SLX4. Together, our data suggest that activation of the DNA damage response, but not SLX4 interaction, is conserved and therefore indicative of an important function of Vpr. Our data also indicate that Vpr activates the DNA damage response through an SLX4-independent mechanism that remains uncharacterized.

  18. Analysis of HIV-1 Vpr determinants responsible for cell growth arrest in Saccharomyces cerevisiae

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    Yao Xiao-Jian

    2004-08-01

    Full Text Available Abstract Background The HIV-1 genome encodes a well-conserved accessory gene product, Vpr, that serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest, and the modulation of HIV-1-induced apoptosis. We previously reported the genetic selection of a panel of di-tryptophan (W-containing peptides capable of interacting with HIV-1 Vpr and inhibiting its cytostatic activity in Saccharomyces cerevisiae (Yao, X.-J., J. Lemay, N. Rougeau, M. Clément, S. Kurtz, P. Belhumeur, and E. A. Cohen, J. Biol. Chem. v. 277, p. 48816–48826, 2002. In this study, we performed a mutagenic analysis of Vpr to identify sequence and/or structural determinants implicated in the interaction with di-W-containing peptides and assessed the effect of mutations on Vpr-induced cytostatic activity in S. cerevisiae. Results Our data clearly shows that integrity of N-terminal α-helix I (17–33 and α-helix III (53–83 is crucial for Vpr interaction with di-W-containing peptides as well as for the protein-induced cytostatic effect in budding yeast. Interestingly, several Vpr mutants, mainly in the N- and C-terminal domains, which were previously reported to be defective for cell-cycle arrest or apoptosis in human cells, still displayed a cytostatic activity in S. cerevisiae and remained sensitive to the inhibitory effect of di-W-containing peptides. Conclusions Vpr-induced growth arrest in budding yeast can be effectively inhibited by GST-fused di-W peptide through a specific interaction of di-W peptide with Vpr functional domain, which includes α-helix I (17–33 and α-helix III (53–83. Furthermore, the mechanism(s underlying Vpr-induced cytostatic effect in budding yeast are likely to be distinct from those implicated in cell-cycle alteration and apoptosis in human cells.

  19. HIV-1 Vpr modulates macrophage metabolic pathways: a SILAC-based quantitative analysis.

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    Carlos A Barrero

    Full Text Available Human immunodeficiency virus type 1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Furthermore, these macrophages are resistant to cell death and are viral reservoir. However, the impact of Vpr on the macrophage proteome is yet to be comprehended. The goal of the present study was to use a stable-isotope labeling by amino acids in cell culture (SILAC coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. Cultured human monocytic cells, U937, were differentiated into macrophages and transduced with adenovirus construct harboring the Vpr gene. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. Quantified proteins were selected and clustered by biological functions, pathway and network analysis using Ingenuity computational pathway analysis. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism was further validated by western blot analysis. In addition, the proteomic data demonstrate down regulation of some key mitochondrial enzymes such as glutamate dehydrogenase 2 (GLUD2, adenylate kinase 2 (AK2 and transketolase (TKT. Based on these observations we postulate that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha axis to induce expression of hexokinase (HK, glucose-6-phosphate dehyrogenase (G6PD and pyruvate kinase muscle type 2 (PKM2 that facilitates viral replication and biogenesis, and long-term survival of macrophages. Furthermore, dysregulation of mitochondrial glutamate metabolism in macrophages can contribute to neurodegeneration via neuroexcitotoxic mechanisms in the context of NeuroAIDS.

  20. Structural characterization of the HIV-1 Vpr N terminus: evidence of cis/trans-proline isomerism.

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    Bruns, Karsten; Fossen, Torgils; Wray, Victor; Henklein, Peter; Tessmer, Uwe; Schubert, Ulrich

    2003-10-31

    The 96-residue human immunodeficiency virus (HIV) accessory protein Vpr serves manifold functions in the retroviral life cycle including augmentation of viral replication in non-dividing host cells, induction of G2 cell cycle arrest, and modulation of HIV-induced apoptosis. Using a combination of dynamic light scattering, circular dichroism, and NMR spectroscopy the N terminus of Vpr is shown to be a unique domain of the molecule that behaves differently from the C-terminal domain in terms of self-association and secondary structure folding. Interestingly, the four highly conserved proline residues in the N terminus are predicted to have a high propensity for cis/trans isomerism. Thus the high resolution structure and folding of a synthetic N-terminal peptide (Vpr1-40) and smaller fragments thereof have been investigated. 1H NMR data indicate Vpr1-40 possesses helical structure between residues 17-32, and for the first time, this helix, which is bound by proline residues, was observed even in aqueous solution devoid of any detergent supplements. In addition, NMR data revealed that all of the proline residues undergo a cis/ trans isomerism to such an extent that approximately 40% of all Vpr molecules possess at least one proline in a cis conformation. This phenomenon of cis/trans isomerism, which is unprecedented for HIV-1 Vpr, not only provides an explanation for the molecular heterogeneity observed in the full-length molecule but also indicates that in vivo the folding and function of Vpr should depend on a cis/trans-proline isomerase activity, particularly as two of the proline residues in positions 14 and 35 show considerable amounts of cis isomers. This prediction correlates well with our recent observation (Zander, K., Sherman, M. P., Tessmer, U., Bruns, K., Wray, V., Prechtel, A. T., Schubert, E., Henklein, P., Luban, J., Neidleman, J., Greene, W. C., and Schubert, U. (2003) J. Biol. Chem. 278, 43170-43181) of a functional interaction between the major

  1. HIV-1 Vpr-mediated G2 arrest involves the DDB1-CUL4AVPRBP E3 ubiquitin ligase.

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    Jean-Philippe Belzile

    2007-07-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 viral protein R (Vpr has been shown to cause G2 cell cycle arrest in human cells by inducing ATR-mediated inactivation of p34cdc2, but factors directly engaged in this process remain unknown. We used tandem affinity purification to isolate native Vpr complexes. We found that damaged DNA binding protein 1 (DDB1, viral protein R binding protein (VPRBP, and cullin 4A (CUL4A--components of a CUL4A E3 ubiquitin ligase complex, DDB1-CUL4A(VPRBP--were able to associate with Vpr. Depletion of VPRBP by small interfering RNA impaired Vpr-mediated induction of G2 arrest. Importantly, VPRBP knockdown alone did not affect normal cell cycle progression or activation of ATR checkpoints, suggesting that the involvement of VPRBP in G2 arrest was specific to Vpr. Moreover, leucine/isoleucine-rich domain Vpr mutants impaired in their ability to interact with VPRBP and DDB1 also produced strongly attenuated G2 arrest. In contrast, G2 arrest-defective C-terminal Vpr mutants were found to maintain their ability to associate with these proteins, suggesting that the interaction of Vpr with the DDB1-VPRBP complex is necessary but not sufficient to block cell cycle progression. Overall, these results point toward a model in which Vpr could act as a connector between the DDB1-CUL4A(VPRBP E3 ubiquitin ligase complex and an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and induce G2 arrest.

  2. The intriguing Cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding

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    Henklein Petra

    2010-10-01

    Full Text Available Abstract Background Cyclophilin A (CypA represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1 replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined. Results Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR exchange spectroscopy and surface plasmon resonance spectroscopy (SPR. NMR data at atomic resolution indicate prolyl cis/trans isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding. Conclusions Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are

  3. Interactions between human immunodeficiency virus (HIV-1 Vpr expression and innate immunity influence neurovirulence

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    Ballanyi Klaus

    2011-06-01

    Full Text Available Abstract Background Viral diversity and abundance are defining properties of human immunodeficiency virus (HIV-1's biology and pathogenicity. Despite the increasing availability of antiretroviral therapy, HIV-associated dementia (HAD continues to be a devastating consequence of HIV-1 infection of the brain although the underlying disease mechanisms remain uncertain. Herein, molecular diversity within the HIV-1 non-structural gene, Vpr, was examined in RNA sequences derived from brain and blood of HIV/AIDS patients with or without HIV-associated dementia (HAD together with the ensuing pathobiological effects. Results Cloned brain- and blood-derived full length vpr alleles revealed that amino acid residue 77 within the brain-derived alleles distinguished HAD (77Q from non-demented (ND HIV/AIDS patients (77R (p vpr transcripts were more frequently detected in HAD brains (p IFN-α, MX1 and BST-2 transcript levels in human glia relative to the 77Q-HAD encoding virus (p Vpr77Q-HAD, 77R (pVpr77R-ND or Vpr null (pVpr(--containing vectors showed that the pVpr77R-ND vector induced higher levels of immune gene expression (p p IFN-α, MX1, PRKRA and BST-2 relative to 77Q-HAD peptide (p p p Conclusions These observations underscored the potent neuropathogenic properties of Vpr but also indicated viral diversity modulates innate neuroimmunity and neurodegeneration.

  4. The DDB1–DCAF1–Vpr–UNG2 crystal structure reveals how HIV-1 Vpr steers human UNG2 toward destruction

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    Wu, Ying; Zhou, Xiaohong; Barnes, Christopher O.; DeLucia, Maria; Cohen, Aina E.; Gronenborn, Angela M.; Ahn, Jinwoo; Calero, Guillermo

    2017-01-01

    The HIV-1 accessory protein Vpr is required for efficient viral infection of macrophages and promotion of viral replication in T cells. Vpr’s biological activities are closely linked to the interaction with human DCAF1, a cellular substrate receptor of the Cullin4–RING E3 ubiquitin ligase (CRL4) of the host ubiquitin–proteasome-mediated protein degradation pathway. The molecular details of how Vpr usurps the protein degradation pathway have not been delineated. Here we present the crystal structure of the DDB1–DCAF1–HIV-1–Vpr–uracil-DNA glycosylase (UNG2) complex. The structure reveals how Vpr engages with DCAF1, creating a binding interface for UNG2 recruitment, in a manner distinct from the recruitment of SAMHD1 by Vpx protein for degradation by Vpx proteins. Vpr and Vpx use similar N-terminal and helical regions to bind the substrate receptor, whereas different regions target the specific cellular substrates. Furthermore, Vpr uses molecular mimicry of DNA by a variable loop for specific recruitment of the UNG2 substrate. PMID:27571178

  5. Direct Vpr-Vpr Interaction in Cells monitored by two Photon Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Imaging

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    Mély Yves

    2008-09-01

    Full Text Available Abstract Background The human immunodeficiency virus type 1 (HIV-1 encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP- and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. Results Results show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three α helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the α-helices could perturb the leucine zipper like motifs. The ΔQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization. Conclusion We report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three α helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.

  6. Molecular characterization of the HIV type 1 vpr gene in infected Chinese former blood/plasma donors at different stages of diseases.

    Science.gov (United States)

    Shen, Chengli; Gupta, Phalguni; Wu, Hao; Chen, Xinyue; Huang, Xiaojie; Zhou, Yusen; Chen, Yue

    2008-04-01

    The HIV-1 vpr regions were PCR amplified and sequenced from eight long-term nonprogressors' (LTNP) and seven AIDS patients' DNA samples of a cohort of HIV-1-infected Chinese plasma/blood donors (PBDs). Sequence analysis revealed that the patients' HIV-1 vpr sequences belong to HIV-1 subtype B and there were no differences in the divergence of vpr sequences between these two groups of patients. Similarly, in the deduced amino acid sequences, no significant differences have been detected in vpr functional domains from patients at different stages of the disease. Moreover, the predicted binding motifs of HLA A2 and A11 were highly conserved among patients' vpr amino acid sequences. These results show that vpr may not play an important role in HIV-1 pathogenesis in different stages of Chinese patients and may have important implications in developing vpr-related treatments suitable for HIV-1-infected PBDs.

  7. Defining the interactions and role of DCAF1/VPRBP in the DDB1-cullin4A E3 ubiquitin ligase complex engaged by HIV-1 Vpr to induce a G2 cell cycle arrest.

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    Francine C A Gérard

    Full Text Available HIV viral protein R (Vpr induces a cell cycle arrest at the G2/M phase by activating the ATR DNA damage/replication stress signalling pathway through engagement of the DDB1-CUL4A-DCAF1 E3 ubiquitin ligase via a direct binding to the substrate specificity receptor DCAF1. Since no high resolution structures of the DDB1-DCAF1-Vpr substrate recognition module currently exist, we used a mutagenesis approach to better define motifs in DCAF1 that are crucial for Vpr and DDB1 binding. Herein, we show that the minimal domain of DCAF1 that retained the ability to bind Vpr and DDB1 was mapped to residues 1041 to 1393 (DCAF1 WD. Mutagenic analyses identified an α-helical H-box motif and F/YxxF/Y motifs located in the N-terminal domain of DCAF1 WD that are involved in exclusive binding to DDB1. While we could not identify elements specifically involved in Vpr binding, overall, the mutagenesis data suggest that the predicted β-propeller conformation of DCAF1 is likely to be critical for Vpr association. Importantly, we provide evidence that binding of Vpr to DCAF1 appears to modulate the formation of a DDB1/DCAF1 complex. Lastly, we show that expression of DCAF1 WD in the absence of endogenous DCAF1 was not sufficient to enable Vpr-mediated G2 arrest activity. Overall, our results reveal that Vpr and DDB1 binding on DCAF1 can be genetically separated and further suggest that DCAF1 contains determinants in addition to the Vpr and DDB1 minimal binding domain, which are required for Vpr to enable the induction of a G2 arrest.

  8. HIV Triggers a cGAS-Dependent, Vpu- and Vpr-Regulated Type I Interferon Response in CD4+ T Cells

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    Jolien Vermeire

    2016-10-01

    Full Text Available Several pattern-recognition receptors sense HIV-1 replication products and induce type I interferon (IFN-I production under specific experimental conditions. However, it is thought that viral sensing and IFN induction are virtually absent in the main target cells of HIV-1 in vivo. Here, we show that activated CD4+ T cells sense HIV-1 infection through the cytosolic DNA sensor cGAS and mount a bioactive IFN-I response. Efficient induction of IFN-I by HIV-1 infection requires proviral integration and is regulated by newly expressed viral accessory proteins: Vpr potentiates, while Vpu suppresses cGAS-dependent IFN-I induction. Furthermore, Vpr also amplifies innate sensing of HIV-1 infection in Vpx-treated dendritic cells. Our results identify cGAS as mediator of an IFN-I response to HIV-1 infection in CD4+ T cells and demonstrate that this response is modulated by the viral accessory proteins Vpr and Vpu. Thus, viral innate immune evasion is incomplete in the main target cells of HIV-1.

  9. 人类免疫缺陷病毒1型vpr基因变异研究%Molecular epidemiology on vpr gene of HIV-1 strains

    Institute of Scientific and Technical Information of China (English)

    王晓辉; 秦彦珉; 程慧; 石向东; 陈琳

    2010-01-01

    目的 分析HIV-1毒株vpr基因变异规律.方法 RT-PCR扩增HIV-1毒株vpr基因,经测序后构建基因进化树并分析其亚型分布;比较Vpr蛋白32~46位关键肽段氨基酸序列的差异;分析国内外Vpr蛋白77位氨基酸多态性的分布差异.结果 01_AE为HIV-1主要亚型(49.53%);B亚型vpr基因组内变异最大;Vpr蛋白77位存在多态性,分别编码谷氨酰胺、精氨酸和组氨酸残基,这种多态性在国内外分布差异无统计学意义(P=0.617).结论 HIV-1毒株vpr基因分型以01_AE亚型为主,vpr基因存在多态性,与国内外报道一致.%Objective To understand the rule of vpr gene variance of HIV-1 strains.Methods RT-PCR was used to amplify vpr gene of HIV-1 strains in Shenzhen. PCR products were sequenced and used for gene phylogenetic analysis and the 32-46 amino acids of Vpr protein were compared. The difference of 77 amino acid polymorphism distribution between domestic region and foreign region was analyzed. Results 01_AE was the major HIV-1 subtype in Shenzhen. The gene distance among subtype B was larger than in other subtypes. 77-amino acid of Vpr protein had three polymorphism forms as Arginin, Glutamine and Histidine, with Glutamine as the wild form. There were no significant differences in the three amino acid distributions between HIV-1 strains from domestic region and foreign region. Conclusion vpr genes of different HIV-1 strains belonged to 01_AE subtype. There was polymorphism seen in the vpr gene which was consistent with both domestic and international HIV-1 strains.

  10. Phylogenetic Insights into the Functional Relationship between Primate Lentiviral Reverse Transcriptase and Accessory Proteins Vpx/Vpr

    Science.gov (United States)

    Sakai, Yosuke; Doi, Naoya; Miyazaki, Yasuyuki; Adachi, Akio; Nomaguchi, Masako

    2016-01-01

    The efficiency of reverse transcription to synthesize viral DNA in infected cells greatly influences replication kinetics of retroviruses. However, viral replication in non-dividing cells such as resting T cells and terminally differentiated macrophages is potently and kinetically restricted by a host antiviral factor designated SAMHD1 (sterile alpha motif and HD-domain containing protein 1). SAMHD1 reduces cellular deoxynucleoside triphosphate (dNTP) pools and affects viral reverse transcription step. Human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency viruses (SIVs) have Vpx or Vpr to efficiently degrade SAMHD1. Interestingly, the reverse transcriptase (RT) derived from HIV-1 that encodes no anti-SAMHD1 proteins has been previously demonstrated to uniquely exhibit a high enzymatic activity. It is thus not irrational to assume that some viruses may have acquired or lost the specific RT property to better adapt themselves to the low dNTP environments confronted in non-dividing cells. This adaptation process may probably be correlated with the SAMHD1-antagonizing ability by viruses. In this report, we asked whether such adaptive events can be inferable from Vpx/Vpr and RT phylogenetic trees overlaid with SAMHD1-degrading capacity of Vpx/Vpr and with kinetic characteristics of RT. Resultant two trees showed substantially similar clustering patterns, and therefore suggested that the properties of RT and Vpx/Vpr can be linked. In other words, HIV/SIVs may possess their own RT proteins to adequately react to various dNTP circumstances in target cells. PMID:27803699

  11. Visualizing Vpr-induced G2 arrest and apoptosis.

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    Tomoyuki Murakami

    Full Text Available Vpr is an accessory protein of human immunodeficiency virus type 1 (HIV-1 with multiple functions. The induction of G2 arrest by Vpr plays a particularly important role in efficient viral replication because the transcriptional activity of the HIV-1 long terminal repeat is most active in G2 phase. The regulation of apoptosis by Vpr is also important for immune suppression and pathogenesis during HIV infection. However, it is not known whether Vpr-induced apoptosis depends on the ability of Vpr to induce G2 arrest, and the dynamics of Vpr-induced G2 arrest and apoptosis have not been visualized. We performed time-lapse imaging to examine the temporal relationship between Vpr-induced G2 arrest and apoptosis using HeLa cells containing the fluorescent ubiquitination-based cell cycle indicator2 (Fucci2. The dynamics of G2 arrest and subsequent long-term mitotic cell rounding in cells transfected with the Vpr-expression vector were visualized. These cells underwent nuclear mis-segregation after prolonged mitotic processes and then entered G1 phase. Some cells subsequently displayed evidence of apoptosis after prolonged mitotic processes and nuclear mis-segregation. Interestingly, Vpr-induced apoptosis was seldom observed in S or G2 phase. Likewise, visualization of synchronized HeLa/Fucci2 cells infected with an adenoviral vector expressing Vpr clearly showed that Vpr arrests the cell cycle at G2 phase, but does not induce apoptosis at S or G2 phase. Furthermore, time-lapse imaging of HeLa/Fucci2 cells expressing SCAT3.1, a caspase-3-sensitive fusion protein, clearly demonstrated that Vpr induces caspase-3-dependent apoptosis. Finally, to examine whether the effects of Vpr on G2 arrest and apoptosis were reversible, we performed live-cell imaging of a destabilizing domain fusion Vpr, which enabled rapid stabilization and destabilization by Shield1. The effects of Vpr on G2 arrest and subsequent apoptosis were reversible. This study is the first to

  12. The progestin-only contraceptive medroxyprogesterone acetate, but not norethisterone acetate, enhances HIV-1 Vpr-mediated apoptosis in human CD4+ T cells through the glucocorticoid receptor.

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    Michele Tomasicchio

    Full Text Available The glucocorticoid receptor (GR regulates several physiological functions, including immune function and apoptosis. The HIV-1 virus accessory protein, viral protein R (Vpr, can modulate the transcriptional response of the GR. Glucocorticoids (GCs and Vpr have been reported to induce apoptosis in various cells, including T-cells. We have previously shown that the injectable contraceptive, medroxyprogesterone acetate (MPA is a partial to full agonist for the GR, unlike norethisterone acetate (NET-A. We investigated the functional cross talk between the GR and Vpr in inducing apoptosis in CD4(+ T-cells, in the absence and presence of GCs and these progestins, as well as progesterone. By using flow cytometry, we show that, in contrast to NET-A and progesterone, the synthetic GR ligand dexamethasone (Dex, cortisol and MPA induce apoptosis in primary CD4(+ T-cells. Furthermore, the C-terminal part of the Vpr peptide, or HIV-1 pseudovirus, together with Dex or MPA further increased the apoptotic phenotype, unlike NET-A and progesterone. By a combination of Western blotting, PCR and the use of receptor- selective agonists, we provide evidence that the GR and the estrogen receptor are the only steroid receptors expressed in peripheral blood mononuclear cells. These results, together with the findings that RU486, a GR antagonist, prevents Dex-, MPA- and Vpr-mediated apoptosis, provide evidence for the first time that GR agonists or partial agonists increase apoptosis in primary CD4(+ T-cells via the GR. We show that apoptotic induction involves differential expression of key apoptotic genes by both Vpr and GCs/MPA. This work suggests that contraceptive doses of MPA but not NET-A or physiological doses of progesterone could potentially accelerate depletion of CD4(+ T-cells in a GR-dependent fashion in HIV-1 positive women, thereby contributing to immunodeficiency. The results imply that choice of progestin used in contraception may be critical to

  13. Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative luciferase-Vpr packaging-based assay.

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    Gaëlle Gonzalez

    Full Text Available The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag into virions or membrane-enveloped virus-like particles (VLP. Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr. VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA derivatives that differed from the leader compound PA-457 (or DSB by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB, showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39

  14. Molecular simulations of conformation change and aggregation of HIV-1 Vpr13-33 on graphene oxide

    Science.gov (United States)

    Zeng, Songwei; Zhou, Guoquan; Guo, Jianzhong; Zhou, Feng; Chen, Junlang

    2016-04-01

    Recent experiments have reported that the fragment of viral protein R (Vpr), Vpr13-33, can assemble and change its conformation after adsorbed on graphene oxide (GO) and then reduce its cytotoxicity. This discovery is of great importance, since the mutation of Vpr13-33 can decrease the viral replication, viral load and delay the disease progression. However, the interactions between Vpr13-33 and GO at atomic level are still unclear. In this study, we performed molecular dynamics simulation to investigate the dynamic process of the adsorption of Vpr13-33 onto GO and the conformation change after aggregating on GO surface. We found that Vpr13-33 was adsorbed on GO surface very quickly and lost its secondary structure. The conformation of peptides-GO complex was highly stable because of π-π stacking and electrostatic interactions. When two peptides aggregated on GO, they did not dimerize, since the interactions between the two peptides were much weaker than those between each peptide and GO.

  15. Human Immunodeficiency Virus Type 1 Vpr Induces DNA Replication Stress In Vitro and In Vivo▿

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    Zimmerman, Erik S.; Sherman, Michael P.; Blackett, Jana L.; Neidleman, Jason A.; Kreis, Christophe; Mundt, Pamela; Williams, Samuel A.; Warmerdam, Maria; Kahn, James; Hecht, Frederick M.; Grant, Robert M.; de Noronha, Carlos M. C.; Weyrich, Andrew S.; Greene, Warner C.; Planelles, Vicente

    2006-01-01

    The human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) causes cell cycle arrest in G2. Vpr-expressing cells display the hallmarks of certain forms of DNA damage, specifically activation of the ataxia telangiectasia mutated and Rad3-related kinase, ATR. However, evidence that Vpr function is relevant in vivo or in the context of viral infection is still lacking. In the present study, we demonstrate that HIV-1 infection of primary, human CD4+ lymphocytes causes G2 arrest in a Vpr-dependent manner and that this response requires ATR, as shown by RNA interference. The event leading to ATR activation in CD4+ lymphocytes is the accumulation of replication protein A in nuclear foci, an indication that Vpr likely induces stalling of replication forks. Primary macrophages are refractory to ATR activation by Vpr, a finding that is consistent with the lack of detectable ATR, Rad17, and Chk1 protein expression in these nondividing cells. These observations begin to explain the remarkable resilience of macrophages to HIV-1-induced cytopathicity. To study the in vivo consequences of Vpr function, we isolated CD4+ lymphocytes from HIV-1-infected individuals and interrogated the cell cycle status of anti-p24Gag-immunoreactive cells. We report that infected cells in vivo display an aberrant cell cycle profile whereby a majority of cells have a 4N DNA content, consistent with the onset of G2 arrest. PMID:16956949

  16. Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

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    Liang Dong

    2010-07-01

    Full Text Available Abstract Background Cell cycle G2 arrest induced by HIV-1 Vpr is thought to benefit viral proliferation by providing an optimized cellular environment for viral replication and by skipping host immune responses. Even though Vpr-induced G2 arrest has been studied extensively, how Vpr triggers G2 arrest remains elusive. Results To examine this initiation event, we measured the Vpr effect over a single cell cycle. We found that even though Vpr stops the cell cycle at the G2/M phase, but the initiation event actually occurs in the S phase of the cell cycle. Specifically, Vpr triggers activation of Chk1 through Ser345 phosphorylation in an S phase-dependent manner. The S phase-dependent requirement of Chk1-Ser345 phosphorylation by Vpr was confirmed by siRNA gene silencing and site-directed mutagenesis. Moreover, downregulation of DNA replication licensing factors Cdt1 by siRNA significantly reduced Vpr-induced Chk1-Ser345 phosphorylation and G2 arrest. Even though hydroxyurea (HU and ultraviolet light (UV also induce Chk1-Ser345 phosphorylation in S phase under the same conditions, neither HU nor UV-treated cells were able to pass through S phase, whereas vpr-expressing cells completed S phase and stopped at the G2/M boundary. Furthermore, unlike HU/UV, Vpr promotes Chk1- and proteasome-mediated protein degradations of Cdc25B/C for G2 induction; in contrast, Vpr had little or no effect on Cdc25A protein degradation normally mediated by HU/UV. Conclusions These data suggest that Vpr induces cell cycle G2 arrest through a unique molecular mechanism that regulates host cell cycle regulation in an S-phase dependent fashion.

  17. Development of a robust cell-based high-throughput screening assay to identify targets of HIV-1 viral protein R dimerization

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    Zych C

    2013-05-01

    Full Text Available Courtney Zych,1 Alexander Domling,2 Velpandi Ayyavoo11Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA; 2Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA, USAAbstract: Targeting protein–protein interactions (PPI is an emerging field in drug discovery. Dimerization and PPI are essential properties of human immunodeficiency virus (HIV-1 proteins, their mediated functions, and virus biology. Additionally, dimerization is required for the functional interaction of HIV-1 proteins with many host cellular components. In this study, a bimolecular fluorescence complementation (BiFC-based screening assay was developed that can quantify changes in dimerization, using HIV-1 viral protein R (Vpr dimerization as a "proof of concept." Results demonstrated that Venus Vpr (generated by BiFC Vpr constructs could be competed off in a dose-dependent manner using untagged, full-length Vpr as a competitor molecule. The change in signal intensity was measured quantitatively through flow cytometry and fluorescence microscopy in a high content screening assay. High content imaging was used to screen a library of small molecules for an effect on Vpr dimerization. Among the tested molecules, a few of the small molecules demonstrate an effect on Vpr dimerization in a dose-dependent manner.Keywords: BiFC, protein–protein interaction, HIV-1 Vpr, dimerization, drug targets

  18. 重组慢病毒表达载体介导HIV-1 Vpr蛋白调控KSHV潜伏感染的初探%Preliminary study on the regulation of KSHV latent infection by HIV-1 Vpr medicated by the recombinant lentivirus

    Institute of Scientific and Technical Information of China (English)

    贾雪梅; 王平; 秦娣; 卢春

    2010-01-01

    Objective To package the recombinant lentivirus containing HIV-1 Vpr gene and detect the effect of Vpr protein expression on the latent infection and lyric replication of KSHV.Methods The fragment of Vpr gene from expression plasmid pCI-neo-Vpr was cloned into the lentivirus vector pHAGE-CMV-MCS-IzsGreen,then the recombinant plasmid pHAGE-Vpr,package vector psPAX2 and envelope vector pMD2.G were cotransfected into 293T cells.GFP expression was observed by fluorescent microscopy.Culture media of 293T cells were harvested and filtered through 0.45 μm filter.After 293T cells were infected by a series of diluted lentivirus,the virus titer was checked by observing GFP expression.Vpr mRNA transcripts in 293T cells were detected by RT-PCR.Then BCBL-1 cells were infected by the recombinant lentivirus with 1 MOI,GFP expression was observed by fluorescent microscopy,and the mRNA transcripts and protein expression of Vpr in BCBL-1 cells were detected by RT-PCR and Western blot.Meanwhile,the mRNA transcripts and protein expression of KSHV lytic cycle gene Rta were detected by RT-PCR and Western blot,respectively.Results The recombinant lentivirus carrying HIV-1 Vpr was packaged successfully with the virus titer of 4 × 107 TU/ml.After infected with lentivirus,BCBL-1 cells could express GFP,and the exact band of Vpr was detectable by RT-PCR and Western blot.Moreover,the expression of KSHV Rta mRNA and protein were downregulated by Vpr protein.Conclusion Overexpression of HIV-1 Vpr mediated by the recombinant lentivirus could inhibit KSHV lytic replication and enhance KSHV latent infection.%目的 利用慢病毒载体系统包装含人类免疫缺陷病毒1型(HIV-1)病毒蛋白R(Vpr)的重组慢病毒,使之感染原发性渗出性淋巴瘤(PEL)细胞BCBL-1,并检测Vpr蛋白对细胞中卡波济肉瘤相关疱疹病毒(KSHV)潜伏感染与裂解性复制的影响.方法 从实验室先前构建的重组真核表达质粒pCI-neo-Vpr中扩增出Vpr基因,插入到pHAGE-CMV-MCS

  19. Evolutionary toggling of Vpx/Vpr specificity results in divergent recognition of the restriction factor SAMHD1.

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    Oliver I Fregoso

    Full Text Available SAMHD1 is a host restriction factor that blocks the ability of lentiviruses such as HIV-1 to undergo reverse transcription in myeloid cells and resting T-cells. This restriction is alleviated by expression of the lentiviral accessory proteins Vpx and Vpr (Vpx/Vpr, which target SAMHD1 for proteasome-mediated degradation. However, the precise determinants within SAMHD1 for recognition by Vpx/Vpr remain unclear. Here we show that evolution of Vpx/Vpr in primate lentiviruses has caused the interface between SAMHD1 and Vpx/Vpr to alter during primate lentiviral evolution. Using multiple HIV-2 and SIV Vpx proteins, we show that Vpx from the HIV-2 and SIVmac lineage, but not Vpx from the SIVmnd2 and SIVrcm lineage, require the C-terminus of SAMHD1 for interaction, ubiquitylation, and degradation. On the other hand, the N-terminus of SAMHD1 governs interactions with Vpx from SIVmnd2 and SIVrcm, but has little effect on Vpx from HIV-2 and SIVmac. Furthermore, we show here that this difference in SAMHD1 recognition is evolutionarily dynamic, with the importance of the N- and C-terminus for interaction of SAMHD1 with Vpx and Vpr toggling during lentiviral evolution. We present a model to explain how the head-to-tail conformation of SAMHD1 proteins favors toggling of the interaction sites by Vpx/Vpr during this virus-host arms race. Such drastic functional divergence within a lentiviral protein highlights a novel plasticity in the evolutionary dynamics of viral antagonists for restriction factors during lentiviral adaptation to its hosts.

  20. Molecular dynamics simulations of conformation changes of HIV-1 regulatory protein on graphene

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    Zhao, Daohui; Li, Libo; He, Daohang; Zhou, Jian

    2016-07-01

    The fragment of viral protein R (Vpr), Vpr13-33, plays an important role in regulating nuclear importing of HIV genes through channel formation in which it adopts a leucine-zipper-like alpha-helical conformation. A recent experimental study reported that helical Vpr13-33 would transform to β-sheet or random coil structures and aggregate on the surface of graphene or graphene oxide through hydrophobic interactions. Due to experimental limitations, however, there is still a considerable lack of understanding on the adsorption dynamics at the early stage of the conformational transition at water-graphene interface and the underlying driving force at molecular level. In this study, atomistic molecular dynamics simulations were used to explore the conformation transition phenomena. Vpr13-33 kept α-helical structure in solution, but changed to β-sheet structure when strongly adsorbed onto graphene. Preferential adsorption of Vpr13-33 on graphene is dominated by hydrophobic interactions. The cluster analysis identified the most significant populated conformation and the early stage of structure conversion from α-helical to β-sheet was found, but the full β-sheet propagation was not observed. Free energy landscape analysis further complemented the transformation analysis of peptide conformations. These findings are consistent with experimental results, and give a molecular level interpretation for the reduced cytotoxicity of Vpr13-33 to some extent upon graphene exposure. Meanwhile, this study provides some significant insights into the detailed mechanism of graphene-induced protein conformation transition.

  1. Molecular dynamics simulations of conformation changes of HIV-1 regulatory protein on graphene

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    Zhao, Daohui; Li, Libo; He, Daohang; Zhou, Jian, E-mail: jianzhou@scut.edu.cn

    2016-07-30

    Graphical abstract: Preferential adsorption of Vpr13-33 on graphene accompanied by early conformational change from α-helix to β-sheet structures was observed by molecular simulations. This work presents the molecular mechanism of graphene-induced peptide conformational alteration and sheds light on developing graphene-based materials to inhibit HIV. - Highlights: • Graphene induced early structural transition of Vpr13-33 is studied by MD simulations. • Both π-π stacking and hydrophobic interactions orchestrate the peptide adsorption. • Vpr has an increased propensity of β-sheet content on graphene surface. • To develop graphene-based materials to inhibit HIV is possible. - Abstract: The fragment of viral protein R (Vpr), Vpr13-33, plays an important role in regulating nuclear importing of HIV genes through channel formation in which it adopts a leucine-zipper-like alpha-helical conformation. A recent experimental study reported that helical Vpr13-33 would transform to β-sheet or random coil structures and aggregate on the surface of graphene or graphene oxide through hydrophobic interactions. Due to experimental limitations, however, there is still a considerable lack of understanding on the adsorption dynamics at the early stage of the conformational transition at water-graphene interface and the underlying driving force at molecular level. In this study, atomistic molecular dynamics simulations were used to explore the conformation transition phenomena. Vpr13-33 kept α-helical structure in solution, but changed to β-sheet structure when strongly adsorbed onto graphene. Preferential adsorption of Vpr13-33 on graphene is dominated by hydrophobic interactions. The cluster analysis identified the most significant populated conformation and the early stage of structure conversion from α-helical to β-sheet was found, but the full β-sheet propagation was not observed. Free energy landscape analysis further complemented the transformation analysis of

  2. Cellular phenotype impacts human immunodeficiency virus type 1 viral protein R subcellular localization

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    Ferrucci Adriano

    2011-08-01

    Full Text Available Abstract Background Human immunodeficiency virus type 1 (HIV-1 viral protein R (Vpr is a virion-associated regulatory protein that functions at several points within the viral life cycle and has been shown to accumulate primarily in the nucleus and at the nuclear envelope. However, most studies have investigated Vpr localization employing cell types irrelevant to HIV-1 pathogenesis. To gain a better understanding of how cellular phenotype might impact HIV-1 Vpr intracellular localization, Vpr localization was examined in several cell lines representing major cellular targets for HIV-1 infection within the peripheral blood, bone marrow, and central nervous system (CNS. Results Utilizing a green fluorescent protein-tagged Vpr, we detected Vpr mainly in foci inside the nucleus, at the nuclear envelope, and around the nucleoli, with dispersed accumulation in the cytoplasm of human endothelial kidney 293T cells. No differences were observed in Vpr localization pattern with respect to either the location of the tag (N- or C-terminus or the presence of other viral proteins. Subsequently, the Vpr localization pattern was explored in two primary HIV-1 target cells within the peripheral blood: the CD4+ T lymphocyte (represented by the Jurkat CD4+ T-cell line and the monocyte-macrophage (represented by the U-937 cell line. Vpr was found primarily in speckles within the cytoplasm of the Jurkat T cells, whereas it accumulated predominantly intranuclearly in U-937 monocytic cells. These patterns differ from that observed in a bone marrow progenitor cell line (TF-1, wherein Vpr localized mainly at the nuclear envelope with some intranuclear punctuate staining. Within the CNS, we examined two astroglioma cell lines and found that Vpr displayed a perinuclear and cytoplasmic distribution. Conclusions The results suggest that the pattern of Vpr localization depends on cellular phenotype, probably owing to interactions between Vpr and cell type-specific host

  3. Inhibition of HIV-1 in Vitro by Combination of Vpr and Tat Specific Short Hairpin RNA via Lentiviral Vectors%慢病毒介导双shRNA靶向于HIV的抑制作用研究

    Institute of Scientific and Technical Information of China (English)

    郝彦哲; 滕智平; 杨怡姝; 孙晓娜; 马晶; 金孝华; 曾毅

    2013-01-01

    RNA干扰可以有效地抑制特定基因的表达,在HIV基因治疗中成为一种重要的方法.为了防止病毒逃逸株的出现,本研究分别构建了靶向于HIV-1调控蛋白基因vpr和tat的shRNA重组慢病毒载体以及含有这两种shRNA的双表达重组慢病毒载体,分别由U6和H1两种启动子启动,通过与靶向基因tat和vpr表达质粒在293T细胞中进行共转染,采用荧光定量PCR方法测定细胞内靶向基因的表达丰度,结果显示双表达shRNA的重组慢病毒载体对vpr和tat的表达抑制率分别可以达到89.20%和62.00%.与pNL4-3病毒包装质粒共转染,P24ELISA显示plvx-vpr-tat shRNA对病毒包装产量的抑制率可以达到99.05%,比单个shRNA的抑制率都要强,HIV-1 NL4-3体外对MT4细胞攻毒实验表明双shRNA重组慢病毒载体具有很强的抑制病毒复制的能力.%Gene therapy offers the promise of curing the HIV-infected patients. Specific, potent, and sustained short hairpin RNA (shRNA)-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. To reduce the probability of viral escape mutants, in this study, we constructed lentiviral vector containing vpr and tat shRNA, respectively, furthermore the bispecific lentiviral vector harboring vpr and tat shRNA expression cassettes from U6 pro-motor and H1 promotor was cotransfected with recombinant plasmid expressing the vpr and tat gene. The result showed that the bispecific lentiviral vector plvx-vpr-tatshRNA could inhibit the vpr and tat effectively, with ratios of 89. 20% and 62. 00% respectively. When cotransfected with pNL4-3 in 293T cell, plvx-vpr-tatshRNA showed higher efficacy in down regulating the HIV NL4-3 packaging production than the plvx-vprshRNA or plvx-tatshRNA individually. MT4 cell clones transduced with recombinant lentiviral vectors were screened and challenged with HIV NL4-3. P24 ELISA test showed that MT4 transduced with the

  4. Lentiviral Vpx accessory factor targets VprBP/DCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligase to enable macrophage infection.

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    Smita Srivastava

    2008-05-01

    Full Text Available Vpx is a small virion-associated adaptor protein encoded by viruses of the HIV-2/SIVsm lineage of primate lentiviruses that enables these viruses to transduce monocyte-derived cells. This probably reflects the ability of Vpx to overcome an as yet uncharacterized block to an early event in the virus life cycle in these cells, but the underlying mechanism has remained elusive. Using biochemical and proteomic approaches, we have found that Vpx protein of the pathogenic SIVmac 239 strain associates with a ternary protein complex comprising DDB1 and VprBP subunits of Cullin 4-based E3 ubiquitin ligase, and DDA1, which has been implicated in the regulation of E3 catalytic activity, and that Vpx participates in the Cullin 4 E3 complex comprising VprBP. We further demonstrate that the ability of SIVmac as well as HIV-2 Vpx to interact with VprBP and its associated Cullin 4 complex is required for efficient reverse transcription of SIVmac RNA genome in primary macrophages. Strikingly, macrophages in which VprBP levels are depleted by RNA interference resist SIVmac infection. Thus, our observations reveal that Vpx interacts with both catalytic and regulatory components of the ubiquitin proteasome system and demonstrate that these interactions are critical for Vpx ability to enable efficient SIVmac replication in primary macrophages. Furthermore, they identify VprBP/DCAF1 substrate receptor for Cullin 4 E3 ubiquitin ligase and its associated protein complex as immediate downstream effector of Vpx for this function. Together, our findings suggest a model in which Vpx usurps VprBP-associated Cullin 4 ubiquitin ligase to enable efficient reverse transcription and thereby overcome a block to lentivirus replication in monocyte-derived cells, and thus provide novel insights into the underlying molecular mechanism.

  5. Strategies for inhibiting function of HIV-1 accessory proteins: a necessary route to AIDS therapy?

    Science.gov (United States)

    Richter, S N; Frasson, I; Palù, G

    2009-01-01

    The Human Immunodeficiency Virus (HIV) genome encodes three major structural proteins common to all retroviruses (Gag, Pol and Env), two regulatory proteins (Tat and Rev) that are essential for viral replication, and four accessory proteins (Nef, Vif, Vpu, Vpr). While accessory proteins were initially reported to be unnecessary for viral growth, their importance as virulence factors is now being more and more appreciated: they can dramatically alter the course and severity of viral infection, replication and disease progression. None of the HIV accessory proteins display enzymatic activity: they rather act altering cellular pathways via multiple protein-protein interactions with a number of host cell factors. All currently approved anti-HIV drugs target pol and env encoded proteins. Therefore, widening the molecular targets of HIV therapy by additionally targeting accessory proteins may expand treatment options, resulting in high impact effective new therapy. In this review we present the state of the art of compounds that target HIV accessory proteins. Most of the research has focused on the inhibition of specific accessory proteins/host cell partner interactions. Promising compounds have been found within different classes of molecules: small natural and synthetic molecules, peptides and proteins, oligonucleotides, in particular those used as RNA interference (RNAi) tools. With the assortment of compounds available, especially against Nef and Vif functions, the demonstration of the clinical efficacy of the new anti-HIV-1 drugs targeting accessory proteins is next challenge.

  6. Anti-cancer effect of HIV-1 viral protein R on doxorubicin resistant neuroblastoma.

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    Richard Y Zhao

    Full Text Available Several unique biological features of HIV-1 Vpr make it a potentially powerful agent for anti-cancer therapy. First, Vpr inhibits cell proliferation by induction of cell cycle G2 arrest. Second, it induces apoptosis through multiple mechanisms, which could be significant as it may be able to overcome apoptotic resistance exhibited by many cancerous cells, and, finally, Vpr selectively kills fast growing cells in a p53-independent manner. To demonstrate the potential utility of Vpr as an anti-cancer agent, we carried out proof-of-concept studies in vitro and in vivo. Results of our preliminary studies demonstrated that Vpr induces cell cycle G2 arrest and apoptosis in a variety of cancer types. Moreover, the same Vpr effects could also be detected in some cancer cells that are resistant to anti-cancer drugs such as doxorubicin (DOX. To further illustrate the potential value of Vpr in tumor growth inhibition, we adopted a DOX-resistant neuroblastoma model by injecting SK-N-SH cells into C57BL/6N and C57BL/6J-scid/scid mice. We hypothesized that Vpr is able to block cell proliferation and induce apoptosis regardless of the drug resistance status of the tumors. Indeed, production of Vpr via adenoviral delivery to neuroblastoma cells caused G2 arrest and apoptosis in both drug naïve and DOX-resistant cells. In addition, pre-infection or intratumoral injection of vpr-expressing adenoviral particles into neuroblastoma tumors in SCID mice markedly inhibited tumor growth. Therefore, Vpr could possibly be used as a supplemental viral therapeutic agent for selective inhibition of tumor growth in anti-cancer therapy especially when other therapies stop working.

  7. Impacts of Humanized Mouse Models on the Investigation of HIV-1 Infection: Illuminating the Roles of Viral Accessory Proteins in Vivo

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    Eri Yamada

    2015-03-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 encodes four accessory genes: vif, vpu, vpr, and nef. Recent investigations using in vitro cell culture systems have shed light on the roles of these HIV-1 accessory proteins, Vif, Vpr, Vpu, and Nef, in counteracting, modulating, and evading various cellular factors that are responsible for anti-HIV-1 intrinsic immunity. However, since humans are the exclusive target for HIV-1 infection, conventional animal models are incapable of mimicking the dynamics of HIV-1 infection in vivo. Moreover, the effects of HIV-1 accessory proteins on viral infection in vivo remain unclear. To elucidate the roles of HIV-1 accessory proteins in the dynamics of viral infection in vivo, humanized mouse models, in which the mice are xenotransplanted with human hematopoietic stem cells, has been utilized. This review describes the current knowledge of the roles of HIV-1 accessory proteins in viral infection, replication, and pathogenicity in vivo, which are revealed by the studies using humanized mouse models.

  8. Modulation of HIV-1 virulence via the host glucocorticoid receptor: towards further understanding the molecular mechanisms of HIV-1 pathogenesis.

    Science.gov (United States)

    Hapgood, Janet Patricia; Tomasicchio, Michele

    2010-07-01

    The glucocorticoid receptor (GR) is a steroid receptor that regulates diverse functions, which include the immune response. In humans, the GR acts via binding to cortisol, resulting in the transcriptional modulation of key host genes. Several lines of evidence suggest that the host GR could be a key protein exploited by HIV at multiple levels to ensure its pathogenic success. Endogenous and therapeutic glucocorticoids play important roles in patients with HIV due to their well-established effects on immune function. AIDS patients develop glucocorticoid hypersensitivity, consistent with a mechanism involving an HIV-1-induced increase in expression or activity of the GR. Both the HIV-1 accessory protein Vpr and the host GR affect transcription of viral proteins from the long terminal repeat (LTR) region of the HIV-1 promoter. In addition, Vpr modulates host GR function to affect transcription of host genes, most likely via direct interaction with the GR. Vpr appears to regulate GR function by acting as a co-activator for the GR. Since both the GR and Vpr are involved in apoptosis in T cells and dendritic cells, crosstalk between these proteins may also regulate apoptosis in these and other cells. Given that cortisol is not the only ligand that activates the GR, other endogenous as well as synthetic GR ligands such as progestins may also modulate HIV pathogenesis, in particular in the cervicovaginal environment. Investigating the molecular determinants, ligand-selectivity and role in HIV pathogenesis of the GR-Vpr interaction may lead to new strategies for development of anti-HIV drugs.

  9. The Natural Killer Cell Cytotoxic Function Is Modulated by HIV-1 Accessory Proteins

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    Edward Barker

    2011-07-01

    Full Text Available Natural killer (NK cells’ major role in the control of viruses is to eliminate established infected cells. The capacity of NK cells to kill virus-infected cells is dependent on the interactions between ligands on the infected cell and receptors on the NK cell surface. Because of the importance of ligand-receptor interactions in modulating the NK cell cytotoxic response, HIV has developed strategies to regulate various NK cell ligands making the infected cell surprisingly refractory to NK cell lysis. This is perplexing because the HIV-1 accessory protein Vpr induces expression of ligands for the NK cell activating receptor, NKG2D. In addition, the accessory protein Nef removes the inhibitory ligands HLA-A and -B. The reason for the ineffective killing by NK cells despite the strong potential to eliminate infected cells is due to HIV-1 Vpu’s ability to down modulate the co-activation ligand, NTB-A, from the cell surface. Down modulation of NTB-A prevents efficient NK cell degranulation. This review will focus on the mechanisms through which the HIV-1 accessory proteins modulate their respective ligands, and its implication for NK cell killing of HIV-infected cells.

  10. Roles of HIV-1 auxiliary proteins in viral pathogenesis and host-pathogen interactions

    Institute of Scientific and Technical Information of China (English)

    Lin; LI; Hai; Shan; LI; C.David; PAUZA; Michael; BUKRINSKY; Richard; Y; ZHAO

    2005-01-01

    Active host-pathogen interactions take place during infection of human immunodeficiency virus type 1 (HIV-1).Outcomes of these interactions determine the efficiency of viral infection and subsequent disease progression. HIV-infected cells respond to viral invasion with various defensive strategies such as innate, cellular and humoral immune antiviral mechanisms. On the other hand, the virus has also developed various offensive tactics to suppress these host cellular responses. Among many of the viral offensive strategies, HIV- 1 viral auxiliary proteins (Tat, Rev, Nef, Vif, Vpr and Vpu) play important roles in the host-pathogen interaction and thus have significant impacts on the outcome of HIV infection. One of the best examples is the interaction of Vif with a host cytidine deaminase APOBEC3G. Although specific roles of other auxiliary proteins are not as well described as Vif-APOBEC3G interaction, it is the goal of this brief review to summarize some of the preliminary findings with the hope to stimulate further discussion and investigation in this exhilarating area of research.

  11. An inducible packaging cell system for safe, efficient lentiviral vector production in the absence of HIV-1 accessory proteins.

    Science.gov (United States)

    Pacchia, A L; Adelson, M E; Kaul, M; Ron, Y; Dougherty, J P

    2001-03-30

    Lentiviral vectors based on human immunodeficiency virus type 1 (HIV-1) possess the ability to deliver exogenous genes to both dividing and nondividing cells and to subsequently establish a stable provirus in these target cells, which can allow long-term expression of the transferred gene. Herein we describe a stable packaging cell line that is devoid of HIV-1 tat, vif, vpr, vpu, and nef. In order to avoid any risk of cytotoxicity associated with constitutive expression of HIV-1 protease or the VSV-G envelope protein, transcription of the packaging and envelope constructs was tightly controlled by employing the ecdysone-inducible system. Using this cell line, we have been able to consistently generate concentrated pseudotyped vector virus stocks with titers in the range of 10(8) IU/ml, which can efficiently transduce actively dividing and growth-arrested cells in vitro. This novel packaging cell line for lentiviral vectors facilitates the production of high-titer virus stocks in the absence of replication-competent virus and provides us with an important tool for use in future gene transfer studies.

  12. Tat RNA silencing suppressor activity contributes to perturbation of lymphocyte miRNA by HIV-1

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    Yu Lianbo

    2011-05-01

    Full Text Available Abstract Background MicroRNA (miRNA-mediated RNA silencing is integral to virtually every cellular process including cell cycle progression and response to virus infection. The interplay between RNA silencing and HIV-1 is multifaceted, and accumulating evidence posits a strike-counterstrike interface that alters the cellular environment to favor virus replication. For instance, miRNA-mediated RNA silencing of HIV-1 translation is antagonized by HIV-1 Tat RNA silencing suppressor activity. The activity of HIV-1 accessory proteins Vpr/Vif delays cell cycle progression, which is a process prominently modulated by miRNA. The expression profile of cellular miRNA is altered by HIV-1 infection in both cultured cells and clinical samples. The open question stands of what, if any, is the contribution of Tat RNA silencing suppressor activity or Vpr/Vif activity to the perturbation of cellular miRNA by HIV-1. Results Herein, we compared the perturbation of miRNA expression profiles of lymphocytes infected with HIV-1NL4-3 or derivative strains that are deficient in Tat RNA silencing suppressor activity (Tat K51A substitution or ablated of the vpr/vif open reading frames. Microarrays recapitulated the perturbation of the cellular miRNA profile by HIV-1 infection. The miRNA expression trends overlapped ~50% with published microarray results on clinical samples from HIV-1 infected patients. Moreover, the number of miRNA perturbed by HIV-1 was largely similar despite ablation of Tat RSS activity and Vpr/Vif; however, the Tat RSS mutation lessened HIV-1 downregulation of twenty-two miRNAs. Conclusions Our study identified miRNA expression changes attributable to Tat RSS activity in HIV-1NL4-3. The results accomplish a necessary step in the process to understand the interface of HIV-1 with host RNA silencing activity. The overlap in miRNA expression trends observed between HIV-1 infected CEMx174 lymphocytes and primary cells supports the utility of cultured

  13. MAS NMR of HIV-1 protein assemblies

    Science.gov (United States)

    Suiter, Christopher L.; Quinn, Caitlin M.; Lu, Manman; Hou, Guangjin; Zhang, Huilan; Polenova, Tatyana

    2015-04-01

    The negative global impact of the AIDS pandemic is well known. In this perspective article, the utility of magic angle spinning (MAS) NMR spectroscopy to answer pressing questions related to the structure and dynamics of HIV-1 protein assemblies is examined. In recent years, MAS NMR has undergone major technological developments enabling studies of large viral assemblies. We discuss some of these evolving methods and technologies and provide a perspective on the current state of MAS NMR as applied to the investigations into structure and dynamics of HIV-1 assemblies of CA capsid protein and of Gag maturation intermediates.

  14. Large Isoform of Mammalian Relative of DnaJ is a Major Determinant of Human Susceptibility to HIV-1 Infection

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    Yu-Ping Chiang

    2014-12-01

    Full Text Available Individual differences in susceptibility to human immunodeficiency virus type 1 (HIV-1 infection have been of interest for decades. We aimed to determine the contribution of large isoform of Mammalian DnaJ (MRJ-L, a HIV-1 Vpr-interacting cellular protein, to this natural variation. Expression of MRJ-L in monocyte-derived macrophages was significantly higher in HIV-infected individuals (n = 31 than their uninfected counterparts (n = 27 (p = 0.009. Fifty male homosexual subjects (20 of them are HIV-1 positive were further recruited to examine the association between MRJ-L levels and occurrence of HIV infection. Bayesian multiple logistic regression revealed that playing a receptive role and increased levels of MRJ-L in macrophages were two risk factors for HIV-1 infection. A 1% rise in MRJ-L expression was associated with a 1.13 fold (95% CrI 1.06–1.29 increase in odds of contracting HIV-1 infection. Ex vivo experiments revealed that MRJ-L facilitated Vpr-dependent nuclear localization of virus. Infection of macrophage-tropic strain is a critical step in HIV-1 transmission. MRJ-L is a critical factor in this process; hence, subjects with higher macrophage MRJ-L levels are more vulnerable to HIV-1 infection.

  15. Hepatitis C virus core protein induces neuroimmune activation and potentiates Human Immunodeficiency Virus-1 neurotoxicity.

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    Pornpun Vivithanaporn

    Full Text Available BACKGROUND: Hepatitis C virus (HCV genomes and proteins are present in human brain tissues although the impact of HIV/HCV co-infection on neuropathogenesis remains unclear. Herein, we investigate HCV infectivity and effects on neuronal survival and neuroinflammation in conjunction with HIV infection. METHODOLOGY: Human microglia, astrocyte and neuron cultures were infected with cell culture-derived HCV or exposed to HCV core protein with or without HIV-1 infection or HIV-1 Viral Protein R (Vpr exposure. Host immune gene expression and cell viability were measured. Patch-clamp studies of human neurons were performed in the presence or absence of HCV core protein. Neurobehavioral performance and neuropathology were examined in HIV-1 Vpr-transgenic mice in which stereotaxic intrastriatal implants of HCV core protein were performed. PRINCIPAL FINDINGS: HCV-encoded RNA as well as HCV core and non-structural 3 (NS3 proteins were detectable in human microglia and astrocytes infected with HCV. HCV core protein exposure induced expression of pro-inflammatory cytokines including interleukin-1β, interleukin-6 and tumor necrosis factor-α in microglia (p<0.05 but not in astrocytes while increased chemokine (e.g. CXCL10 and interleukin-8 expression was observed in both microglia and astrocytes (p<0.05. HCV core protein modulated neuronal membrane currents and reduced both β-III-tubulin and lipidated LC3-II expression (p<0.05. Neurons exposed to supernatants from HCV core-activated microglia exhibited reduced β-III-tubulin expression (p<0.05. HCV core protein neurotoxicity and interleukin-6 induction were potentiated by HIV-1 Vpr protein (p<0.05. HIV-1 Vpr transgenic mice implanted with HCV core protein showed gliosis, reduced neuronal counts together with diminished LC3 immunoreactivity. HCV core-implanted animals displayed neurobehavioral deficits at days 7 and 14 post-implantation (p<0.05. CONCLUSIONS: HCV core protein exposure caused neuronal injury

  16. Das Nukleoprotein SPOC1 erhöht die HIV-1 Integration unterdrückt aber die virale Genexpression

    OpenAIRE

    Hofmann, Stephan

    2015-01-01

    In this study, the role of the nuclear protein SPOC1 for HIV-1 replication was investigated. SPOC1 was shown to increase proviral integration and hence the total number of infected cells. Nonetheless, SPOC1 was degraded by the viral accessory protein Vpr post integration. This degradation evolved most likely due to repressive effects of SPOC1 on viral gene expression. Hence, SPOC1 is not a bona-fide antiviral restriction factor but is important for efficient HIV-1 integration. At later steps,...

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

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    Lydia Kobbi

    2016-06-01

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

  18. HIV-1 accessory proteins: Vpu and Vif.

    Science.gov (United States)

    Andrew, Amy; Strebel, Klaus

    2014-01-01

    HIV-1 Vif and Vpu are accessory factors involved in late stages of viral replication. Vif regulates viral infectivity by preventing virion incorporation of APOBEC3G and other members of the family of cytidine deaminases, while Vpu causes degradation of CD4 and promotes virus release by functionally inactivating the host factor BST-2. This chapter described techniques used for the characterization of Vif and Vpu and their functional interaction with host factors. Many of the techniques are, however, applicable to the functional analysis of other viral proteins.

  19. Sequence alignment reveals possible MAPK docking motifs on HIV proteins.

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    Perry Evans

    Full Text Available Over the course of HIV infection, virus replication is facilitated by the phosphorylation of HIV proteins by human ERK1 and ERK2 mitogen-activated protein kinases (MAPKs. MAPKs are known to phosphorylate their substrates by first binding with them at a docking site. Docking site interactions could be viable drug targets because the sequences guiding them are more specific than phosphorylation consensus sites. In this study we use multiple bioinformatics tools to discover candidate MAPK docking site motifs on HIV proteins known to be phosphorylated by MAPKs, and we discuss the possibility of targeting docking sites with drugs. Using sequence alignments of HIV proteins of different subtypes, we show that MAPK docking patterns previously described for human proteins appear on the HIV matrix, Tat, and Vif proteins in a strain dependent manner, but are absent from HIV Rev and appear on all HIV Nef strains. We revise the regular expressions of previously annotated MAPK docking patterns in order to provide a subtype independent motif that annotates all HIV proteins. One revision is based on a documented human variant of one of the substrate docking motifs, and the other reduces the number of required basic amino acids in the standard docking motifs from two to one. The proposed patterns are shown to be consistent with in silico docking between ERK1 and the HIV matrix protein. The motif usage on HIV proteins is sufficiently different from human proteins in amino acid sequence similarity to allow for HIV specific targeting using small-molecule drugs.

  20. Inhibition of renin activity slows down the progression of HIV-associated nephropathy.

    Science.gov (United States)

    Kumar, Dileep; Plagov, Andrei; Yadav, Iti; Torri, Deepti D; Sayeneni, Swapna; Sagar, Ankita; Rai, Partab; Adabala, Madhuri; Lederman, Rivka; Chandel, Nirupama; Ding, Guohua; Malhotra, Ashwani; Singhal, Pravin C

    2012-09-01

    In the present study, we evaluated the effect of inhibition of renin activity (aliskiren) on the progression of renal lesions in two different mouse models (Vpr and Tg26) of human immunodeficiency virus (HIV)-associated nephropathy (HIVAN). In protocol A, Vpr mice were fed either water (C-VprA) or doxycycline [Doxy (D-VprA)] in their drinking water for 6 wk. In protocols B and C, Vpr mice received either normal saline (C-VprB/C), Doxy + normal saline (D-VprB/C), or Doxy + aliskiren (AD-VprB/C) for 6 wk (protocol B) or 12 wk (protocol C). In protocols D and E, Vpr mice were fed Doxy for 6 wk followed by kidney biopsy. Subsequently, half of the mice were administered either normal saline (D-VprD/E) or aliskiren (AD-VprD/E) for 4 wk (protocol D) or 8 (protocol E) wk. All D-VprA mice showed renal lesions in the form of focal segmental glomerular sclerosis and dilatation of tubules. In protocols B and C, aliskiren diminished both progression of renal lesions and proteinuria. In protocol C, aliskiren also diminished (P Doxy-treated mice displayed increased serum ANG I levels (the product of plasma renin activity); on the other hand, all aliskiren-treated mice displayed diminished serum ANG I levels. Renal tissues of D-VprC displayed increased ANG II content; however, aliskiren attenuated renal tissue ANG II production in AD-VprC. In protocol D, AD-VprD showed a 24.2% increase in the number of sclerosed glomeruli compared with 139.2% increase in sclerosed glomeruli in D-VprD (P < 0.01) from their baseline. The attenuating effect of aliskiren on the progression of renal lesions continued in AD-VprE. Aliskiren also diminished blood pressure, proteinuria, and progression of renal lesions in Tg26 mice. These findings indicate that inhibition of renin activity has a potential to slow down the progression of HIVAN.

  1. TIM-family proteins inhibit HIV-1 release.

    Science.gov (United States)

    Li, Minghua; Ablan, Sherimay D; Miao, Chunhui; Zheng, Yi-Min; Fuller, Matthew S; Rennert, Paul D; Maury, Wendy; Johnson, Marc C; Freed, Eric O; Liu, Shan-Lu

    2014-09-02

    Accumulating evidence indicates that T-cell immunoglobulin (Ig) and mucin domain (TIM) proteins play critical roles in viral infections. Herein, we report that the TIM-family proteins strongly inhibit HIV-1 release, resulting in diminished viral production and replication. Expression of TIM-1 causes HIV-1 Gag and mature viral particles to accumulate on the plasma membrane. Mutation of the phosphatidylserine (PS) binding sites of TIM-1 abolishes its ability to block HIV-1 release. TIM-1, but to a much lesser extent PS-binding deficient mutants, induces PS flipping onto the cell surface; TIM-1 is also found to be incorporated into HIV-1 virions. Importantly, TIM-1 inhibits HIV-1 replication in CD4-positive Jurkat cells, despite its capability of up-regulating CD4 and promoting HIV-1 entry. In addition to TIM-1, TIM-3 and TIM-4 also block the release of HIV-1, as well as that of murine leukemia virus (MLV) and Ebola virus (EBOV); knockdown of TIM-3 in differentiated monocyte-derived macrophages (MDMs) enhances HIV-1 production. The inhibitory effects of TIM-family proteins on virus release are extended to other PS receptors, such as Axl and RAGE. Overall, our study uncovers a novel ability of TIM-family proteins to block the release of HIV-1 and other viruses by interaction with virion- and cell-associated PS. Our work provides new insights into a virus-cell interaction that is mediated by TIMs and PS receptors.

  2. The HIV Protein gp120 Alters Mitochondrial Dynamics in Neurons

    Science.gov (United States)

    Castellano, Paul; Dedoni, Simona; Palchik, Guillermo; Trejo, Margarita; Adame, Anthony; Rockenstein, Edward; Eugenin, Eliseo; Masliah, Eliezer; Mocchetti, Italo

    2016-01-01

    Neurotoxicity of human immunodeficiency virus-1 (HIV) includes synaptic simplification and neuronal apoptosis. However, the mechanisms of HIV-associated neurotoxicity remain unclear, thus precluding an effective treatment of the neurological complications. The present study was undertaken to characterize novel mechanisms of HIV neurotoxicity that may explain how HIV subjects develop neuronal degeneration. Several neurodegenerative disorders are characterized by mitochondrial dysfunction; therefore, we hypothesized that HIV promotes mitochondrial damage. We first analyzed brains from HIV encephalitis (HIVE) by electron microscopy. Several sections of HIVE subjects contained enlarged and damaged mitochondria compared to brains from HIV subjects with no neurological complications. Similar pathologies were observed in mice overexpressing the HIV protein gp120, suggesting that this viral protein may be responsible for mitochondrial pathology found in HIVE. To gain more information about the cellular mechanisms of gp120 neurotoxicity, we exposed rat cortical neurons to gp120 and we determined cellular oxygen consumption rate, mitochondrial distribution, and trafficking. Our data show that gp120 evokes impairment in mitochondrial function and distribution. These data suggest that one of the mechanisms of HIV neurotoxicity includes altered mitochondrial dynamics in neurons. PMID:26936603

  3. Determining protein biomarkers for DLBCL using FFPE tissues from HIV negative and HIV positive patients.

    Science.gov (United States)

    Magangane, Pumza; Sookhayi, Raveendra; Govender, Dhirendra; Naidoo, Richard

    2016-12-01

    DLBCL is the most common lymphoma subtype occurring in older populations as well as in younger HIV infected patients. The current treatment options for DLBCL are effective for most patients yet the relapse rate is high. While many biomarkers for DLBCL exist, they are not in clinical use due to low sensitivity and specificity. In addition, these biomarkers have not been studied in the HIV context. Therefore, the identification of new biomarkers for HIV negative and HIV positive DLBCL, may lead to a better understanding of the disease pathology and better therapeutic design. Protein biomarkers for DLBCL were determined using MALDI imaging mass spectrometry (IMS) and characterised using LC-MS. The expression of one of the biomarkers, heat shock protein (Hsp) 70, was confirmed on a separate cohort of samples using immunohistochemistry. The biomarkers identified in the study consisted of four protein clusters including glycolytic enzymes, ribosomal proteins, histones and collagen. These proteins could differentiate between control and tumour tissue, and the DLBCL immunohistochemical subtypes in both cohorts. The majority (41/52) of samples in the confirmation cohort were negative for Hsp70 expression. The HIV positive DLBCL cases had a higher percentage of cases expressing Hsp70 than their HIV negative counterparts. The non-GC subtype also frequently overexpressed Hsp70, confirming MALDI IMS data. The expression of Hsp70 did not correlate with survival in both the HIV negative and HIV positive cohort. This study identified potential biomarkers for HIV negative and HIV positive DLBCL from FFPE tissue sections. These may be used as diagnostic and prognostic markers complementary to current clinical management programmes for DLBCL.

  4. The Vibrio cholerae VprA-VprB two-component system controls virulence through endotoxin modification.

    Science.gov (United States)

    Herrera, Carmen M; Crofts, Alexander A; Henderson, Jeremy C; Pingali, S Cassandra; Davies, Bryan W; Trent, M Stephen

    2014-12-23

    The bacterial cell surface is the first structure the host immune system targets to prevent infection. Cationic antimicrobial peptides of the innate immune system bind to the membrane of Gram-negative pathogens via conserved, surface-exposed lipopolysaccharide (LPS) molecules. We recently reported that modern strains of the global intestinal pathogen Vibrio cholerae modify the anionic lipid A domain of LPS with a novel moiety, amino acids. Remarkably, glycine or diglycine addition to lipid A alters the surface charge of the bacteria to help evade the cationic antimicrobial peptide polymyxin. However, the regulatory mechanisms of lipid A modification in V. cholerae are unknown. Here, we identify a novel two-component system that regulates lipid A glycine modification by responding to important biological cues associated with pathogenesis, including bile, mildly acidic pH, and cationic antimicrobial peptides. The histidine kinase Vc1319 (VprB) and the response regulator Vc1320 (VprA) respond to these signals and are required for the expression of the almEFG operon that encodes the genes essential for glycine modification of lipid A. Importantly, both the newly identified two-component system and the lipid A modification machinery are required for colonization of the mammalian host. This study demonstrates how V. cholerae uses a previously unknown regulatory network, independent of well-studied V. cholerae virulence factors and regulators, to respond to the host environment and cause infection. Vibrio cholerae, the etiological agent of cholera disease, infects millions of people every year. V. cholerae El Tor and classical biotypes have been responsible for all cholera pandemics. The El Tor biotype responsible for the current seventh pandemic has displaced the classical biotype worldwide and is highly resistant to cationic antimicrobial peptides, like polymyxin B. This resistance arises from the attachment of one or two glycine residues to the lipid A domain of

  5. Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy.

    Science.gov (United States)

    Imamichi, Hiromi; Dewar, Robin L; Adelsberger, Joseph W; Rehm, Catherine A; O'Doherty, Una; Paxinos, Ellen E; Fauci, Anthony S; Lane, H Clifford

    2016-08-02

    Despite years of plasma HIV-RNA levels <40 copies per milliliter during combination antiretroviral therapy (cART), the majority of HIV-infected patients exhibit persistent seropositivity to HIV-1 and evidence of immune activation. These patients also show persistence of proviruses of HIV-1 in circulating peripheral blood mononuclear cells. Many of these proviruses have been characterized as defective and thus thought to contribute little to HIV-1 pathogenesis. By combining 5'LTR-to-3'LTR single-genome amplification and direct amplicon sequencing, we have identified the presence of "defective" proviruses capable of transcribing novel unspliced HIV-RNA (usHIV-RNA) species in patients at all stages of HIV-1 infection. Although these novel usHIV-RNA transcripts had exon structures that were different from those of the known spliced HIV-RNA variants, they maintained translationally competent ORFs, involving elements of gag, pol, env, rev, and nef to encode a series of novel HIV-1 chimeric proteins. These novel usHIV-RNAs were detected in five of five patients, including four of four patients with prolonged viral suppression of HIV-RNA levels <40 copies per milliliter for more than 6 y. Our findings suggest that the persistent defective proviruses of HIV-1 are not "silent," but rather may contribute to HIV-1 pathogenesis by stimulating host-defense pathways that target foreign nucleic acids and proteins.

  6. Solid-phase synthesis and screening of N-acylated polyamine (NAPA) combinatorial libraries for protein binding.

    Science.gov (United States)

    Iera, Jaclyn A; Jenkins, Lisa M Miller; Kajiyama, Hiroshi; Kopp, Jeffrey B; Appella, Daniel H

    2010-11-15

    Inhibitors for protein-protein interactions are challenging to design, in part due to the unique and complex architectures of each protein's interaction domain. Most approaches to develop inhibitors for these interactions rely on rational design, which requires prior structural knowledge of the target and its ligands. In the absence of structural information, a combinatorial approach may be the best alternative to finding inhibitors of a protein-protein interaction. Current chemical libraries, however, consist mostly of molecules designed to inhibit enzymes. In this manuscript, we report the synthesis and screening of a library based on an N-acylated polyamine (NAPA) scaffold that we designed to have specific molecular features necessary to inhibit protein-protein interactions. Screens of the library identified a member with favorable binding properties to the HIV viral protein R (Vpr), a regulatory protein from HIV, that is involved in numerous interactions with other proteins critical for viral replication.

  7. HIV-specific T cell immunity across the entire HIV genome in Chinese men who have sex with men

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiao-yan; SHAO Yi-ming; HUANG Xiang-gang; XU Jian-qing; LI Shen-wei; JIANG Shu-lin; ZHANG Xiao-xi; LI Dong-liang; RUAN Yu-hua; XING Hui

    2006-01-01

    Background Man who has sex with man (MSM) is one of the high risk groups for spreading HIV/AIDS. It was reported that the most prevalent human immunodeficiency virus type 1 (HIV-1) strain among MSM is subtype B; however, T cell immunity remains unknown across the HIV-1 B genome in this population.Methods Using Elispot assay with synthetic peptides spanning the sequence of HIV-1 consensus B,HIV-1-specific cytotoxic T-cell lymphocyte responses were quantified among 3 treated and 19 untreated HIV-1 infected MSM from Beijing, China. Cross-sectional association between viral loads and cellular immune responses were analyzed.Results Peptide pools corresponding to each HIV-1 protein were used for Env, Gag, Pol, Nef, Tat/Rev, Vpr/Vpu and Vif. The results showed that the magnitude of T cell responses in the 3 treated HIV+ MSM group [median,770 spot forming cells (SFCs) per 106 peripheral blood mononuclear cells (PBMCs)] might be significantly lower than that in the 19 untreated HIV+ MSM group (median, 6175 SFCs per 106 PBMCs). Nef, Gag and Pol are the most frequently targeted HIV-1 antigens; and 16 subjects (73%) were identified with vigorous T cell immunity against each of these three proteins. The overall magnitude of T cell immunity closely related to its breadth (r=-0.72, P<0.05) and was inversely but weakly associated with viral loads (r=-0.15). Further analysis showed that both Gag (r=-0.24) and Pol specific T cells (r=-0.12) contributed to this inverse association whereas Nef specific T cells showed no association with viral loads.Conclusions The magnitude of HIV-1 specific T cells is inversely but weakly associated with viral loads among MSM; HIV-specific T cell responses against conservative sequences (Gag and Poi) are the main contributors to this association among Chinese HIV+ MSM. These findings have important implications for vaccine design.

  8. Surfactant protein D binds to human immunodeficiency virus (HIV) envelope protein gp120 and inhibits HIV replication

    DEFF Research Database (Denmark)

    Meschi, Joseph; Crouch, Erika C; Skolnik, Paul;

    2005-01-01

    The envelope protein (gp120) of human immunodeficiency virus (HIV) contains highly conserved mannosylated oligosaccharides. These glycoconjugates contribute to resistance to antibody neutralization, and binding to cell surface lectins on macrophages and dendritic cells. Mannose-binding lectin (MBL......) binds to gp120 and plays a role in defence against the virus. In this study it is demonstrated that surfactant protein D (SP-D) binds to gp120 and inhibits HIV infectivity at significantly lower concentrations than MBL. The binding of SP-D was mediated by its calcium-dependent carbohydrate...... defence against HIV. A chimeric protein containing the N-terminal and collagen domains of SP-D linked to the neck and carbohydrate-recognition domains of MBL (called SP-D/MBL(neck+CRD)) had greater ability to bind to gp120 and inhibit virus replication than either SP-D or MBL. The enhanced binding of SP...

  9. In vitro nuclear interactome of the HIV-1 Tat protein.

    LENUS (Irish Health Repository)

    Gautier, Virginie W

    2009-01-01

    BACKGROUND: One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86-101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry. RESULTS: Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied in silico analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture. CONCLUSION: We have completed the in vitro Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will

  10. HIV-1 Vpr促进Vif和APOBEC3G蛋白表达的研究%Enhanced protein production of Vif and APOBEC3G by HIV-1 Vpr

    Institute of Scientific and Technical Information of China (English)

    李林; 梁栋; 李敬云; 赵玉琪

    2008-01-01

    目的 观察人获得性免疫缺陷病毒蛋白Vpr对Vif蛋白和APOBEC3G蛋白表达水平的影响.方法 使用电转化法将携带Vif基因的酵母表达载体转化到裂殖酵母中,使用脂质体转染法将表达Vif蛋白、APOBEC3G蛋白的哺乳动物表达载体转染到可诱导稳定表达Vpr蛋白的哺乳动物HEK293细胞中;使用Western Blot方法检测目标蛋白表达水平的变化.结果 在裂殖酵母和哺乳动物细胞中,Vpr蛋白的表达能够提高细胞内的Vif蛋白的表达水平,在哺乳动物细胞中,Vpr蛋白对APOBEC3G蛋白的表达亦有促进作用,Vpr蛋白引起的Vif蛋白量的提高并没有导致APOBEC3G蛋白的减少.结论 Vpr蛋白具有调节细胞内蛋白表达的功能.

  11. Prediction of HIV-1 virus-host protein interactions using virus and host sequence motifs

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2009-05-01

    Full Text Available Abstract Background Host protein-protein interaction networks are altered by invading virus proteins, which create new interactions, and modify or destroy others. The resulting network topology favors excessive amounts of virus production in a stressed host cell network. Short linear peptide motifs common to both virus and host provide the basis for host network modification. Methods We focused our host-pathogen study on the binding and competing interactions of HIV-1 and human proteins. We showed that peptide motifs conserved across 70% of HIV-1 subtype B and C samples occurred in similar positions on HIV-1 proteins, and we documented protein domains that interact with these conserved motifs. We predicted which human proteins may be targeted by HIV-1 by taking pairs of human proteins that may interact via a motif conserved in HIV-1 and the corresponding interacting protein domain. Results Our predictions were enriched with host proteins known to interact with HIV-1 proteins ENV, NEF, and TAT (p-value Conclusion A list of host proteins highly enriched with those targeted by HIV-1 proteins can be obtained by searching for host protein motifs along virus protein sequences. The resulting set of host proteins predicted to be targeted by virus proteins will become more accurate with better annotations of motifs and domains. Nevertheless, our study validates the role of linear binding motifs shared by virus and host proteins as an important part of the crosstalk between virus and host.

  12. Recognition of HIV-1 peptides by host CTL is related to HIV-1 similarity to human proteins.

    Directory of Open Access Journals (Sweden)

    Morgane Rolland

    Full Text Available BACKGROUND: While human immunodeficiency virus type 1 (HIV-1-specific cytotoxic T lymphocytes preferentially target specific regions of the viral proteome, HIV-1 features that contribute to immune recognition are not well understood. One hypothesis is that similarities between HIV and human proteins influence the host immune response, i.e., resemblance between viral and host peptides could preclude reactivity against certain HIV epitopes. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the extent of similarity between HIV-1 and the human proteome. Proteins from the HIV-1 B consensus sequence from 2001 were dissected into overlapping k-mers, which were then probed against a non-redundant database of the human proteome in order to identify segments of high similarity. We tested the relationship between HIV-1 similarity to host encoded peptides and immune recognition in HIV-infected individuals, and found that HIV immunogenicity could be partially modulated by the sequence similarity to the host proteome. ELISpot responses to peptides spanning the entire viral proteome evaluated in 314 individuals showed a trend indicating an inverse relationship between the similarity to the host proteome and the frequency of recognition. In addition, analysis of responses by a group of 30 HIV-infected individuals against 944 overlapping peptides representing a broad range of individual HIV-1B Nef variants, affirmed that the degree of similarity to the host was significantly lower for peptides with reactive epitopes than for those that were not recognized. CONCLUSIONS/SIGNIFICANCE: Our results suggest that antigenic motifs that are scarcely represented in human proteins might represent more immunogenic CTL targets not selected against in the host. This observation could provide guidance in the design of more effective HIV immunogens, as sequences devoid of host-like features might afford superior immune reactivity.

  13. Identification of HIV-1 specific T lymphocyte responses in highly exposed persistently seronegative Chinese

    Institute of Scientific and Technical Information of China (English)

    LIU Hong-wei; SHAO Yi-ming; HONG Kun-xue; MA Jun; YUAN Lin; LIU Sha; CHEN Jian-ping; ZHANG Yuan-zhi; RUAN Yu-hua; XU Jian-qing

    2006-01-01

    Background Studies of highly exposed persistently seronegative (HEPS) individuals may provide valuable information on mechanisms of protection and on vaccine design. Cellular immune responses play a critical role in containing human immunodeficiency virus. However, the cellular immune responses in HEPS individuals have not been thoroughly assessed at the entire viral genome level.Methods Ten HEPS Chinese with a history of frequent penetrative vaginal intercourse (mean frequency, at least once a week), with some unprotected sexual contact occurring in the weeks or days immediately before enrollment, 25 HIV-1 seropositive individuals, 10 HIV-1-seronegative healthy individuals with low-risk sexual behavior and no history suggestive of exposure to HIV-1 infection were enrolled. HIV-1-specific T cell responses were comprehensively analyzed by an interferon- γ Elispot assay against 770 overlapping peptides spanning all HIV-1 proteins.Results HIV-1-specific T-cell responses of interferon- γ secretion were identified in 3 (30%) out of 10 HEPS individuals; the specific cytotoxic T lymphocytes were targeted at Pol (2/10), Env (2/10), and Tat (1/10).HIV-1-specific T-cell responses of interferon- γ secretion were identified in 20 (80%) out of 25 seropositive intravenous drug users (IDUs), revealing that all HIV-1 proteins and protein subunits could serve as targets for HIV-1-specific CD8+ T cell responses with 85% recognizing Gag, 80% recognizing Nef, 75% recognizing Pol,60% recognizing Env, 55% recognizing Vpu, 45% recognizing Vpr, 20% recognizing Vif, 20% recognizing Tat and 15% recognizing Rev in these seropositive individuals. None of the seronegative healthy individuals gave the positive T-cell responses.Conclusions About 30% of HEPS Chinese mounted HIV-1 specific T cell immune responses. Cell-mediated immunity against HIV-1 may be developed through non-productive infections.

  14. APOBEC3 proteins can copackage and comutate HIV-1 genomes

    OpenAIRE

    Desimmie, Belete A.; Burdick, Ryan C.; Izumi, Taisuke; Doi, Hibiki; Shao, Wei; Alvord, W. Gregory; Sato, Kei; Koyanagi, Yoshio; Jones, Sara; Wilson, Eleanor; Hill, Shawn; Maldarelli, Frank; Hu, Wei-Shau; Pathak, Vinay K.

    2016-01-01

    Although APOBEC3 cytidine deaminases A3G, A3F, A3D and A3H are packaged into virions and inhibit viral replication by inducing G-to-A hypermutation, it is not known whether they are copackaged and whether they can act additively or synergistically to inhibit HIV-1 replication. Here, we showed that APOBEC3 proteins can be copackaged by visualization of fluorescently-tagged APOBEC3 proteins using single-virion fluorescence microscopy. We further determined that viruses produced in the presence ...

  15. HIV Tat protein affects circadian rhythmicity by interfering with the circadian system.

    Science.gov (United States)

    Wang, T; Jiang, Z; Hou, W; Li, Z; Cheng, S; Green, L A; Wang, Y; Wen, X; Cai, L; Clauss, M; Wang, Z

    2014-10-01

    Sleep disorders are common in patients with HIV/AIDS, and can lead to poor quality of life. Although many studies have investigated the aetiology of these disorders, it is still unclear whether impaired sleep quality is associated with HIV itself, social problems, or side effects of antiretroviral therapy (ART). Moreover, despite its known neurological associations, little is known about the role of the trans-activator of transcription (Tat) protein in sleep disorders in patients with HIV/AIDS. The purpose of this study was to test the hypothesis that the sleep quality of patients with HIV/AIDS affected by an altered circadian rhythm correlates with cerebrospinal HIV Tat protein concentration. Ninety-six patients with HIV/AIDS between 20 and 69 years old completed the Pittsburgh Sleep Quality Index. Their circadian rhythm parameters of blood pressure, Tat concentration in cerebrospinal fluid, melatonin concentration, CD4 cell count and HIV RNA viral load in serum were measured. The circadian amplitude of systolic blood pressure and the score for sleep quality (Pittsburgh Sleep Quality Index) were negatively correlated with HIV Tat protein concentration, while the melatonin value was positively correlated with Tat protein concentration. The HIV Tat protein affects circadian rhythmicity by interfering with the circadian system in patients with HIV/AIDS and further increases the melatonin excretion value. A Tat protein-related high melatonin value may counteract HIV-related poor sleep quality during the progression of HIV infection. This study provides the first clinical evidence offering an explanation for why sleep quality did not show an association with progression of HIV infection in previous studies. © 2014 The Authors. HIV Medicine published by John Wiley & Sons Ltd on behalf of British HIV Association.

  16. Proteomic analysis of saliva in HIV-positive heroin addicts reveals proteins correlated with cognition.

    Science.gov (United States)

    Dominy, Stephen S; Brown, Joseph N; Ryder, Mark I; Gritsenko, Marina; Jacobs, Jon M; Smith, Richard D

    2014-01-01

    The prevalence of HIV-associated neurocognitive disorders (HAND) remains high despite effective antiretroviral therapies. Multiple etiologies have been proposed over the last several years to account for this phenomenon, including the neurotoxic effects of antiretrovirals and co-morbid substance abuse; however, no underlying molecular mechanism has been identified. Emerging evidence in several fields has linked the gut to brain diseases, but the effect of the gut on the brain during HIV infection has not been explored. Saliva is the most accessible gut biofluid, and is therefore of great scientific interest for diagnostic and prognostic purposes. This study presents a longitudinal, liquid chromatography-mass spectrometry-based quantitative proteomics study investigating saliva samples taken from 8 HIV-positive (HIV+), 11 -negative (HIV-) heroin addicts. In addition, saliva samples were investigated from 11 HIV-, non-heroin addicted healthy controls. In the HIV+ group, 58 proteins were identified that show significant correlations with cognitive scores, implicating disruption of protein quality control pathways by HIV. Notably, only one protein from the HIV- heroin addict cohort showed a significant correlation with cognitive scores, and no proteins correlated with cognitive scores in the healthy control group. In addition, the majority of correlated proteins have been shown to be associated with exosomes, allowing us to propose that the salivary glands and/or oral epithelium may modulate brain function during HIV infection through the release of discrete packets of proteins in the form of exosomes.

  17. Proteomic analysis of saliva in HIV-positive heroin addicts reveals proteins correlated with cognition.

    Directory of Open Access Journals (Sweden)

    Stephen S Dominy

    Full Text Available The prevalence of HIV-associated neurocognitive disorders (HAND remains high despite effective antiretroviral therapies. Multiple etiologies have been proposed over the last several years to account for this phenomenon, including the neurotoxic effects of antiretrovirals and co-morbid substance abuse; however, no underlying molecular mechanism has been identified. Emerging evidence in several fields has linked the gut to brain diseases, but the effect of the gut on the brain during HIV infection has not been explored. Saliva is the most accessible gut biofluid, and is therefore of great scientific interest for diagnostic and prognostic purposes. This study presents a longitudinal, liquid chromatography-mass spectrometry-based quantitative proteomics study investigating saliva samples taken from 8 HIV-positive (HIV+, 11 -negative (HIV- heroin addicts. In addition, saliva samples were investigated from 11 HIV-, non-heroin addicted healthy controls. In the HIV+ group, 58 proteins were identified that show significant correlations with cognitive scores, implicating disruption of protein quality control pathways by HIV. Notably, only one protein from the HIV- heroin addict cohort showed a significant correlation with cognitive scores, and no proteins correlated with cognitive scores in the healthy control group. In addition, the majority of correlated proteins have been shown to be associated with exosomes, allowing us to propose that the salivary glands and/or oral epithelium may modulate brain function during HIV infection through the release of discrete packets of proteins in the form of exosomes.

  18. HIV-1 Tat interacts with LIS1 protein

    Directory of Open Access Journals (Sweden)

    Turner Willie

    2005-02-01

    Full Text Available Abstract Background HIV-1 Tat activates transcription of HIV-1 viral genes by inducing phosphorylation of the C-terminal domain (CTD of RNA polymerase II (RNAPII. Tat can also disturb cellular metabolism by inhibiting proliferation of antigen-specific T lymphocytes and by inducing cellular apoptosis. Tat-induced apoptosis of T-cells is attributed, in part, to the distortion of microtubules polymerization. LIS1 is a microtubule-associated protein that facilitates microtubule polymerization. Results We identified here LIS1 as a Tat-interacting protein during extensive biochemical fractionation of T-cell extracts. We found several proteins to co-purify with a Tat-associated RNAPII CTD kinase activity including LIS1, CDK7, cyclin H, and MAT1. Tat interacted with LIS1 but not with CDK7, cyclin H or MAT1 in vitro. LIS1 also co-immunoprecipitated with Tat expressed in HeLa cells. Further, LIS1 interacted with Tat in a yeast two-hybrid system. Conclusion Our results indicate that Tat interacts with LIS1 in vitro and in vivo and that this interaction might contribute to the effect of Tat on microtubule formation.

  19. Identifying potential survival strategies of HIV-1 through virus-host protein interaction networks

    NARCIS (Netherlands)

    van Dijk, David; Ertaylan, Gokhan; Boucher, Charles Ab; Sloot, Peter Ma

    2010-01-01

    BACKGROUND: The National Institute of Allergy and Infectious Diseases has launched the HIV-1 Human Protein Interaction Database in an effort to catalogue all published interactions between HIV-1 and human proteins. In order to systematically investigate these interactions functionally and dynamicall

  20. IFITM Proteins Restrict HIV-1 Infection by Antagonizing the Envelope Glycoprotein

    Directory of Open Access Journals (Sweden)

    Jingyou Yu

    2015-10-01

    Full Text Available The interferon-induced transmembrane (IFITM proteins have been recently shown to restrict HIV-1 and other viruses. Here, we provide evidence that IFITM proteins, particularly IFITM2 and IFITM3, specifically antagonize the HIV-1 envelope glycoprotein (Env, thereby inhibiting viral infection. IFITM proteins interact with HIV-1 Env in viral producer cells, leading to impaired Env processing and virion incorporation. Notably, the level of IFITM incorporation into HIV-1 virions does not strictly correlate with the extent of inhibition. Prolonged passage of HIV-1 in IFITM-expressing T lymphocytes leads to emergence of Env mutants that overcome IFITM restriction. The ability of IFITMs to inhibit cell-to-cell infection can be extended to HIV-1 primary isolates, HIV-2 and SIVs; however, the extent of inhibition appears to be virus-strain dependent. Overall, our study uncovers a mechanism by which IFITM proteins specifically antagonize HIV-1 Env to restrict HIV-1 infection and provides insight into the specialized role of IFITMs in HIV infection.

  1. Interaction between Tat and Drugs of Abuse during HIV-1 Infection and Central Nervous System Disease

    Directory of Open Access Journals (Sweden)

    Monique E Maubert

    2016-01-01

    Full Text Available In many individuals, drug abuse is intimately linked with HIV-1 infection. In addition to being associated with one-third of all HIV-1 infections in the United States, drug abuse also plays a role in disease progression and severity in HIV-1-infected patients, including adverse effects on the central nervous system (CNS. Specific systems within the brain are known to be damaged in HIV-1-infected individuals and this damage is similar to that observed in drug abuse. Even in the era of anti-retroviral therapy (ART, CNS pathogenesis occurs with HIV-1 infection, with a broad range of cognitive impairment observed, collectively referred to as HIV-1-associated neurocognitive disorders (HAND. A number of HIV-1 proteins (Tat, gp120, Nef, Vpr have been implicated in the etiology of pathogenesis and disease as a result of the biologic activity of the extracellular form of each of the proteins in a number of tissues, including the CNS, even in ART-suppressed patients. In this review, we have made Tat the center of attention for a number of reasons. First, it has been shown to be synthesized and secreted by HIV-1-infected cells in the CNS, despite the most effective suppression therapies available to date. Second, Tat has been shown to alter the functions of several host factors, disrupting the molecular and biochemical balance of numerous pathways contributing to cellular toxicity, dysfunction, and death. In addition, the advantages and disadvantages of ART suppression with regard to controlling the genesis and progression of neurocognitive impairment are currently under debate in the field and are yet to be fully determined. In this review, we discuss the individual and concerted contributions of HIV-1 Tat, drug abuse, and ART with respect to damage in the CNS, and how these factors contribute to the development of HAND in HIV-1-infected patients.

  2. Identification of host proteins associated with HIV-1 preintegration complexes isolated from infected CD4+ cells.

    Science.gov (United States)

    Raghavendra, Nidhanapati K; Shkriabai, Nikolozi; Graham, Robert Lj; Hess, Sonja; Kvaratskhelia, Mamuka; Wu, Li

    2010-08-11

    An integrated HIV-1 genomic DNA leads to an infected cell becoming either an active or a latent virus-producing cell. Upon appropriate activation, a latently infected cell can result in production of progeny viruses that spread the infection to uninfected cells. The host proteins influence several steps of HIV-1 infection including formation of the preintegration complex (PIC), a key nucleoprotein intermediate essential for integration of reverse transcribed viral DNA into the chromosome. Much effort has gone into the identification of host proteins contributing to the assembly of functional PICs. Experimental approaches included the use of yeast two-hybrid system, co-immunoprecipitation, affinity tagged HIV-1 viral proteins and in vitro reconstitution of salt-stripped PIC activity. Several host proteins identified using these approaches have been shown to affect HIV-1 replication in cells and influence catalytic activities of recombinant IN in vitro. However, the comprehensive identification and characterization of host proteins associated with HIV-1 PICs of infected cells have been hindered in part by the technical limitation in acquiring sufficient amount of catalytically active PICs. To efficiently identify additional host factors associated with PICs in infected cells, we have developed the following novel approach. The catalytically active PICs from HIV-1-infected CD4+ cells were isolated using biotinylated target DNA, and the proteins selectively co-purifying with PICs have been analyzed by mass spectrometry. This technology enabled us to reveal at least 19 host proteins that are associated with HIV-1 PICs, of which 18 proteins have not been described previously with respect to HIV-1 integration. Physiological functions of the identified proteins range from chromatin organization to protein transport. A detailed characterization of these host proteins could provide new insights into the mechanism of HIV-1 integration and uncover new antiviral targets to

  3. Proteomic Analysis of Saliva in HIV-positive Heroin Addicts Reveals Proteins Correlated with Cognition

    Energy Technology Data Exchange (ETDEWEB)

    Dominy, Stephen; Brown, Joseph N.; Ryder, Mark I.; Gritsenko, Marina A.; Jacobs, Jon M.; Smith, Richard D.

    2014-04-01

    The prevalence of HIV-associated neurocognitive disorders (HAND) remains high despite effective antiretroviral therapies. Multiple etiologies have been proposed over the last few years to account for this phenomenon, including the neurotoxic effects of antiretrovirals and co-morbid substance abuse. However, no underlying molecular mechanism has been identified. Emerging evidence in several fields has linked the gut to brain diseases, but the effect of the gut on the brain during HIV infection has not been explored. Saliva is the most accessible gut biofluid, and is therefore of great scientific interest for diagnostic and prognostic purposes. This study presents a longitudinal, liquid chromatography-mass spectrometry-based quantitative proteomics study investigating saliva samples taken from 8 HIV-positive (HIV+) and 11 -negative (HIV-) heroin addicts. In the HIV+ group, 58 proteins were identified that show significant correlations with cognitive scores and that implicate disruption of protein quality control pathways by HIV. Notably, no proteins from the HIV- heroin addict cohort showed significant correlations with cognitive scores. In addition, the majority of correlated proteins have been shown to be associated with exosomes, allowing us to propose that the salivary glands and/or oral epithelium may modulate brain function during HIV infection through the release of discrete packets of proteins in the form of exosomes.

  4. HIVed, a knowledgebase for differentially expressed human genes and proteins during HIV infection, replication and latency

    Science.gov (United States)

    Li, Chen; Ramarathinam, Sri H.; Revote, Jerico; Khoury, Georges; Song, Jiangning; Purcell, Anthony W.

    2017-01-01

    Measuring the altered gene expression level and identifying differentially expressed genes/proteins during HIV infection, replication and latency is fundamental for broadening our understanding of the mechanisms of HIV infection and T-cell dysfunction. Such studies are crucial for developing effective strategies for virus eradication from the body. Inspired by the availability and enrichment of gene expression data during HIV infection, replication and latency, in this study, we proposed a novel compendium termed HIVed (HIV expression database; http://hivlatency.erc.monash.edu/) that harbours comprehensive functional annotations of proteins, whose genes have been shown to be dysregulated during HIV infection, replication and latency using different experimental designs and measurements. We manually curated a variety of third-party databases for structural and functional annotations of the protein entries in HIVed. With the goal of benefiting HIV related research, we collected a number of biological annotations for all the entries in HIVed besides their expression profile, including basic protein information, Gene Ontology terms, secondary structure, HIV-1 interaction and pathway information. We hope this comprehensive protein-centric knowledgebase can bridge the gap between the understanding of differentially expressed genes and the functions of their protein products, facilitating the generation of novel hypotheses and treatment strategies to fight against the HIV pandemic. PMID:28358052

  5. Characterization of the anti-HIV effects of native lactoferrin and other milk proteins and protein-derived peptides

    NARCIS (Netherlands)

    Berkhout, B; van Wamel, JLB; Beljaars, L; Meijer, DKF; Visser, Servaas; Floris, R

    2002-01-01

    In a search for natural proteins with anti-HIV activity, we screened a large set of purified proteins from bovine milk and peptide fragments thereof. Because several charged proteins and peptides are known to inhibit the process of virus entry, we selected proteins with an unusual charge composition

  6. Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

    Science.gov (United States)

    Barros, Marilia; Nanda, Hirsh

    2016-01-01

    ABSTRACT By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane

  7. Immunogenic compositions comprising human immunodeficiency virus (HIV) mosaic Nef proteins

    Science.gov (United States)

    Korber, Bette T [Los Alamos, NM; Perkins, Simon [Los Alamos, NM; Bhattacharya, Tanmoy [Los Alamos, NM; Fischer, William M [Los Alamos, NM; Theiler, James [Los Alamos, NM; Letvin, Norman [Boston, MA; Haynes, Barton F [Durham, NC; Hahn, Beatrice H [Birmingham, AL; Yusim, Karina [Los Alamos, NM; Kuiken, Carla [Los Alamos, NM

    2012-02-21

    The present invention relates to mosaic clade M HIV-1 Nef polypeptides and to compositions comprising same. The polypeptides of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

  8. HIV-1 envelope trimer fusion proteins and their applications

    NARCIS (Netherlands)

    Sliepen, K.H.E.W.J.

    2016-01-01

    HIV-1 is a major threat to global health and a vaccine is not yet on the horizon. A successful HIV-1 vaccine should probably induce HIV-1 neutralizing antibodies that target the envelope glycoprotein (Env) trimer on the outside of the virion. A possible starting point for such a vaccine are soluble

  9. hiv and serum protein electrophoresis patterns in kwazulu

    African Journals Online (AJOL)

    2011-04-01

    Apr 1, 2011 ... in the effects of HIV on immunoglobulin production.1 The prevalence of monoclonal ... link between multiple myeloma and HIV infection.3,4,6. However, the ... antigens of HIV, viruses such as hepatitis C, or other opportunistic ...

  10. Inhibition of HIV-1 replication by small interfering RNAs directed against Glioma Pathogenesis Related Protein (GliPR expression

    Directory of Open Access Journals (Sweden)

    Ottmann Oliver G

    2010-03-01

    Full Text Available Abstract Background Previously, we showed that glioma pathogenesis related protein (GliPR is induced in CEM T cells upon HIV-1 infection in vitro. To examine whether GliPR plays a role as HIV dependency factor (HDF, we tested the effect of GliPR suppression by siRNA on HIV-1 replication. Results Induction of GliPR expression by HIV-1 was confirmed in P4-CCR5 cells. When GliPR was suppressed by siRNA, HIV-1 replication was significantly reduced as measured by HIV-1 transcript levels, HIV-1 p24 protein levels, and HIV-1 LTR-driven reporter gene expression, suggesting that GliPR is a cellular co-factor of HIV-1. Microarray analysis of uninfected HeLa cells following knockdown of GliPR revealed, among a multitude of gene expression alterations, a down-regulation of syndecan-1, syndecan-2, protein kinase C alpha (PRKCA, the catalytic subunit β of cAMP-dependent protein kinase (PRKACB, nuclear receptor co-activator 3 (NCOA3, and cell surface protein CD59 (protectin, all genes having relevance for HIV-1 pathology. Conclusions The up-regulation of GliPR by HIV-1 and the early significant inhibition of HIV-1 replication mediated by knockdown of GliPR reveal GliPR as an important HIV-1 dependency factor (HDF, which may be exploited for HIV-1 inhibition.

  11. Identifying potential survival strategies of HIV-1 through virus-host protein interaction networks

    Directory of Open Access Journals (Sweden)

    Boucher Charles AB

    2010-07-01

    Full Text Available Abstract Background The National Institute of Allergy and Infectious Diseases has launched the HIV-1 Human Protein Interaction Database in an effort to catalogue all published interactions between HIV-1 and human proteins. In order to systematically investigate these interactions functionally and dynamically, we have constructed an HIV-1 human protein interaction network. This network was analyzed for important proteins and processes that are specific for the HIV life-cycle. In order to expose viral strategies, network motif analysis was carried out showing reoccurring patterns in virus-host dynamics. Results Our analyses show that human proteins interacting with HIV form a densely connected and central sub-network within the total human protein interaction network. The evaluation of this sub-network for connectivity and centrality resulted in a set of proteins essential for the HIV life-cycle. Remarkably, we were able to associate proteins involved in RNA polymerase II transcription with hubs and proteasome formation with bottlenecks. Inferred network motifs show significant over-representation of positive and negative feedback patterns between virus and host. Strikingly, such patterns have never been reported in combined virus-host systems. Conclusions HIV infection results in a reprioritization of cellular processes reflected by an increase in the relative importance of transcriptional machinery and proteasome formation. We conclude that during the evolution of HIV, some patterns of interaction have been selected for resulting in a system where virus proteins preferably interact with central human proteins for direct control and with proteasomal proteins for indirect control over the cellular processes. Finally, the patterns described by network motifs illustrate how virus and host interact with one another.

  12. Total protein, albumin and low-molecular-weight protein excretion in HIV-positive patients

    Directory of Open Access Journals (Sweden)

    Campbell Lucy J

    2012-08-01

    Full Text Available Abstract Background Chronic kidney disease is common in HIV positive patients and renal tubular dysfunction has been reported in those receiving combination antiretroviral therapy (cART. Tenofovir (TFV in particular has been linked to severe renal tubular disease as well as proximal tubular dysfunction. Markedly elevated urinary concentrations of retinal-binding protein (RBP have been reported in patients with severe renal tubular disease, and low-molecular-weight proteins (LMWP such as RBP may be useful in clinical practice to assess renal tubular function in patients receiving TFV. We analysed 3 LMWP as well as protein and albumin in the urine of a sample of HIV positive patients. Methods In a cross-sectional fashion, total protein, albumin, RBP, cystatin C, and neutrophil gelatinase-associated lipocalin (NGAL were quantified in random urine samples of 317 HIV positive outpatients and expressed as the ratio-to-creatinine (RBPCR, CCR and NGALCR. Exposure to cART was categorised as none, cART without TFV, and cART containing TFV and a non-nucleoside reverse-transcriptase-inhibitor (TFV/NNRTI or TFV and a protease-inhibitor (TFV/PI. Results Proteinuria was present in 10.4 % and microalbuminuria in 16.7 % of patients. Albumin accounted for approximately 10 % of total urinary protein. RBPCR was within the reference range in 95 % of patients while NGALCR was elevated in 67 % of patients. No overall differences in urine protein, albumin, and LMWP levels were observed among patients stratified by cART exposure, although a greater proportion of patients exposed to TFV/PI had RBPCR >38.8 μg/mmol (343 μg/g (p = 0.003. In multivariate analyses, black ethnicity (OR 0.43, 95 % CI 0.24, 0.77 and eGFR 2 (OR 3.54, 95 % CI 1.61, 7.80 were independently associated with upper quartile (UQ RBPCR. RBPCR correlated well to CCR (r2 = 0.71, but not to NGALCR, PCR or ACR. Conclusions In HIV positive patients, proteinuria was predominantly of

  13. Transmembrane protein aptamers that inhibit CCR5 expression and HIV coreceptor function.

    Science.gov (United States)

    Scheideman, Elizabeth H; Marlatt, Sara A; Xie, Yanhua; Hu, Yani; Sutton, Richard E; DiMaio, Daniel

    2012-10-01

    We have exploited the ability of transmembrane domains to engage in highly specific protein-protein interactions to construct a new class of small proteins that inhibit HIV infection. By screening a library encoding hundreds of thousands of artificial transmembrane proteins with randomized transmembrane domains (termed "traptamers," for transmembrane aptamers), we isolated six 44- or 45-amino-acid proteins with completely different transmembrane sequences that inhibited cell surface and total expression of the HIV coreceptor CCR5. The traptamers inhibited transduction of human T cells by HIV reporter viruses pseudotyped with R5-tropic gp120 envelope proteins but had minimal effects on reporter viruses with X4-tropic gp120. Optimization of two traptamers significantly increased their activity and resulted in greater than 95% inhibition of R5-tropic reporter virus transduction without inhibiting expression of CD4, the primary HIV receptor, or CXCR4, another HIV coreceptor. In addition, traptamers inhibited transduction mediated by a mutant R5-tropic gp120 protein resistant to maraviroc, a small-molecule CCR5 inhibitor, and they dramatically inhibited replication of an R5-tropic laboratory strain of HIV in a multicycle infection assay. Genetic experiments suggested that the active traptamers specifically interacted with the transmembrane domains of CCR5 and that some of the traptamers interacted with different portions of CCR5. Thus, we have constructed multiple proteins not found in nature that interfere with CCR5 expression and inhibit HIV infection. These proteins may be valuable tools to probe the organization of the transmembrane domains of CCR5 and their relationship to its biological activities, and they may serve as starting points to develop new strategies to inhibit HIV infection.

  14. Stem-loop binding protein is a multifaceted cellular regulator of HIV-1 replication

    Science.gov (United States)

    Tucker, Lynne D.; Asara, John M.; Cheruiyot, Collins K.; Lu, Huafei; Wu, Zhijin J.; Newstein, Michael C.; Dooner, Mark S.; Friedman, Jennifer; Lally, Michelle A.; Ramratnam, Bharat

    2016-01-01

    A rare subset of HIV-1–infected individuals is able to maintain plasma viral load (VL) at low levels without antiretroviral treatment. Identifying the mechanisms underlying this atypical response to infection may lead to therapeutic advances for treating HIV-1. Here, we developed a proteomic analysis to compare peripheral blood cell proteomes in 20 HIV-1–infected individuals who maintained either high or low VL with the aim of identifying host factors that impact HIV-1 replication. We determined that the levels of multiple histone proteins were markedly decreased in cohorts of individuals with high VL. This reduction was correlated with lower levels of stem-loop binding protein (SLBP), which is known to control histone metabolism. Depletion of cellular SLBP increased promoter engagement with the chromatin structures of the host gene high mobility group protein A1 (HMGA1) and viral long terminal repeat (LTR), which led to higher levels of HIV-1 genomic integration and proviral transcription. Further, we determined that TNF-α regulates expression of SLBP and observed that plasma TNF-α levels in HIV-1–infected individuals correlated directly with VL levels and inversely with cellular SLBP levels. Our findings identify SLBP as a potentially important cellular regulator of HIV-1, thereby establishing a link between histone metabolism, inflammation, and HIV-1 infection. PMID:27454292

  15. HIV and schistosomiasis in rural Zimbabwe: the association of Retinol-binding protein with disease progression, inflammation and mortality

    OpenAIRE

    Sebastian Ranzi Kotzé; Rutendo Zinyama-Gutsire; Per Kallestrup; Christine Stabell Benn; Exnevia Gomo; Jan Gerstoft; Govert van Dam; Ole Hartvig Mortensen; Henrik Ullum; Christian Erikstrup

    2015-01-01

    Background: Vitamin A has widespread effects on immune function and is therefore interesting in HIV-infection. Retinol-binding protein (RBP or RBP4) is a negative acute-phase protein and a marker of vitamin A status. Our aim was to investigate the association of RBP with HIV progression, infection with schistosomiasis, inflammatory cytokines, and mortality. Methods: The study included 192 HIV-infected and 177 HIV-uninfected individuals from Mupfure in rural Zimbabwe. Of these, 208 were inf...

  16. RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

    Energy Technology Data Exchange (ETDEWEB)

    López, Claudia S., E-mail: lopezcl@ohsu.edu [Departments of Molecular Microbiology and Immunology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97239 (United States); Sloan, Rachel; Cylinder, Isabel [Departments of Molecular Microbiology and Immunology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97239 (United States); Kozak, Susan L.; Kabat, David [Biochemistry and Molecular Biology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97239 (United States); Barklis, Eric, E-mail: barklis@ohsu.edu [Departments of Molecular Microbiology and Immunology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97239 (United States)

    2014-08-15

    The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export.

  17. Nonhuman Primate IFITM Proteins Are Potent Inhibitors of HIV and SIV.

    Directory of Open Access Journals (Sweden)

    Jordan Wilkins

    Full Text Available Interferon-induced transmembrane (IFITM proteins are potent antiviral factors shown to restrict the infection of many enveloped viruses, including HIV. Here we report cloning and characterization of a panel of nonhuman primate IFITMs. We show that, similar to human IFITM, nonhuman primate IFITM proteins inhibit HIV and other primate lentiviruses. While some nonhuman primate IFITM proteins are more potent than human counterparts to inhibit HIV-1, they are generally not effective against HIV-2 similar to that of human IFITMs. Notably, depending on SIV strains and also IFITM species tested, nonhuman primate IFITM proteins exhibit distinct activities against SIVs; no correlation was found to support the notion that IFITM proteins are most active in non-natural primate hosts. Consistent with our recent findings for human IFITMs, nonhuman primate IFITM proteins interact with HIV-1 Env and strongly act in viral producer cells to impair viral infectivity and block cell-to-cell transmission. Accordingly, knockdown of primate IFITM3 increases HIV-1 replication in nohuman primate cells. Interestingly, analysis of DNA sequences of human and nonhuman primate IFITMs suggest that IFITM proteins have been undergoing purifying selection, rather than positive selection typical for cellular restriction factors. Overall, our study reveals some new and unexpected features of IFITMs in restricting primate lentiviruses, which enhances our understanding of virus-host interaction and AIDS pathogenesis.

  18. Construction of Recombinant Fowlpox Virus (rFpv) Expressing HIV-1 Gag-pol Protein

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The induction of human immunodeficiency virus(HIV)-specific T-cell response is generally considered as critical to the development of effective immunity to HIV type 1 ( HIV-1 ). Recombinant Avipoxvirus vectors are used widely for vaccination against HIV-1, where the induction of a cytotoxic CD8 + T-cell(CTL) response seems to be an important component of protective immunity. A recombinant fowlpox virus(rFPV/Gag-pol) expressing the Gag-pol protein of HIV was constructed and characterized. The specific expression protein in CEF cells infected by recombinant fowlpox and the specific antibody in the sera of mice immunized with rFPV were analyzed via Westeru-blot.

  19. Effect of HIV-1-related protein expression on cardiac and skeletal muscles from transgenic rats

    Directory of Open Access Journals (Sweden)

    Guidot David M

    2008-04-01

    Full Text Available Abstract Background Human immunodeficiency virus type 1 (HIV-1 infection and the consequent acquired immunodeficiency syndrome (AIDS has protean manifestations, including muscle wasting and cardiomyopathy, which contribute to its high morbidity. The pathogenesis of these myopathies remains partially understood, and may include nutritional deficiencies, biochemical abnormalities, inflammation, and other mechanisms due to viral infection and replication. Growing evidence has suggested that HIV-1-related proteins expressed by the host in response to viral infection, including Tat and gp120, may also be involved in the pathophysiology of AIDS, particularly in cells or tissues that are not directly infected with HIV-1. To explore the potentially independent effects of HIV-1-related proteins on heart and skeletal muscles, we used a transgenic rat model that expresses several HIV-1-related proteins (e.g., Tat, gp120, and Nef. Outcome measures included basic heart and skeletal muscle morphology, glutathione metabolism and oxidative stress, and gene expressions of atrogin-1, muscle ring finger protein-1 (MuRF-1 and Transforming Growth Factor-β1 (TGFβ1, three factors associated with muscle catabolism. Results Consistent with HIV-1 associated myopathies in humans, HIV-1 transgenic rats had increased relative heart masses, decreased relative masses of soleus, plantaris and gastrocnemius muscles, and decreased total and myosin heavy chain type-specific plantaris muscle fiber areas. In both tissues, the levels of cystine (Cyss, the oxidized form of the anti-oxidant cysteine (Cys, and Cyss:Cys ratios were significantly elevated, and cardiac tissue from HIV-1 transgenic rats had altered glutathione metabolism, all reflective of significant oxidative stress. In HIV-1 transgenic rat hearts, MuRF-1 gene expression was increased. Further, HIV-1-related protein expression also increased atrogin-1 (~14- and ~3-fold and TGFβ1 (~5-fold and ~3-fold in heart and

  20. Detection of human immunodeficiency virus type 1 (HIV-1) Tat protein by aptamer-based biosensors

    Science.gov (United States)

    Hashim, Uda; Fatin, M. F.; Ruslinda, A. R.; Gopinath, Subash C. B.; Uda, M. N. A.

    2017-03-01

    A study was conducted to detect the human immunodeficiency virus (HIV-1) Tat protein using interdigitated electrodes. The measurements and images of the IDEs' finger gaps and the images of chitosan-carbon nanotubes deposited on top of the interdigitated electrodes were taken using the Scanning Electron Microscope. The detection of HIV-1 Tat protein was done using split aptamers and aptamer tail. Biosensors were chosen as diagnostic equipment due to their rapid diagnostic capabilities.

  1. Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures.

    Science.gov (United States)

    Rao, Vasudev R; Eugenin, Eliseo A; Prasad, Vinayaka R

    2016-01-01

    Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood-brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes.

  2. Optimizing HIV-1 protease production in Escherichia coli as fusion protein

    Directory of Open Access Journals (Sweden)

    Piubelli Luciano

    2011-06-01

    Full Text Available Abstract Background Human immunodeficiency virus (HIV is the etiological agent in AIDS and related diseases. The aspartyl protease encoded by the 5' portion of the pol gene is responsible for proteolytic processing of the gag-pol polyprotein precursor to yield the mature capsid protein and the reverse transcriptase and integrase enzymes. The HIV protease (HIV-1Pr is considered an attractive target for designing inhibitors which could be used to tackle AIDS and therefore it is still the object of a number of investigations. Results A recombinant human immunodeficiency virus type 1 protease (HIV-1Pr was overexpressed in Escherichia coli cells as a fusion protein with bacterial periplasmic protein dithiol oxidase (DsbA or glutathione S-transferase (GST, also containing a six-histidine tag sequence. Protein expression was optimized by designing a suitable HIV-1Pr cDNA (for E. coli expression and to avoid autoproteolysis and by screening six different E. coli strains and five growth media. The best expression yields were achieved in E. coli BL21-Codon Plus(DE3-RIL host and in TB or M9 medium to which 1% (w/v glucose was added to minimize basal expression. Among the different parameters assayed, the presence of a buffer system (based on phosphate salts and a growth temperature of 37°C after adding IPTG played the main role in enhancing protease expression (up to 10 mg of chimeric DsbA:HIV-1Pr/L fermentation broth. GST:HIVPr was in part (50% produced as soluble protein while the overexpressed DsbA:HIV-1Pr chimeric protein largely accumulated in inclusion bodies as unprocessed fusion protein. A simple refolding procedure was developed on HiTrap Chelating column that yielded a refolded DsbA:HIV-1Pr with a > 80% recovery. Finally, enterokinase digestion of resolubilized DsbA:HIV-1Pr gave more than 2 mg of HIV-1Pr per liter of fermentation broth with a purity ≤ 80%, while PreScission protease cleavage of soluble GST:HIVPr yielded ~ 0.15 mg of pure HIV-1

  3. Shutdown of HIV-1 Transcription in T Cells by Nullbasic, a Mutant Tat Protein

    Directory of Open Access Journals (Sweden)

    Hongping Jin

    2016-07-01

    Full Text Available Nullbasic is a derivative of the HIV-1 transactivator of transcription (Tat protein that strongly inhibits HIV-1 replication in lymphocytes. Here we show that lentiviral vectors that constitutively express a Nullbasic-ZsGreen1 (NB-ZSG1 fusion protein by the eEF1α promoter led to robust long-term inhibition of HIV-1 replication in Jurkat cells. Although Jurkat-NB-ZSG1 cells were infected by HIV-1, no virus production could be detected and addition of phorbol ester 12-myristate 13-acetate (PMA and JQ1 had no effect, while suberanilohydroxamic acid (SAHA modestly stimulated virus production but at levels 300-fold lower than those seen in HIV-1-infected Jurkat-ZSG1 cells. Virus replication was not recovered by coculture of HIV-1-infected Jurkat-NB-ZSG1 cells with uninfected Jurkat cells. Latently infected Jurkat latent 6.3 and ACH2 cells treated with latency-reversing agents produced measurable viral capsid (CA, but little or none was made when they expressed NB-ZSG1. When Jurkat cells chronically infected with HIV-1 were transduced with lentiviral virus-like particles conveying NB-ZSG1, a >3-log reduction in CA production was observed. Addition of PMA increased virus CA production but at levels 500-fold lower than those seen in nontransduced Jurkat cells. Transcriptome sequencing analysis confirmed that HIV-1 mRNA was strongly inhibited by NB-ZSG1 but indicated that full-length viral mRNA was made. Analysis of HIV-1-infected Jurkat cells expressing NB-ZSG1 by chromatin immunoprecipitation assays indicated that recruitment of RNA polymerase II (RNAPII and histone 3 lysine 9 acetylation were inhibited. The reduction of HIV-1 promoter-associated RNAPII and epigenetic changes in viral nucleosomes indicate that Nullbasic can inhibit HIV-1 replication by enforcing viral silencing in cells.

  4. HIV-1 nucleocapsid protein localizes efficiently to the nucleus and nucleolus

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Kyung Lee; Lee, Sun Hee; Lee, Eun Soo; You, Ji Chang, E-mail: jiyou@catholic.ac.kr

    2016-05-15

    The HIV-1 nucleocapsid (NC) is an essential viral protein containing two highly conserved retroviral-type zinc finger (ZF) motifs, which functions in multiple stages of the HIV-1 life cycle. Although a number of functions for NC either in its mature form or as a domain of Gag have been revealed, little is known about the intracellular localization of NC and, moreover, its role in Gag protein trafficking. Here, we have investigated various forms of HIV-1 NC protein for its cellular localization and found that the NC has a strong nuclear and nucleolar localization activity. The linker region, composed of a stretch of basic amino acids between the two ZF motifs, was necessary and sufficient for the activity. - Highlights: • HIV-1 NC possess a NLS and leads to nuclear and nucleolus localization. • Mutations in basic residues between two ZFs in NC decrease the nucleus localization. • ZFs of NC affect cytoplasmic organelles localization rather than nucleus localization.

  5. Dual Mechanisms of Translation Initiation of the Full-Length HIV-1 mRNA Contribute to Gag Synthesis

    Science.gov (United States)

    Rivero, Matias; Cohen, Éric A.; Lopez-Lastra, Marcelo; Mouland, Andrew J.

    2013-01-01

    The precursor group-specific antigen (pr55Gag) is central to HIV-1 assembly. Its expression alone is sufficient to assemble into virus-like particles. It also selects the genomic RNA for encapsidation and is involved in several important virus-host interactions for viral assembly and restriction, making its synthesis essential for aspects of viral replication. Here, we show that the initiation of translation of the HIV-1 genomic RNA is mediated through both a cap-dependent and an internal ribosome entry site (IRES)-mediated mechanisms. In support of this notion, pr55Gag synthesis was maintained at 70% when cap-dependent translation initiation was blocked by the expression of eIF4G- and PABP targeting viral proteases in two in vitro systems and in HIV-1-expressing cells directly infected with poliovirus. While our data reveal that IRES-dependent translation of the viral genomic RNA ensures pr55Gag expression, the synthesis of other HIV-1 proteins, including that of pr160Gag/Pol, Vpr and Tat is suppressed early during progressive poliovirus infection. The data presented herein implies that the unspliced HIV-1 genomic RNA utilizes both cap-dependent and IRES-dependent translation initiation to supply pr55Gag for virus assembly and production. PMID:23861855

  6. Dual mechanisms of translation initiation of the full-length HIV-1 mRNA contribute to gag synthesis.

    Directory of Open Access Journals (Sweden)

    Anne Monette

    Full Text Available The precursor group-specific antigen (pr55(Gag is central to HIV-1 assembly. Its expression alone is sufficient to assemble into virus-like particles. It also selects the genomic RNA for encapsidation and is involved in several important virus-host interactions for viral assembly and restriction, making its synthesis essential for aspects of viral replication. Here, we show that the initiation of translation of the HIV-1 genomic RNA is mediated through both a cap-dependent and an internal ribosome entry site (IRES-mediated mechanisms. In support of this notion, pr55(Gag synthesis was maintained at 70% when cap-dependent translation initiation was blocked by the expression of eIF4G- and PABP targeting viral proteases in two in vitro systems and in HIV-1-expressing cells directly infected with poliovirus. While our data reveal that IRES-dependent translation of the viral genomic RNA ensures pr55(Gag expression, the synthesis of other HIV-1 proteins, including that of pr160(Gag/Pol, Vpr and Tat is suppressed early during progressive poliovirus infection. The data presented herein implies that the unspliced HIV-1 genomic RNA utilizes both cap-dependent and IRES-dependent translation initiation to supply pr55(Gag for virus assembly and production.

  7. South African mutations of the CCR5 coreceptor for HIV modify interaction with chemokines and HIV Envelope protein.

    Science.gov (United States)

    Folefoc, Asongna T; Fromme, Bernhard J; Katz, Arieh A; Flanagan, Colleen A

    2010-08-01

    The CCR5 chemokine receptor is the major coreceptor for HIV-1 and the receptor for CC-chemokines, MIP-1alpha, MIP-1beta, and regulated upon activation normal T-cell-expressed and secreted. Individuals, who are homozygous for the nonfunctional CCR5Delta32 allele, are largely resistant to HIV-1 infection. Four unique mutations that affect the amino acid sequence of CCR5 have been identified in South Africa. We have assessed the effect of these mutations on CCR5 interactions with chemokines and HIV Envelope protein. The LeuPhe mutation did not affect CCR5 expression, chemokine binding, intracellular signaling, or interaction with Envelope. The ArgGln mutant was similar to wild-type CCR5, but ligand-independent intracellular signaling suggests that it is partially constitutively active. The AspVal mutation decreased chemokine-binding affinity, chemokine-stimulated intracellular signaling, and receptor expression. It also decreased HIV Envelope-mediated cell fusion. The ArgStop mutant showed no measurable chemokine binding or signaling and no measurable expression of CCR5 at the cell surface or within the cell. Consistent with lack of cell surface expression, it did not support envelope-mediated cell fusion. These results show that South African CCR5 variants have a range of phenotypes in vitro that may reflect altered chemokine responses and susceptibility to HIV infection in individuals who carry these alleles.

  8. Boosting of HIV-1 neutralizing antibody responses by a distally related retroviral envelope protein.

    Science.gov (United States)

    Uchtenhagen, Hannes; Schiffner, Torben; Bowles, Emma; Heyndrickx, Leo; LaBranche, Celia; Applequist, Steven E; Jansson, Marianne; De Silva, Thushan; Back, Jaap Willem; Achour, Adnane; Scarlatti, Gabriella; Fomsgaard, Anders; Montefiori, David; Stewart-Jones, Guillaume; Spetz, Anna-Lena

    2014-06-15

    Our knowledge of the binding sites for neutralizing Abs (NAb) that recognize a broad range of HIV-1 strains (bNAb) has substantially increased in recent years. However, gaps remain in our understanding of how to focus B cell responses to vulnerable conserved sites within the HIV-1 envelope glycoprotein (Env). In this article, we report an immunization strategy composed of a trivalent HIV-1 (clade B envs) DNA prime, followed by a SIVmac239 gp140 Env protein boost that aimed to focus the immune response to structurally conserved parts of the HIV-1 and simian immunodeficiency virus (SIV) Envs. Heterologous NAb titers, primarily to tier 1 HIV-1 isolates, elicited during the trivalent HIV-1 env prime, were significantly increased by the SIVmac239 gp140 protein boost in rabbits. Epitope mapping of Ab-binding reactivity revealed preferential recognition of the C1, C2, V2, V3, and V5 regions. These results provide a proof of concept that a distally related retroviral SIV Env protein boost can increase pre-existing NAb responses against HIV-1.

  9. Human Milk Galectin-3 Binding Protein and Breastfeeding-Associated HIV Transmission

    Science.gov (United States)

    Chan, Christina S.; Kim, Hae-Young; Autran, Chloe; Kim, Jae H.; Sinkala, Moses; Kankasa, Chipepo; Mwiya, Mwiya; Thea, Donald M.; Aldrovandi, Grace M.; Kuhn, Louise; Bode, Lars

    2013-01-01

    Analysis of milk from 247 HIV-infected Zambian mothers showed that Galectin-3 Binding Protein (Gal3BP) concentrations were significantly higher among HIV-infected mothers who transmitted HIV through breastfeeding (6.51±2.12 ug/mL) than among non-transmitters but were also correlated with higher milk and plasma HIV RNA copies/ml and lower CD4+ cell counts. The association between Gal3BP and postnatal transmission was attenuated after adjustment for milk and plasma HIV load and CD4+ cell counts. This suggests that although milk Gal3BP is a marker of advanced maternal disease, it does not independently modify transmission risk. PMID:23899964

  10. The level of DING proteins is increased in HIV-infected patients: in vitro and in vivo studies.

    Directory of Open Access Journals (Sweden)

    Ahmed Djeghader

    Full Text Available DING proteins constitute an interesting family, owing to their intriguing and important activities. However, after a decade of research, little is known about these proteins. In humans, at least five different DING proteins have been identified, which were implicated in important biological processes and diseases, including HIV. Indeed, recent data from different research groups have highlighted the anti-HIV activity of some DING representatives. These proteins share the ability to inhibit the transcriptional step of HIV-1, a key step of the viral cycle that is not yet targeted by the current therapies. Since such proteins have been isolated from humans, we undertook a comprehensive study that focuses on the relationship between these proteins and HIV-infection in an infectious context. Hence, we developed a home-made ELISA for the quantification of the concentration of DING proteins in human serum. Using this method, we were able to determine the concentration of DING proteins in healthy and HIV-infected patients. Interestingly, we observed a significant increase of the concentration of DING proteins in non treated and treated HIV-infected patients compared to controls. In addition, cell cultures infected with HIV also show an increased expression of DING proteins, ruling out the possible role of antiretroviral treatment in the increase of the expression of DING proteins. In conclusion, results from this study show that the organism reacts to HIV-infection by an overexpression of DING proteins.

  11. Super-resolution imaging of ESCRT-proteins at HIV-1 assembly sites.

    Directory of Open Access Journals (Sweden)

    Jens Prescher

    2015-02-01

    Full Text Available The cellular endosomal sorting complex required for transport (ESCRT machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane associated lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be observed, the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resolution fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%. All colocalizing ESCRT clusters exhibited closed, circular structures with an average size (full-width at half-maximum between 45 and 60 nm or a diameter (determined using a Ripley's L-function analysis of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices

  12. Solution Properties of Murine Leukemia Virus Gag Protein: Differences from HIV-1 Gag

    Energy Technology Data Exchange (ETDEWEB)

    Datta, Siddhartha A.K.; Zuo, Xiaobing; Clark, Patrick K.; Campbell, Stephen J.; Wang, Yun-Xing; Rein, Alan (SAIC); (NCI)

    2012-05-09

    Immature retrovirus particles are assembled from the multidomain Gag protein. In these particles, the Gag proteins are arranged radially as elongated rods. We have previously characterized the properties of HIV-1 Gag in solution. In the absence of nucleic acid, HIV-1 Gag displays moderately weak interprotein interactions, existing in monomer-dimer equilibrium. Neutron scattering and hydrodynamic studies suggest that the protein is compact, and biochemical studies indicate that the two ends can approach close in three-dimensional space, implying the need for a significant conformational change during assembly. We now describe the properties of the Gag protein of Moloney murine leukemia virus (MLV), a gammaretrovirus. We found that this protein is very different from HIV-1 Gag: it has much weaker protein-protein interaction and is predominantly monomeric in solution. This has allowed us to study the protein by small-angle X-ray scattering and to build a low-resolution molecular envelope for the protein. We found that MLV Gag is extended in solution, with an axial ratio of {approx}7, comparable to its dimensions in immature particles. Mutational analysis suggests that runs of prolines in its matrix and p12 domains and the highly charged stretch at the C terminus of its capsid domain all contribute to this extended conformation. These differences between MLV Gag and HIV-1 Gag and their implications for retroviral assembly are discussed.

  13. Effects of opiates and HIV proteins on neurons: the role of ferritin heavy chain and a potential for synergism.

    Science.gov (United States)

    Festa, Lindsay; Meucci, Olimpia

    2012-07-01

    Human immunodeficiency virus 1 (HIV-1) and its associated proteins can have a profound impact on the central nervous system. Co-morbid abuse of opiates, such as morphine and heroin, is often associated with rapid disease progression and greater neurological dysfunction. The mechanisms by which HIV proteins and opiates cause neuronal damage on their own and together are unclear. The emergence of ferritin heavy chain (FHC) as a negative regulator of the chemokine receptor CXCR4, a co-receptor for HIV, may prove to be important in elucidating the interaction between HIV proteins and opiates. This review summarizes our current knowledge of central nervous system damage inflicted by HIV and opiates, as well as the regulation of CXCR4 by opiate-induced changes in FHC protein levels. We propose that HIV proteins and opiates exhibit an additive or synergistic effect on FHC/CXCR4, thereby decreasing neuronal signaling and function.

  14. HIV Blocks Interferon Induction in Human Dendritic Cells and Macrophages by Dysregulation of TBK1

    Science.gov (United States)

    Harman, Andrew N.; Nasr, Najla; Feetham, Alexandra; Galoyan, Ani; Alshehri, Abdullateef A.; Rambukwelle, Dharshini; Botting, Rachel A.; Hiener, Bonnie M.; Diefenbach, Eve; Diefenbach, Russell J.; Kim, Min; Mansell, Ashley

    2015-01-01

    ABSTRACT Dendritic cells (DCs) and macrophages are present in the tissues of the anogenital tract, where HIV-1 transmission occurs in almost all cases. These cells are both target cells for HIV-1 and represent the first opportunity for the virus to interfere with innate recognition. Previously we have shown that both cell types fail to produce type I interferons (IFNs) in response to HIV-1 but that, unlike T cells, the virus does not block IFN induction by targeting IFN regulatory factor 3 (IRF3) for cellular degradation. Thus, either HIV-1 inhibits IFN induction by an alternate mechanism or, less likely, these cells fail to sense HIV-1. Here we show that HIV-1 (but not herpes simplex virus 2 [HSV-2] or Sendai virus)-exposed DCs and macrophages fail to induce the expression of all known type I and III IFN genes. These cells do sense the virus, and pattern recognition receptor (PRR)-induced signaling pathways are triggered. The precise stage in the IFN-inducing signaling pathway that HIV-1 targets to block IFN induction was identified; phosphorylation but not K63 polyubiquitination of TANK-binding kinase 1 (TBK1) was completely inhibited. Two HIV-1 accessory proteins, Vpr and Vif, were shown to bind to TBK1, and their individual deletion partly restored IFN-β expression. Thus, the inhibition of TBK1 autophosphorylation by binding of these proteins appears to be the principal mechanism by which HIV-1 blocks type I and III IFN induction in myeloid cells. IMPORTANCE Dendritic cells (DCs) and macrophages are key HIV target cells. Therefore, definition of how HIV impairs innate immune responses to initially establish infection is essential to design preventative interventions, especially by restoring initial interferon production. Here we demonstrate how HIV-1 blocks interferon induction by inhibiting the function of a key kinase in the interferon signaling pathway, TBK1, via two different viral accessory proteins. Other viral proteins have been shown to target the

  15. Human-Phosphate-Binding-Protein inhibits HIV-1 gene transcription and replication

    Directory of Open Access Journals (Sweden)

    Candolfi Ermanno

    2011-07-01

    Full Text Available Abstract The Human Phosphate-Binding protein (HPBP is a serendipitously discovered lipoprotein that binds phosphate with high affinity. HPBP belongs to the DING protein family, involved in various biological processes like cell cycle regulation. We report that HPBP inhibits HIV-1 gene transcription and replication in T cell line, primary peripherical blood lymphocytes and primary macrophages. We show that HPBP is efficient in naïve and HIV-1 AZT-resistant strains. Our results revealed HPBP as a new and potent anti HIV molecule that inhibits transcription of the virus, which has not yet been targeted by HAART and therefore opens new strategies in the treatment of HIV infection.

  16. The HIV matrix protein p17 induces hepatic lipid accumulation via modulation of nuclear receptor transcriptoma.

    Science.gov (United States)

    Renga, Barbara; Francisci, Daniela; Carino, Adriana; Marchianò, Silvia; Cipriani, Sabrina; Chiara Monti, Maria; Del Sordo, Rachele; Schiaroli, Elisabetta; Distrutti, Eleonora; Baldelli, Franco; Fiorucci, Stefano

    2015-10-15

    Liver disease is the second most common cause of mortality in HIV-infected persons. Exactly how HIV infection per se affects liver disease progression is unknown. Here we have investigated mRNA expression of 49 nuclear hormone receptors (NRs) and 35 transcriptional coregulators in HepG2 cells upon stimulation with the HIV matrix protein p17. This viral protein regulated mRNA expression of some NRs among which LXRα and its transcriptional co-activator MED1 were highly induced at mRNA level. Dissection of p17 downstream intracellular pathway demonstrated that p17 mediated activation of Jak/STAT signaling is responsible for the promoter dependent activation of LXR. The treatment of both HepG2 as well as primary hepatocytes with HIV p17 results in the transcriptional activation of LXR target genes (SREBP1c and FAS) and lipid accumulation. These effects are lost in HepG2 cells pre-incubated with a serum from HIV positive person who underwent a vaccination with a p17 peptide as well as in HepG2 cells pre-incubated with the natural LXR antagonist gymnestrogenin. These results suggest that HIV p17 affects NRs and their related signal transduction thus contributing to the progression of liver disease in HIV infected patients.

  17. Inhibition of HIV type 1 infection with a RANTES-IgG3 fusion protein.

    Science.gov (United States)

    Challita-Eid, P M; Klimatcheva, E; Day, B T; Evans, T; Dreyer, K; Rimel, B J; Rosenblatt, J D; Planelles, V

    1998-12-20

    The natural ligands for the chemokine receptors CCR5 (RANTES, MIP-1alpha, and MIP-1beta) and CXCR4 (SDF-1) can act as potent inhibitors of infection by the human immunodeficiency virus type 1 (HIV-1) at the level of viral entry. Unlike antibody-mediated inhibition, chemokine-mediated inhibition is broadly effective. Different HIV-1 strains can utilize the same coreceptor(s) for viral entry and, therefore, can be blocked by the same chemokine(s). HIV-1 strains that are highly resistant to neutralization by V3-specific antibodies are sensitive to inhibition by chemokines. Therefore, the use of chemokine-derived molecules constitutes a potential therapeutic approach to prevent infection by HIV-1. We have generated a fusion protein between RANTES and human IgG3 (RANTES-IgG3). The effectiveness of RANTES-IgG3 inhibition of infection by HIV-1 was similar to that of rRANTES. Inhibition of HIV-1 by RANTES-IgG3 was specific for CCR5-dependent but not CXCR4-dependent HIV-1 isolates. Fusion of a chemokine to an IgG moiety offers two desirable properties with respect to the recombinant chemokine alone. First, IgG fusion proteins have extended half-lives in vivo. Second, molecules with IgG heavy chain moieties may be able to cross the placenta and potentially induce fetal protection.

  18. Nucleic acids encoding mosaic HIV-1 gag proteins

    Science.gov (United States)

    Korber, Bette T.; Perkins, Simon; Bhattacharya, Tanmoy; Fischer, William M.; Theiler, James; Letvin, Norman; Haynes, Barton F.; Hahn, Beatrice H.; Yusim, Karina; Kuiken, Carla

    2016-11-15

    The disclosure generally relates to an immunogenic composition (e.g., a vaccine) and, in particular, to a polyvalent immunogenic composition, such as a polyvalent HIV vaccine, and to methods of using same.

  19. Nucleic acids encoding mosaic HIV-1 gag proteins

    Energy Technology Data Exchange (ETDEWEB)

    Korber, Bette T.; Perkins, Simon; Bhattacharya, Tanmoy; Fischer, William M.; Theiler, James; Letvin, Norman; Haynes, Barton F.; Hahn, Beatrice H.; Yusim, Karina; Kuiken, Carla

    2016-11-15

    The disclosure generally relates to an immunogenic composition (e.g., a vaccine) and, in particular, to a polyvalent immunogenic composition, such as a polyvalent HIV vaccine, and to methods of using same.

  20. Mimicking protein-protein interactions through peptide-peptide interactions: HIV-1 gp120 and CXCR4

    Directory of Open Access Journals (Sweden)

    Andrea eGross

    2013-09-01

    Full Text Available We have recently designed a soluble synthetic peptide that functionally mimics the HIV-1 coreceptor CXCR4, which is a chemokine receptor that belongs to the family of seven-transmembrane GPCRs. This CXCR4 mimetic peptide, termed CX4-M1, presents the three extracellular loops (ECLs of the receptor. In binding assays involving recombinant proteins, as well as in cellular infection assays, CX4-M1 was found to selectively recognize gp120 from HIV-1 strains that use CXCR4 for cell entry (X4 tropic HIV-1. Furthermore, anti-HIV-1 antibodies modulate this interaction in a molecular mechanism related to that of their impact on the gp120-CXCR4 interaction. We could now show that the selectivity of CX4-M1 pertains not only to gp120 from X4 tropic HIV-1, but also to synthetic peptides presenting the V3 loops of these gp120 proteins. The V3 loop is thought to be an essential part of the coreceptor binding site of gp120 that contacts the second ECL of the coreceptor. We were able to experimentally confirm this notion in binding assays using substitution analogs of CX4-M1 and the V3 loop peptides, respectively, as well as in cellular infection assays. These results indicate that interactions of the HIV-1 Env with coreceptors can be mimicked by synthetic peptides, which may be useful to explore these interactions at the molecular level in more detail.

  1. Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag.

    Science.gov (United States)

    Friedrich, Melanie; Setz, Christian; Hahn, Friedrich; Matthaei, Alina; Fraedrich, Kirsten; Rauch, Pia; Henklein, Petra; Traxdorf, Maximilian; Fossen, Torgils; Schubert, Ulrich

    2016-04-25

    The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (L-) domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane.

  2. Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag

    Directory of Open Access Journals (Sweden)

    Melanie Friedrich

    2016-04-01

    Full Text Available The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (l- domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane.

  3. Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals

    Science.gov (United States)

    Achkar, Jacqueline M.; Cortes, Laetitia; Croteau, Pascal; Yanofsky, Corey; Mentinova, Marija; Rajotte, Isabelle; Schirm, Michael; Zhou, Yiyong; Junqueira-Kipnis, Ana Paula; Kasprowicz, Victoria O.; Larsen, Michelle; Allard, René; Hunter, Joanna; Paramithiotis, Eustache

    2015-01-01

    Biomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV−) and co-infected (HIV+) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV− individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV+ individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC = 0.96 for HIV− TB, 0.95 for HIV+ TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB. PMID:26501113

  4. Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals

    Directory of Open Access Journals (Sweden)

    Jacqueline M. Achkar

    2015-09-01

    Full Text Available Biomarkers for active tuberculosis (TB are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI, uninfected states, or respiratory diseases other than TB (ORD. Serum samples from 209 HIV uninfected (HIV− and co-infected (HIV+ individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV− individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1, or 10 host proteins in HIV+ individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6, respectively, distinguished TB from ORD with excellent accuracy (AUC = 0.96 for HIV− TB, 0.95 for HIV+ TB. These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB.

  5. The human immunodeficiency virus (HIV) Rev-binding protein (HRB) is a co-factor for HIV-1 Nef-mediated CD4 downregulation.

    Science.gov (United States)

    Landi, Alessia; Timermans, Cristina Garcia; Naessens, Evelien; Vanderstraeten, Hanne; Stove, Veronique; Verhasselt, Bruno

    2016-03-01

    Human immunodeficiency virus type 1 (HIV-1)-mediated CD4 downregulation is an important determinant of viral replication in vivo. Research on cellular co-factors involved in this process could lead to the identification of potential therapeutic targets. We found that CD4 surface levels were significantly higher in HIV-1-infected cells knocked-down for the HIV Rev-binding protein (HRB) compared with control cells. HRB knock-down affected CD4 downregulation induced by Nef but not by HIV-1 Vpu. Interestingly, the knock-down of the related protein HRBL (HRB-like), but not of the HRB interaction partner EPS15 (epidermal growth factor receptor pathway substrate 15), increased CD4 levels in Vpu-expressing cells significantly. Both of these proteins are known to be involved in HIV-1-mediated CD4 downregulation as co-factors of HIV-1 Nef. These results identify HRB as a previously unknown co-factor for HIV-1 Nef-mediated CD4 downregulation and highlight differences with the related protein HRBL, which affects the CD4 downregulation in a dual role as co-factor of both HIV-1 Nef and Vpu.

  6. Alterations in the expression of DEAD-box and other RNA binding proteins during HIV-1 replication

    Directory of Open Access Journals (Sweden)

    Zeichner Steven L

    2004-12-01

    Full Text Available Abstract Recent results showed that certain DEAD box protein RNA helicases, DDX3 and DDX1, play an important role in the HIV infection cycle by facilitating the export of long, singly spliced or unspliced HIV RNAs from the nucleus via the CRM1-Rev pathway. Close examination of an extensive microarray expression profiling dataset obtained from cells latently infected with HIV induced to undergo lytic viral replication indicated that additional DEAD box proteins, beyond DDX3 and DDX1, exhibit differential expression during lytic HIV replication, and in latently infected cells prior to induction into active replication. This finding provides additional evidence that the involvement of DEAD box proteins and other RNA-binding proteins may play roles in active HIV replication and in the control of viral latency. Agents targeting these functions may offer new approaches to antiretroviral therapy and the therapeutic manipulation of HIV latency.

  7. The enhancement of astrocytic-derived monocyte chemoattractant protein-1 induced by the interaction of opiate and HIV tat in HIV-associated dementia

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    HIV-associated dementia(HAD)is a public health problem and is particularly prevalent in drug abusers.The neuropathogenesis of human immunodeficiency virus(HIV)infection involves a complex cascade of inflammatory events,including monocyte/macrophage infiltration in the brain,glial immune activation and release of neurotoxic substances.In these events,astrocytic-derived monocyte chemoattractant protein-1(MCP-1)plays an important role,whose release is elevated by HIV transactivator of transcription(HIV tat)and...

  8. The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

    Directory of Open Access Journals (Sweden)

    Qingwen Jin

    Full Text Available Insertion of T4 lysozyme (T4L into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed.We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects.Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1 infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5.Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

  9. The enhancement of astrocytic-derived monocyte chemoattractant protein-1 induced by the interaction of opiate and HIV tat in HIV-associated dementia

    Institute of Scientific and Technical Information of China (English)

    Xiao Han

    2009-01-01

    HIV-assodated dementia (HAD) is a public health problem and is particularly prevalent in drug abusers. The neuropathogenesis of human immunodeficiency virus (HIV) infection involves a complex cascade of inflammatory events, including monocyte/macrophage infiltration in the brain, glial immune activation and release of neurotoxic substances. In these events, astrocytic-derived monocyte chemoattractant protein-1 (MCP-1) plays an important role, whose release is elevated by HIV transactivator of transcription (HIV tat) and could be further elevated by opiates. This review will also consider some critical factors and events in MCP-1 enhancement induced by the interactions of opiate and HIV tat, including the mediating role of mu opioid receptor (MOR) and CCR2 as well as the possible signal transduction pathways within the cells. Finally, it will make some future perspectives on the exact pathways, new receptors and target cells, and the vulnerability to neurodegeneration with HIV and opiates.

  10. HIV-1 nucleocapsid protein localizes efficiently to the nucleus and nucleolus.

    Science.gov (United States)

    Yu, Kyung Lee; Lee, Sun Hee; Lee, Eun Soo; You, Ji Chang

    2016-05-01

    The HIV-1 nucleocapsid (NC) is an essential viral protein containing two highly conserved retroviral-type zinc finger (ZF) motifs, which functions in multiple stages of the HIV-1 life cycle. Although a number of functions for NC either in its mature form or as a domain of Gag have been revealed, little is known about the intracellular localization of NC and, moreover, its role in Gag protein trafficking. Here, we have investigated various forms of HIV-1 NC protein for its cellular localization and found that the NC has a strong nuclear and nucleolar localization activity. The linker region, composed of a stretch of basic amino acids between the two ZF motifs, was necessary and sufficient for the activity.

  11. Antibody Response is More Likely to Pneumococcal Proteins Than to Polysaccharide After HIV-associated Invasive Pneumococcal Disease

    DEFF Research Database (Denmark)

    Kantsø, Bjørn; Green, Nicola; Goldblatt, David;

    2015-01-01

    BACKGROUND: Human immunodeficiency virus (HIV)-infected individuals are at increased risk of invasive pneumococcal disease (IPD). In order to assess the immunogenicity of pneumococcal proteins and polysaccharide, we investigated protein and serotype-specific antibody responses after HIV-associate...

  12. Cytoplasmic HIV-1 RNA is mainly transported by diffusion in the presence or absence of Gag protein

    DEFF Research Database (Denmark)

    Chen, Jianbo; Grunwald, David; Sardo, Luca

    2014-01-01

    Full-length HIV-1 RNA plays a central role in viral replication by serving as the mRNA for essential viral proteins and as the genome packaged into infectious virions. Proper RNA trafficking is required for the functions of RNA and its encoded proteins; however, the mechanism by which HIV-1 RNA...

  13. Protein Kinase C: One Pathway towards the Eradication of Latent HIV-1 Reservoirs

    Directory of Open Access Journals (Sweden)

    Lisa N. McKernan

    2012-01-01

    Full Text Available An effective means to eradicate latent reservoirs in HIV-1-infected individuals remains elusive. Attempts to purge these reservoirs were undertaken over a decade ago without success. The subsequent lapse in further clinical attempts since may have been justified as our knowledge of the mechanisms which underpin the latent state still evolves. Although additional novel molecular antagonists of HIV-1 latency have subsequently been reported, these candidate agents have not been tested in human trials for reservoir ablation. This review provides an overview of the protein kinase C (PKC pathway which can be modulated by small molecular agents to induce the expression of latent HIV-1 from within infected reservoir cells. Some of these agents have been tested against select cancers with seemingly tolerable side effects. As such, modulation of the PKC pathway may yet be a viable mechanism toward HIV-1 reservoir eradication.

  14. Sequence- and interactome-based prediction of viral protein hotspots targeting host proteins: a case study for HIV Nef.

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    Mahdi Sarmady

    Full Text Available Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk.

  15. Brain transcriptome-wide screen for HIV-1 Nef protein interaction partners reveals various membrane-associated proteins.

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    Ellen C Kammula

    Full Text Available HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

  16. Brain transcriptome-wide screen for HIV-1 Nef protein interaction partners reveals various membrane-associated proteins.

    Science.gov (United States)

    Kammula, Ellen C; Mötter, Jessica; Gorgels, Alexandra; Jonas, Esther; Hoffmann, Silke; Willbold, Dieter

    2012-01-01

    HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

  17. Solid-State NMR Studies of HIV-1 Capsid Protein Assemblies

    OpenAIRE

    HAN, YUN; Ahn, Jinwoo; Concel, Jason; Byeon, In-Ja L.; Gronenborn, Angela M.; YANG, Jun; Polenova, Tatyana

    2010-01-01

    In mature HIV-1 virions, a 26.6 kDa CA protein is assembled into a characteristic cone shaped core (capsid) that encloses the RNA viral genome. The assembled capsid structure is best described by a fullerene cone model that is made up from a hexameric lattice containing a variable number of CA pentamers, thus allowing for closure of tubular or conical structures. In this report, we present a solid-state NMR analysis of the wild type HIV-1 CA protein, prepared as conical and spherical assembli...

  18. [The role of structural protein Gag and related gene (protein) in late stages of the HIV-1 replication cycle and the inhibitors].

    Science.gov (United States)

    Jiang, Yan; Liu, Xin-yong

    2010-02-01

    The late stages of the HIV-1 replication cycle are important to the overall replication cycle. During the late stages, HIV-1 replication undergoes the processes of assembly, release, and maturation, resulting in the production of a mature virus particle capable of infecting a new target cell. The structural protein Gag and its related gene (protein) play a central role in these pathways. The different regions of Gag worked in concert to drive production of a mature infectious particle through protein-protein, protein-RNA and protein-lipid interactions. The designed drug aimed directly at these stages can efficiently block the maturation and infectivity of HIV-1. In this article, the role of structural protein Gag and related gene (protein) in late stages of the HIV-1 replication cycle and related inhibitors is reviewed.

  19. A High Throughput Protein Microarray Approach to Classify HIV Monoclonal Antibodies and Variant Antigens.

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    Emmanuel Y Dotsey

    Full Text Available In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs has been applied to greatly advance our understanding of the specificity, and functional activity of antibodies against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip comprised of the HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001 with concentrations from ELISA binding measurements. Polyclonal immune responses in plasma samples from HIV-1 infected subjects exhibited different binding patterns, and reactivity against printed proteins. Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV.

  20. Running Loose or Getting Lost: How HIV-1 Counters and Capitalizes on APOBEC3-Induced Mutagenesis through Its Vif Protein

    OpenAIRE

    Christel Kamp; Dieter Häussinger; Jörg Zielonka; Carsten Münk; Jensen, Björn-Erik O.

    2012-01-01

    Human immunodeficiency virus-1 (HIV-1) dynamics reflect an intricate balance within the viruses’ host. The virus relies on host replication factors, but must escape or counter its host’s antiviral restriction factors. The interaction between the HIV-1 protein Vif and many cellular restriction factors from the APOBEC3 protein family is a prominent example of this evolutionary arms race. The viral infectivity factor (Vif) protein largely neutralizes APOBEC3 proteins, which c...

  1. Improved innate and adaptive immunostimulation by genetically modified HIV-1 protein expressing NYVAC vectors.

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    Esther D Quakkelaar

    Full Text Available Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs, RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines.

  2. P body-associated protein Mov10 inhibits HIV-1 replication at multiple stages.

    Science.gov (United States)

    Burdick, Ryan; Smith, Jessica L; Chaipan, Chawaree; Friew, Yeshitila; Chen, Jianbo; Venkatachari, Narasimhan J; Delviks-Frankenberry, Krista A; Hu, Wei-Shau; Pathak, Vinay K

    2010-10-01

    Recent studies have shown that APOBEC3G (A3G), a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, is localized to cytoplasmic mRNA-processing bodies (P bodies). However, the functional relevance of A3G colocalization with P body marker proteins has not been established. To explore the relationship between HIV-1, A3G, and P bodies, we analyzed the effects of overexpression of P body marker proteins Mov10, DCP1a, and DCP2 on HIV-1 replication. Our results show that overexpression of Mov10, a putative RNA helicase that was previously reported to belong to the DExD superfamily and was recently reported to belong to the Upf1-like group of helicases, but not the decapping enzymes DCP1a and DCP2, leads to potent inhibition of HIV-1 replication at multiple stages. Mov10 overexpression in the virus producer cells resulted in reductions in the steady-state levels of the HIV-1 Gag protein and virus production; Mov10 was efficiently incorporated into virions and reduced virus infectivity, in part by inhibiting reverse transcription. In addition, A3G and Mov10 overexpression reduced proteolytic processing of HIV-1 Gag. The inhibitory effects of A3G and Mov10 were additive, implying a lack of functional interaction between the two inhibitors. Small interfering RNA (siRNA)-mediated knockdown of endogenous Mov10 by 80% resulted in a 2-fold reduction in virus production but no discernible impact on the infectivity of the viruses after normalization for the p24 input, suggesting that endogenous Mov10 was not required for viral infectivity. Overall, these results show that Mov10 can potently inhibit HIV-1 replication at multiple stages.

  3. Human Polycomb group EED protein negatively affects HIV-1 assembly and release

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    Darlix Jean-Luc

    2007-06-01

    Full Text Available Abstract Background The human EED protein, a member of the superfamily of Polycomb group (PcG proteins with WD-40 repeats, has been found to interact with three HIV-1 components, namely the structural Gag matrix protein (MA, the integrase enzyme (IN and the Nef protein. The aim of the present study was to analyze the possible biological role of EED in HIV-1 replication, using the HIV-1-based vector HIV-Luc and EED protein expressed by DNA transfection of 293T cells. Results During the early phase of HIV-1 infection, a slight negative effect on virus infectivity occurred in EED-expressing cells, which appeared to be dependent on EED-MA interaction. At late times post infection, EED caused an important reduction of virus production, from 20- to 25-fold as determined by CAp24 immunoassay, to 10- to 80-fold based on genomic RNA levels, and this decrease was not due to a reduction of Gag protein synthesis. Coexpression of WTNef, or the non-N-myristoylated mutant NefG2A, restored virus yields to levels obtained in the absence of exogenous EED protein. This effect was not observed with mutant NefΔ57 mimicking the Nef core, or with the lipid raft-retargeted fusion protein LAT-Nef. LATAA-Nef, a mutant defective in the lipid raft addressing function, had the same anti-EED effect as WTNef. Cell fractionation and confocal imaging showed that, in the absence of Nef, EED mainly localized in membrane domains different from the lipid rafts. Upon co-expression with WTNef, NefG2A or LATAA-Nef, but not with NefΔ57 or LAT-Nef, EED was found to relocate into an insoluble fraction along with Nef protein. Electron microscopy of HIV-Luc producer cells overexpressing EED showed significant less virus budding at the cell surface compared to control cells, and ectopic assembly and clustering of nuclear pore complexes within the cytoplasm. Conclusion Our data suggested that EED exerted an antiviral activity at the late stage of HIV-1 replication, which included genomic

  4. Nuclear Envelope Protein SUN2 Promotes Cyclophilin-A-Dependent Steps of HIV Replication

    Science.gov (United States)

    Lahaye, Xavier; Satoh, Takeshi; Gentili, Matteo; Cerboni, Silvia; Silvin, Aymeric; Conrad, Cécile; Ahmed-Belkacem, Abdelhakim; Rodriguez, Elisa C.; Guichou, Jean-François; Bosquet, Nathalie; Piel, Matthieu; Le Grand, Roger; King, Megan C.; Pawlotsky, Jean-Michel; Manel, Nicolas

    2016-01-01

    Summary During the early phase of replication, HIV reverse transcribes its RNA and crosses the nuclear envelope while escaping host antiviral defenses. The host factor Cyclophilin A (CypA) is essential for these steps and binds the HIV capsid; however, the mechanism underlying this effect remains elusive. Here, we identify related capsid mutants in HIV-1, HIV-2, and SIVmac that are restricted by CypA. This antiviral restriction of mutated viruses is conserved across species and prevents nuclear import of the viral cDNA. Importantly, the inner nuclear envelope protein SUN2 is required for the antiviral activity of CypA. We show that wild-type HIV exploits SUN2 in primary CD4+ T cells as an essential host factor that is required for the positive effects of CypA on reverse transcription and infection. Altogether, these results establish essential CypA-dependent functions of SUN2 in HIV infection at the nuclear envelope. PMID:27149839

  5. Nuclear Envelope Protein SUN2 Promotes Cyclophilin-A-Dependent Steps of HIV Replication

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    Xavier Lahaye

    2016-04-01

    Full Text Available During the early phase of replication, HIV reverse transcribes its RNA and crosses the nuclear envelope while escaping host antiviral defenses. The host factor Cyclophilin A (CypA is essential for these steps and binds the HIV capsid; however, the mechanism underlying this effect remains elusive. Here, we identify related capsid mutants in HIV-1, HIV-2, and SIVmac that are restricted by CypA. This antiviral restriction of mutated viruses is conserved across species and prevents nuclear import of the viral cDNA. Importantly, the inner nuclear envelope protein SUN2 is required for the antiviral activity of CypA. We show that wild-type HIV exploits SUN2 in primary CD4+ T cells as an essential host factor that is required for the positive effects of CypA on reverse transcription and infection. Altogether, these results establish essential CypA-dependent functions of SUN2 in HIV infection at the nuclear envelope.

  6. Unperturbed posttranscriptional regulatory Rev protein function and HIV-1 replication in astrocytes.

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    Ashok Chauhan

    Full Text Available Astrocytes protect neurons, but also evoke proinflammatory responses to injury and viral infections, including HIV. There is a prevailing notion that HIV-1 Rev protein function in astrocytes is perturbed, leading to restricted viral replication. In earlier studies, our finding of restricted viral entry into astrocytes led us to investigate whether there are any intracellular restrictions, including crippled Rev function, in astrocytes. Despite barely detectable levels of DDX3 (Rev-supporting RNA helicase and TRBP (anti-PKR in primary astrocytes compared to astrocytic cells, Rev function was unperturbed in wild-type, but not DDX3-ablated astrocytes. As in permissive cells, after HIV-1 entry bypass in astrocytes, viral-encoded Tat and Rev proteins had robust regulatory activities, leading to efficient viral replication. Productive HIV-1 infection in astrocytes persisted for several weeks. Our findings on HIV-1 entry bypass in astrocytes demonstrated that the intracellular environment is conducive to viral replication and that Tat and Rev functions are unperturbed.

  7. Identification of the HIV-1 Vif and Human APOBEC3G Protein Interface.

    Science.gov (United States)

    Letko, Michael; Booiman, Thijs; Kootstra, Neeltje; Simon, Viviana; Ooms, Marcel

    2015-12-01

    Human cells express natural antiviral proteins, such as APOBEC3G (A3G), that potently restrict HIV replication. As a counter-defense, HIV encodes the accessory protein Vif, which binds A3G and mediates its proteasomal degradation. Our structural knowledge on how Vif and A3G interact is limited, because a co-structure is not available. We identified specific points of contact between Vif and A3G by using functional assays with full-length A3G, patient-derived Vif variants, and HIV forced evolution. These anchor points were used to model and validate the Vif-A3G interface. The resultant co-structure model shows that the negatively charged β4-α4 A3G loop, which contains primate-specific variation, is the core Vif binding site and forms extensive interactions with a positively charged pocket in HIV Vif. Our data present a functional map of this viral-host interface and open avenues for targeted approaches to block HIV replication by obstructing the Vif-A3G interaction.

  8. Immunotherapy of HIV-infected patients with Gc protein-derived macrophage activating factor (GcMAF).

    Science.gov (United States)

    Yamamoto, Nobuto; Ushijima, Naofumi; Koga, Yoshihiko

    2009-01-01

    Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of HIV-infected patients was lost or reduced because Gc protein is deglycosylated by alpha-N-acetylgalactosaminidase (Nagalase) secreted from HIV-infected cells. Therefore, macrophages of HIV-infected patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Since Nagalase is the intrinsic component of the envelope protein gp120, serum Nagalase activity is the sum of enzyme activities carried by both HIV virions and envelope proteins. These Nagalase carriers were already complexed with anti-HIV immunoglobulin G (IgG) but retained Nagalase activity that is required for infectivity. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF), which produces no side effects in humans. Macrophages activated by administration of 100 ng GcMAF develop a large amount of Fc-receptors as well as an enormous variation of receptors that recognize IgG-bound and unbound HIV virions. Since latently HIV-infected cells are unstable and constantly release HIV virions, the activated macrophages rapidly intercept the released HIV virions to prevent reinfection resulting in exhaustion of infected cells. After less than 18 weekly administrations of 100 ng GcMAF for nonanemic patients, they exhibited low serum Nagalase activities equivalent to healthy controls, indicating eradication of HIV-infection, which was also confirmed by no infectious center formation by provirus inducing agent-treated patient PBMCs. No recurrence occurred and their healthy CD + cell counts were maintained for 7 years.

  9. The HIV-1 antisense protein (ASP) induces CD8 T cell responses during chronic infection.

    Science.gov (United States)

    Bet, Anne; Maze, Emmanuel Atangana; Bansal, Anju; Sterrett, Sarah; Gross, Antoine; Graff-Dubois, Stéphanie; Samri, Assia; Guihot, Amélie; Katlama, Christine; Theodorou, Ioannis; Mesnard, Jean-Michel; Moris, Arnaud; Goepfert, Paul A; Cardinaud, Sylvain

    2015-02-10

    CD8+ T cells recognize HIV-1 epitopes translated from a gene's primary reading frame (F1) and any one of its five alternative reading frames (ARFs) in the forward (F2, F3) or reverse (R1-3) directions. The 3' end of HIV-1's proviral coding strand contains a conserved sequence that is directly overlapping but antiparallel to the env gene (ARF R2) and encodes for a putative antisense HIV-1 protein called ASP. ASP expression has been demonstrated in vitro using HIV-transfected cell lines or infected cells. Although antibodies to ASP were previously detected in patient sera, T cell recognition of ASP-derived epitopes has not been evaluated. We therefore investigated the ex vivo and in vitro induction of ASP-specific T cell responses as a measure of immune recognition and protein expression during HIV-1 infection. A panel of overlapping peptides was initially designed from the full-length ASP sequence to perform a global assessment of T cell responses. Recognition of ASP-derived antigens was evaluated in an IFN-γELISpot assay using PBMCs from HIV-1 seropositive and seronegative individuals. Eight of 25 patients had positive responses to ASP antigens and none of the seronegative donors responded. As a complimentary approach, a second set of antigens was designed using HLA-I binding motifs and affinities. Two ASP-derived peptides with high predicted binding affinities for HLA-A*02 (ASP-YL9) and HLA-B*07 (ASP-TL10) were tested using PBMCs from HIV-1 seropositive and seronegative individuals who expressed the matching HLA-I-restricting allele. We found that HLA-I-restricted ASP peptides were only recognized by CD8+ T cells from patients with the relevant HLA-I and did not induce responses in any of the seronegative donors or patients who do not express the restrictive HLA alleles. Further, ASP-YL9-specific CD8+ T cells had functional profiles that were similar to a previously described HLA-A*02-restricted epitope (Gag-SL9). Specific recognition of ASP-YL9 by CD8+ T cells

  10. BAG3 protein regulates caspase-3 activation in HIV-1-infected human primary microglial cells

    Science.gov (United States)

    Rosati, Alessandra; Khalili, Kamel; Deshmane, Satish L.; Radhakrishnan, Sujatha; Pascale, Maria; Turco, M. Caterina; Marzullo, Liberato

    2015-01-01

    BAG3, a member of the BAG co-chaperones family, is expressed in several cell types subjected to stressful conditions, such as exposure to high temperature, heavy metals, drugs. Furthermore, it is constitutively expressed in some tumors. Among the biological activities of the protein, there is apoptosis downmodulation; this appears to be exerted through BAG3 interaction with the heat shock protein (Hsp) 70, that influences cell apoptosis at several levels. We recently reported that BAG3 protein was detectable in the cytoplasm of reactive astrocytes in HIV-1-associated encephalopathy biopsies. Here we report that downmodulation of BAG3 protein levels allows caspase-3 activation by HIV-1 infection in human primary microglial cells. This is the first reported evidence of a role for BAG3 in the balance of death versus survival during viral infection. PMID:18821563

  11. Molecular characterization of the HIV-1 gag nucleocapsid gene associated with vertical transmission

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    Ramakrishnan Rajesh

    2006-04-01

    Full Text Available Abstract Background The human immunodeficiency virus type 1 (HIV-1 nucleocapsid (NC plays a pivotal role in the viral lifecycle: including encapsulating the viral genome, aiding in strand transfer during reverse transcription, and packaging two copies of the viral genome into progeny virions. Another gag gene product, p6, plays an integral role in successful viral budding from the plasma membrane and inclusion of the accessory protein Vpr within newly budding virions. In this study, we have characterized the gag NC and p6 genes from six mother-infant pairs following vertical transmission by performing phylogenetic analysis and by analyzing the degree of genetic diversity, evolutionary dynamics, and conservation of functional domains. Results Phylogenetic analysis of 168 gag NC and p6 genes sequences revealed six separate subtrees that corresponded to each mother-infant pair, suggesting that epidemiologically linked individuals were closer to each other than epidemiologically unlinked individuals. A high frequency (92.8% of intact open reading frames of NC and p6 with patient and pair specific sequence motifs were conserved in mother-infant pairs' sequences. Nucleotide and amino acid distances showed a lower degree of viral heterogeneity, and a low degree of estimates of genetic diversity was also found in NC and p6 sequences. The NC and p6 sequences from both mothers and infants were found to be under positive selection pressure. The two important functional motifs within NC, the zinc-finger motifs, were highly conserved in most of the sequences, as were the gag p6 Vpr binding, AIP1 and late binding domains. Several CTL recognition epitopes identified within the NC and p6 genes were found to be mostly conserved in 6 mother-infant pairs' sequences. Conclusion These data suggest that the gag NC and p6 open reading frames and functional domains were conserved in mother-infant pairs' sequences following vertical transmission, which confirms the

  12. HLA-associated immune escape pathways in HIV-1 subtype B Gag, Pol and Nef proteins.

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    Zabrina L Brumme

    Full Text Available BACKGROUND: Despite the extensive genetic diversity of HIV-1, viral evolution in response to immune selective pressures follows broadly predictable mutational patterns. Sites and pathways of Human Leukocyte-Antigen (HLA-associated polymorphisms in HIV-1 have been identified through the analysis of population-level data, but the full extent of immune escape pathways remains incompletely characterized. Here, in the largest analysis of HIV-1 subtype B sequences undertaken to date, we identify HLA-associated polymorphisms in the three HIV-1 proteins most commonly considered in cellular-based vaccine strategies. Results are organized into protein-wide escape maps illustrating the sites and pathways of HLA-driven viral evolution. METHODOLOGY/PRINCIPAL FINDINGS: HLA-associated polymorphisms were identified in HIV-1 Gag, Pol and Nef in a multicenter cohort of >1500 chronically subtype-B infected, treatment-naïve individuals from established cohorts in Canada, the USA and Western Australia. At q< or =0.05, 282 codons commonly mutating under HLA-associated immune pressures were identified in these three proteins. The greatest density of associations was observed in Nef (where close to 40% of codons exhibited a significant HLA association, followed by Gag then Pol (where approximately 15-20% of codons exhibited HLA associations, confirming the extensive impact of immune selection on HIV evolution and diversity. Analysis of HIV codon covariation patterns identified over 2000 codon-codon interactions at q< or =0.05, illustrating the dense and complex networks of linked escape and secondary/compensatory mutations. CONCLUSIONS/SIGNIFICANCE: The immune escape maps and associated data are intended to serve as a user-friendly guide to the locations of common escape mutations and covarying codons in HIV-1 subtype B, and as a resource facilitating the systematic identification and classification of immune escape mutations. These resources should facilitate

  13. Defining differential genetic signatures in CXCR4- and the CCR5-utilizing HIV-1 co-linear sequences.

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    Benjamas Aiamkitsumrit

    Full Text Available The adaptation of human immunodeficiency virus type-1 (HIV-1 to an array of physiologic niches is advantaged by the plasticity of the viral genome, encoded proteins, and promoter. CXCR4-utilizing (X4 viruses preferentially, but not universally, infect CD4+ T cells, generating high levels of virus within activated HIV-1-infected T cells that can be detected in regional lymph nodes and peripheral blood. By comparison, the CCR5-utilizing (R5 viruses have a greater preference for cells of the monocyte-macrophage lineage; however, while R5 viruses also display a propensity to enter and replicate in T cells, they infect a smaller percentage of CD4+ T cells in comparison to X4 viruses. Additionally, R5 viruses have been associated with viral transmission and CNS disease and are also more prevalent during HIV-1 disease. Specific adaptive changes associated with X4 and R5 viruses were identified in co-linear viral sequences beyond the Env-V3. The in silico position-specific scoring matrix (PSSM algorithm was used to define distinct groups of X4 and R5 sequences based solely on sequences in Env-V3. Bioinformatic tools were used to identify genetic signatures involving specific protein domains or long terminal repeat (LTR transcription factor sites within co-linear viral protein R (Vpr, trans-activator of transcription (Tat, or LTR sequences that were preferentially associated with X4 or R5 Env-V3 sequences. A number of differential amino acid and nucleotide changes were identified across the co-linear Vpr, Tat, and LTR sequences, suggesting the presence of specific genetic signatures that preferentially associate with X4 or R5 viruses. Investigation of the genetic relatedness between X4 and R5 viruses utilizing phylogenetic analyses of complete sequences could not be used to definitively and uniquely identify groups of R5 or X4 sequences; in contrast, differences in the genetic diversities between X4 and R5 were readily identified within these co

  14. Defining differential genetic signatures in CXCR4- and the CCR5-utilizing HIV-1 co-linear sequences.

    Science.gov (United States)

    Aiamkitsumrit, Benjamas; Dampier, Will; Martin-Garcia, Julio; Nonnemacher, Michael R; Pirrone, Vanessa; Ivanova, Tatyana; Zhong, Wen; Kilareski, Evelyn; Aldigun, Hazeez; Frantz, Brian; Rimbey, Matthew; Wojno, Adam; Passic, Shendra; Williams, Jean W; Shah, Sonia; Blakey, Brandon; Parikh, Nirzari; Jacobson, Jeffrey M; Moldover, Brian; Wigdahl, Brian

    2014-01-01

    The adaptation of human immunodeficiency virus type-1 (HIV-1) to an array of physiologic niches is advantaged by the plasticity of the viral genome, encoded proteins, and promoter. CXCR4-utilizing (X4) viruses preferentially, but not universally, infect CD4+ T cells, generating high levels of virus within activated HIV-1-infected T cells that can be detected in regional lymph nodes and peripheral blood. By comparison, the CCR5-utilizing (R5) viruses have a greater preference for cells of the monocyte-macrophage lineage; however, while R5 viruses also display a propensity to enter and replicate in T cells, they infect a smaller percentage of CD4+ T cells in comparison to X4 viruses. Additionally, R5 viruses have been associated with viral transmission and CNS disease and are also more prevalent during HIV-1 disease. Specific adaptive changes associated with X4 and R5 viruses were identified in co-linear viral sequences beyond the Env-V3. The in silico position-specific scoring matrix (PSSM) algorithm was used to define distinct groups of X4 and R5 sequences based solely on sequences in Env-V3. Bioinformatic tools were used to identify genetic signatures involving specific protein domains or long terminal repeat (LTR) transcription factor sites within co-linear viral protein R (Vpr), trans-activator of transcription (Tat), or LTR sequences that were preferentially associated with X4 or R5 Env-V3 sequences. A number of differential amino acid and nucleotide changes were identified across the co-linear Vpr, Tat, and LTR sequences, suggesting the presence of specific genetic signatures that preferentially associate with X4 or R5 viruses. Investigation of the genetic relatedness between X4 and R5 viruses utilizing phylogenetic analyses of complete sequences could not be used to definitively and uniquely identify groups of R5 or X4 sequences; in contrast, differences in the genetic diversities between X4 and R5 were readily identified within these co-linear sequences in

  15. Tenascin-C is an innate broad-spectrum, HIV-1–neutralizing protein in breast milk

    Science.gov (United States)

    Fouda, Genevieve G.; Jaeger, Frederick H.; Amos, Joshua D.; Ho, Carrie; Kunz, Erika L.; Anasti, Kara; Stamper, Lisa W.; Liebl, Brooke E.; Barbas, Kimberly H.; Ohashi, Tomoo; Moseley, Martin Arthur; Liao, Hua-Xin; Erickson, Harold P.; Alam, S. Munir; Permar, Sallie R.

    2013-01-01

    Achieving an AIDS-free generation will require elimination of postnatal transmission of HIV-1 while maintaining the nutritional and immunologic benefits of breastfeeding for infants in developing regions. Maternal/infant antiretroviral prophylaxis can reduce postnatal HIV-1 transmission, yet toxicities and the development of drug-resistant viral strains may limit the effectiveness of this strategy. Interestingly, in the absence of antiretroviral prophylaxis, greater than 90% of infants exposed to HIV-1 via breastfeeding remain uninfected, despite daily mucosal exposure to the virus for up to 2 y. Moreover, milk of uninfected women inherently neutralizes HIV-1 and prevents virus transmission in animal models, yet the factor(s) responsible for this anti-HIV activity is not well-defined. In this report, we identify a primary HIV-1–neutralizing protein in breast milk, Tenascin-C (TNC). TNC is an extracellular matrix protein important in fetal development and wound healing, yet its antimicrobial properties have not previously been established. Purified TNC captured and neutralized multiclade chronic and transmitted/founder HIV-1 variants, and depletion of TNC abolished the HIV-1–neutralizing activity of milk. TNC bound the HIV-1 Envelope protein at a site that is induced upon engagement of its primary receptor, CD4, and is blocked by V3 loop- (19B and F39F) and chemokine coreceptor binding site-directed (17B) monoclonal antibodies. Our results demonstrate the ability of an innate mucosal host protein found in milk to neutralize HIV-1 via binding to the chemokine coreceptor site, potentially explaining why the majority of HIV-1–exposed breastfed infants are protected against mucosal HIV-1 transmission. PMID:24145401

  16. HIV-1 Tat protein increases microglial outward K(+ current and resultant neurotoxic activity.

    Directory of Open Access Journals (Sweden)

    Jianuo Liu

    Full Text Available Microglia plays a crucial role in the pathogenesis of HIV-1-associated neurocognitive disorders. Increasing evidence indicates the voltage-gated potassium (Kv channels are involved in the regulation of microglia function, prompting us to hypothesize Kv channels may also be involved in microglia-mediated neurotoxic activity in HIV-1-infected brain. To test this hypothesis, we investigated the involvement of Kv channels in the response of microglia to HIV-1 Tat protein. Treatment of rat microglia with HIV-1 Tat protein (200 ng/ml resulted in pro-inflammatory microglial activation, as indicated by increases in TNF-α, IL-1β, reactive oxygen species, and nitric oxide, which were accompanied by enhanced outward K(+ current and Kv1.3 channel expression. Suppression of microglial Kv1.3 channel activity, either with Kv1.3 channel blockers Margatoxin, 5-(4-Phenoxybutoxypsoralen, or broad-spectrum K(+ channel blocker 4-Aminopyridine, or by knockdown of Kv1.3 expression via transfection of microglia with Kv1.3 siRNA, was found to abrogate the neurotoxic activity of microglia resulting from HIV-1 Tat exposure. Furthermore, HIV-1 Tat-induced neuronal apoptosis was attenuated with the application of supernatant collected from K(+ channel blocker-treated microglia. Lastly, the intracellular signaling pathways associated with Kv1.3 were investigated and enhancement of microglial Kv1.3 was found to correspond with an increase in Erk1/2 mitogen-activated protein kinase activation. These data suggest targeting microglial Kv1.3 channels may be a potential new avenue of therapy for inflammation-mediated neurological disorders.

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

    Directory of Open Access Journals (Sweden)

    Ming-Chih Lai

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

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

    Science.gov (United States)

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

    2013-01-01

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

  19. Retinoblastoma protein induction by HIV viremia or CCR5 in monocytes exposed to HIV-1 mediates protection from activation-induced apoptosis: ex vivo and in vitro study.

    Science.gov (United States)

    Gekonge, Bethsebah; Raymond, Andrea D; Yin, Xiangfan; Kostman, Jay; Mounzer, Karam; Collman, Ronald G; Showe, Louise; Montaner, Luis J

    2012-08-01

    We have previously described an antiapoptotic steady-state gene expression profile in circulating human monocytes from asymptomatic viremic HIV(+) donors, but the mechanism associated with this apoptosis resistance remains to be fully elucidated. Here, we show that Rb1 activation is a dominant feature of apoptosis resistance in monocytes exposed to HIV-1 in vivo (as measured ex vivo) and in vitro. Monocytes from asymptomatic viremic HIV(+) individuals show a positive correlation between levels of hypophosphorylated (active) Rb1 and VL in conjunction with increases in other p53-inducible proteins associated with antiapoptosis regulation, such as p21 and PAI-1 (SERPINE1), when compared with circulating monocytes from uninfected donors. Monocytes exposed in vitro to HIV-1 R5 isolates but not X4 isolates showed lower caspase-3 activation after apoptosis induction, indicating a role for the CCR5 signaling pathway. Moreover, monocytes exposed to R5 HIV-1 or MIP-1 β induced Rb1 and p21 expression and an accumulation of autophagy markers, LC3 and Beclin. The inhibition of Rb1 activity in HIV-1 R5 viral-exposed monocytes using siRNA led to increased apoptosis sensitivity, thereby confirming a central role for Rb1 in the antiapoptotic phenotype. Our data identify Rb1 induction in chronic asymptomatic HIV-1 infection as a mediator of apoptosis resistance in monocytes in association with protective autophagy and contributing to monocyte survival during immune activation and/or HIV-1 viremia.

  20. Gnidimacrin, a Potent Anti-HIV Diterpene, Can Eliminate Latent HIV-1 Ex Vivo by Activation of Protein Kinase C β.

    Science.gov (United States)

    Lai, Weihong; Huang, Li; Zhu, Lei; Ferrari, Guido; Chan, Cliburn; Li, Wei; Lee, Kuo-Hsiung; Chen, Chin-Ho

    2015-11-12

    HIV-1-latency-reversing agents, such as histone deacetylase inhibitors (HDACIs), were ineffective in reducing latent HIV-1 reservoirs ex vivo using CD4 cells from patients as a model. This deficiency poses a challenge to current pharmacological approaches for HIV-1 eradication. The results of this study indicated that gnidimacrin (GM) was able to markedly reduce the latent HIV-1 DNA level and the frequency of latently infected cells in an ex vivo model using patients peripheral blood mononuclear cells. GM induced approximately 10-fold more HIV-1 production than the HDACI SAHA or romidepsin, which may be responsible for the effectiveness of GM in reducing latent HIV-1 levels. GM achieved these effects at low picomolar concentrations by selective activation of protein kinase C βI and βII. Notably, GM was able to reduce the frequency of HIV-1 latently infected cells at concentrations without global T cell activation or stimulating inflammatory cytokine production. GM merits further development as a clinical trial candidate for latent HIV-1 eradication.

  1. Sequence and structure requirements for specific recognition of HIV-1 TAR and DIS RNA by the HIV-1 Vif protein.

    Science.gov (United States)

    Freisz, Séverine; Mezher, Joelle; Hafirassou, Lamine; Wolff, Philippe; Nominé, Yves; Romier, Christophe; Dumas, Philippe; Ennifar, Eric

    2012-07-01

    The HIV-1 Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion and in vivo pathogenesis. Vif neutralizes the human DNA-editing enzyme APOBEC3 protein, an antiretroviral cellular factor from the innate immune system, allowing the virus to escape the host defence system. It was shown that Vif is packaged into viral particles through specific interactions with the viral genomic RNA. Conserved and structured sequences from the 5'-noncoding region, such as the Tat-responsive element (TAR) or the genomic RNA dimerization initiation site (DIS), are primary binding sites for Vif. In the present study we used isothermal titration calorimetry to investigate sequence and structure determinants important for Vif binding to short viral RNA corresponding to TAR and DIS stem-loops. We showed that Vif specifically binds TAR and DIS in the low nanomolar range. In addition, Vif primarily binds the TAR UCU bulge, but not the apical loop. Determinants for Vif binding to the DIS loop-loop complex are likely more complex and involve the self-complementary loop together with the upper part of the stem. These results suggest that Tat-TAR inhibitors or DIS small molecule binders might be also effective to disturb Vif-TAR and Vif-DIS binding in order to reduce Vif packaging into virions.

  2. HIV-1 Nef associates with p22-phox, a component of the NADPH oxidase protein complex.

    Science.gov (United States)

    Salmen, Siham; Colmenares, Melisa; Peterson, Darrel L; Reyes, Elbert; Rosales, Jose D; Berrueta, Lisbeth

    2010-01-01

    Altered neutrophil function may contribute to the development of AIDS during the course of HIV infection. It has been described that Nef, a regulatory protein from HIV, can modulate superoxide production in other cells, therefore altered superoxide production in neutrophils from HIV infected patients, could be secondary to a direct effect of Nef on components of the NADPH oxidase complex. In this work, we describe that Nef, was capable of increasing superoxide production in human neutrophils. Furthermore, a specific association between Nef and p22-phox, a membrane component of the NADPH oxidase complex, was found. We propose that this association may reflect a capability of Nef to modulate by direct association, the enzymatic complex responsible for one of the most efficient innate defense mechanisms in phagocytes, contributing to the pathogenesis of the disease.

  3. Detection of two amino acid deletions in HIV-1 Nef protein from Chinese former paid blood donors

    Institute of Scientific and Technical Information of China (English)

    SONG Yan-hui; WEI Min; MA Peng-fei; XING Hui; SI Xue-feng; TANG Hai-li; LI Hui-guang; SHAO Yi-ming

    2007-01-01

    @@ HIV-1 Nef protein plays an important role in HIV-1 pathogenesis, including downregulation of the cell surface CD4 and class Ⅰ major histocompatibility complex (MHC), enhancement of virion infectivity and alteration of a variety of cellular signal transduction pathways.

  4. Clustering patterns of cytotoxic T-lymphocyte epitopes in human immunodeficiency virus type 1 (HIV-1) proteins reveal imprints of immune evasion on HIV-1 global variation

    DEFF Research Database (Denmark)

    Yusim, K.; Kesmir, Can; Gaschen, B.;

    2002-01-01

    for amino acids that do not serve as C-terminal anchor residues. Finally, CTL epitopes are more highly concentrated in alpha-helical regions of proteins. Based on amino acid sequence characteristics, in a blinded fashion, we predicted regions in HIV regulatory and accessory proteins that would be likely...

  5. Viroporin potential of the lentivirus lytic peptide (LLP domains of the HIV-1 gp41 protein

    Directory of Open Access Journals (Sweden)

    Garry Robert F

    2007-11-01

    Full Text Available Abstract Background Mechanisms by which HIV-1 mediates reductions in CD4+ cell levels in infected persons are being intensely investigated, and have broad implications for AIDS drug and vaccine development. Virally induced changes in membrane ionic permeability induced by lytic viruses of many families contribute to cytopathogenesis. HIV-1 induces disturbances in plasma membrane ion transport. The carboxyl terminus of TM (gp41 contains potential amphipathic α-helical motifs identified through their structural similarities to naturally occurring cytolytic peptides. These sequences have been dubbed lentiviral lytic peptides (LLP -1, -2, and -3. Results Peptides corresponding to the LLP domains (from a clade B virus partition into lipid membranes, fold into α-helices and disrupt model membrane permeability. A peptide corresponding to the LLP-1 domain of a clade D HIV-1 virus, LLP-1D displayed similar activity to the LLP-1 domain of the clade B virus in all assays, despite a lack of amino acid sequence identity. Conclusion These results suggest that the C-terminal domains of HIV-1 Env proteins may form an ion channel, or viroporin. Increased understanding of the function of LLP domains and their role in the viral replication cycle could allow for the development of novel HIV drugs.

  6. Quantitative analysis of differentially expressed saliva proteins in human immunodeficiency virus type 1 (HIV-1) infected individuals

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Nawei; Zhang, Zhenyu [Beijing Chaoyang Hospital Affiliated Capital Medical University, Beijing (China); Feng, Shan [MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing (China); Wang, Qingtao [Beijing Chaoyang Hospital Affiliated Capital Medical University, Beijing (China); Malamud, Daniel [NYU College of Dentistry, 345 East 24th Street, New York, NY 10010 (United States); Deng, Haiteng, E-mail: dht@mail.tsinghua.edu.cn [MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing (China)

    2013-04-24

    Highlights: ► A high-throughput method for profiling and quantification of the differentially expressed proteins in saliva samples was developed. ► Identified that DMBT1, S100A7, S100A8, S100A9 and alpha defensin were up-regulated in saliva from HIV-1 seropositive patients. ► Established analytical strategies are translatable to the clinical setting. -- Abstract: In the present study, we have established a new methodology to analyze saliva proteins from HIV-1-seropositive patients before highly active antiretroviral therapy (HAART) and seronegative controls. A total of 593 and 601 proteins were identified in the pooled saliva samples from 5 HIV-1 subjects and 5 controls, respectively. Forty-one proteins were found to be differentially expressed. Bioinformatic analysis of differentially expressed salivary proteins showed an increase of antimicrobial proteins and decrease of protease inhibitors upon HIV-1 infection. To validate some of these differentially expressed proteins, a high-throughput quantitation method was established to determine concentrations of 10 salivary proteins in 40 individual saliva samples from 20 seropositive patients before HAART and 20 seronegative subjects. This method was based on limited protein separation within the zone of the stacking gel of the 1D SDS PAGE and using isotope-coded synthetic peptides as internal standards. The results demonstrated that a combination of protein profiling and targeted quantitation is an efficient method to identify and validate differentially expressed salivary proteins. Expression levels of members of the calcium-binding S100 protein family and deleted in malignant brain tumors 1 protein (DMBT1) were up-regulated while that of Mucin 5B was down-regulated in HIV-1 seropositive saliva samples, which may provide new perspectives for monitoring HIV-infection and understanding the mechanism of HIV-1 infectivity.

  7. Immunogenicity of a polyvalent HIV-1 candidate vaccine based on fourteen wild type gp120 proteins in golden hamsters

    Directory of Open Access Journals (Sweden)

    Ghorbani Masoud

    2006-10-01

    Full Text Available Abstract Background One of the major obstacles in the design of an effective vaccine against HIV-1 is the hypervariability of the HIV-1 envelope glycoprotein. Most HIV-1 vaccine candidates have utilized envelope glycoprotein from a single virus isolate, but to date, none of them elicited broadly reactive humoral immunity. Herein, we hypothesised that a cocktail of HIV-1 gp120 proteins containing multiple epitopes may increase the breadth of immune responses against HIV-1. We compared and evaluated the immunogenicity of HIV-1 vaccines containing either gp120 protein alone or in combinations of four or fourteen gp120s from different primary HIV-1 isolates in immunized hamsters. Results We amplified and characterized 14 different gp120s from primary subtype B isolates with both syncytium and non-syncytium inducing properties, and expressed the proteins in Chinese Hamster Ovary (CHO cell lines. Purified proteins were used either alone or in combinations of four or fourteen different gp120s to vaccinate golden hamsters. The polyvalent vaccine showed higher antibody titers to HIV-1 subtype B isolates MN and SF162 compared to the groups that received one or four gp120 proteins. However, the polyvalent vaccine was not able to show higher neutralizing antibody responses against HIV-1 primary isolates. Interestingly, the polyvalent vaccine group had the highest proliferative immune responses and showed a substantial proportion of cross-subtype CD4 reactivity to HIV-1 subtypes B, C, and A/E Conclusion Although the polyvalent approach achieved only a modest increase in the breadth of humoral and cellular immunity, the qualitative change in the vaccine (14 vs. 1 gp120 resulted in a quantitative improvement in vaccine-induced immunity.

  8. CTL Responses to Regulatory Proteins Tat and Rev in HIV-1 B'/C Virus-Infected Individuals

    Institute of Scientific and Technical Information of China (English)

    MING-MING JIA; KUN-XUE HONG; JIAN-PING CHEN; HONG-WEI LIU; SHA LIU; XIAO-QING ZHANG; HONG-JING ZHAO; YI-MING SHAO

    2008-01-01

    To characterize HIV-1 specific CTL responses to regulatory proteins Tat and Rev in HIV-B'/C vires-infected ART-naive individuals. Methods HIV-1-specific CTL responses were analyzed by IFN-γ ELISPOT assay using overlapping peptides spanning the consensus sequences of HIV-1 clade C Tat and Rev proteins. Statistical analysis and graphical presentation were performed using SIGMAPLOT 10.0 and SIGMASTAT 3.5. For samples with a positive response, the magnitude of CTL responses was compared between HIV-1 C proteins by Wilcoxon rank sum test, and the significance threshold was P<0.05. Results Tat and Rev were frequently recognized, with 23% and 52% of the tested individuals having detectable responses to these proteins, respectively. Several immunodominant regions were detected in Rev. No significant correlation was observed between the magnitude and breadth of CTL responses to regulatory proteins and the control of virus replication in this study. Conclusion Tat and Rev can serve as targets for HIV-1-specific CTL, and several immunodominant regions are detectable in Rev. Further characterization of epitopes and their role in virus control may shed light on pathogenesis of HIV-1 natural infection and also be useful for the design and testing of candidate vaccines.

  9. Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells

    Directory of Open Access Journals (Sweden)

    Gay Bernard

    2011-09-01

    Full Text Available Abstract Background Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. Results We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. Conclusion These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP.

  10. Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells.

    Science.gov (United States)

    Clerc, Isabelle; Laverdure, Sylvain; Torresilla, Cynthia; Landry, Sébastien; Borel, Sophie; Vargas, Amandine; Arpin-André, Charlotte; Gay, Bernard; Briant, Laurence; Gross, Antoine; Barbeau, Benoît; Mesnard, Jean-Michel

    2011-09-19

    Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP.

  11. HIV-related proteins prolong macrophage survival through induction of Triggering receptor expressed on myeloid cells-1

    Science.gov (United States)

    Yuan, Zhihong; Fan, Xian; Staitieh, Bashar; Bedi, Chetna; Spearman, Paul; Guidot, David M; Sadikot, Ruxana T

    2017-01-01

    Triggering receptor expressed on myeloid cells-1(TREM-1) is a member of the superimmunoglobulin receptor family. We have previously shown that TREM-1 prolongs survival of macrophages treated with lipoolysaccharide through Egr2-Bcl2 signaling. Recent studies suggest a role for TREM-1 in viral immunity. Human immunodeficiency virus-1 (HIV) targets the monocyte/macrophage lineage at varying stages of infection. Emerging data suggest that macrophages are key reservoirs for latent HIV even in individuals on antiretroviral therapy. Here, we investigated the potential role of TREM-1 in HIV latency in macrophages. Our data show that human macrophages infected with HIV show an increased expression of TREM-1. In parallel, direct exposure to the HIV-related proteins Tat or gp120 induces TREM-1 expression in macrophages and confers anti-apoptotic attributes.NF-κB p65 silencing identified that these proteins induce TREM-1 in p65-dependent manner. TREM-1 silencing in macrophages exposed to HIV-related proteins led to increased caspase 3 activation and reduced Bcl-2 expression, rendering them susceptible to apotosis. These novel data reveal that TREM-1 may play a critical role in establishing HIV reservoir in macrophages by inhibiting apoptosis. Therefore, targeting TREM-1 could be a novel therapeutic approach to enhance clearance of the HIV reservoir, at least within the macrophage pools. PMID:28181540

  12. Running loose or getting lost: how HIV-1 counters and capitalizes on APOBEC3-induced mutagenesis through its Vif protein.

    Science.gov (United States)

    Münk, Carsten; Jensen, Björn-Erik O; Zielonka, Jörg; Häussinger, Dieter; Kamp, Christel

    2012-11-14

    Human immunodeficiency virus-1 (HIV-1) dynamics reflect an intricate balance within the viruses’ host. The virus relies on host replication factors, but must escape or counter its host’s antiviral restriction factors. The interaction between the HIV-1 protein Vif and many cellular restriction factors from the APOBEC3 protein family is a prominent example of this evolutionary arms race. The viral infectivity factor (Vif) protein largely neutralizes APOBEC3 proteins, which can induce in vivo hypermutations in HIV-1 to the extent of lethal mutagenesis, and ensures the production of viable virus particles. HIV-1 also uses the APOBEC3-Vif interaction to modulate its own mutation rate in harsh or variable environments, and it is a model of adaptation in a coevolutionary setting. Both experimental evidence and the substantiation of the underlying dynamics through coevolutionary models are presented as complementary views of a coevolutionary arms race.

  13. Running Loose or Getting Lost: How HIV-1 Counters and Capitalizes on APOBEC3-Induced Mutagenesis through Its Vif Protein

    Directory of Open Access Journals (Sweden)

    Christel Kamp

    2012-11-01

    Full Text Available Human immunodeficiency virus-1 (HIV-1 dynamics reflect an intricate balance within the viruses’ host. The virus relies on host replication factors, but must escape or counter its host’s antiviral restriction factors. The interaction between the HIV-1 protein Vif and many cellular restriction factors from the APOBEC3 protein family is a prominent example of this evolutionary arms race. The viral infectivity factor (Vif protein largely neutralizes APOBEC3 proteins, which can induce in vivo hypermutations in HIV-1 to the extent of lethal mutagenesis, and ensures the production of viable virus particles. HIV-1 also uses the APOBEC3-Vif interaction to modulate its own mutation rate in harsh or variable environments, and it is a model of adaptation in a coevolutionary setting. Both experimental evidence and the substantiation of the underlying dynamics through coevolutionary models are presented as complementary views of a coevolutionary arms race.

  14. A mouse model based on replication-competent Tiantan vaccinia expressing luciferase/HIV-1 Gag fusion protein for the evaluation of protective efficacy of HIV vaccine

    Institute of Scientific and Technical Information of China (English)

    HUANG Yang; QIU Chao; LIU Lian-xing; FENG Yan-meng; ZHU Ting; XU Jian-qing

    2009-01-01

    Background Developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1) remains a grand challenge after more than two decades of intensive effort. It is partially due to the lack of suitable animal models for screening and prioritizing vaccine candidates. In this study, we aim to develop a mice model to test HIV-1 vaccine efficacy. Methods We constructed a recombinant vaccinia expressing firefly luciferase and HIV-1 Gag fusion protein based on Tiantan strain, an attenuated but replication-competent poxvirus (rTTV-lucgag). By quantifying the luciferase activity as its read out, we defined the biodistribution of Tiantan strain poxvirus in mice inoculated intraperitoneally and attempted to apply this model to evaluate the HIV-1 vaccine efficacy. Results Our data demonstrated that the rTTV-lucgag was able to express high level of luciferase (≤106 relative luciferase units (RLU)/mg protein) and HIV-1 Gag (>3 folds increase comparing to the control). After intraperitoneal inoculation, this virus had dominant replication in the ovary, uterus, and cervix of mice and the luciferase activities in those organs are significantly correlated with viral titers (r2=0.71, P <0.01). Pre-immunization with an HIV gag DNA vaccine reduced the luciferase activity in ovary from (6006+3141) RLU/mg protein in control group to (1538±463) RLU/mg protein in vaccine group (P=0.1969). Conclusions The luciferase activity in ovary could represent viral replication in vivo;, this rTTV-lucgag/mice model may be suitable to assess the protective efficacy of cytotoxic T-cell responses to HIV Gag with less tedious work and high through-put.

  15. Effects of Opiates and HIV Proteins on Neurons: The Role of Ferritin Heavy Chain and a Potential for Synergism

    OpenAIRE

    Festa, Lindsay; Meucci, Olimpia

    2012-01-01

    Human immunodeficiency virus 1 (HIV-1) and its associated proteins can have a profound impact on the central nervous system. Co-morbid abuse of opiates, such as morphine and heroin, is often associated with rapid disease progression and greater neurological dysfunction. The mechanisms by which HIV proteins and opiates cause neuronal damage on their own and together are unclear. The emergence of ferritin heavy chain (FHC) as a negative regulator of the chemokine receptor CXCR4, a co-receptor f...

  16. Inhibition of HIV-1 replication by balsamin, a ribosome inactivating protein of Momordica balsamina.

    Science.gov (United States)

    Kaur, Inderdeep; Puri, Munish; Ahmed, Zahra; Blanchet, Fabien P; Mangeat, Bastien; Piguet, Vincent

    2013-01-01

    Ribosome-inactivating proteins (RIPs) are endowed with several medicinal properties, including antiviral activity. We demonstrate here that the recently identified type I RIP from Momordica balsamina also possesses antiviral activity, as determined by viral growth curve assays and single-round infection experiments. Importantly, this activity is at play even as doses where the RIP has no cytotoxic effect. In addition, balsamin inhibits HIV-1 replication not only in T cell lines but also in human primary CD4(+) T cells. This antiviral compound exerts its activity at a viral replicative step occurring later than reverse-transcription, most likely on viral protein translation, prior to viral budding and release. Finally, we demonstrate that balsamin antiviral activity is broad since it also impedes influenza virus replication. Altogether our results demonstrate that type I RIP can exert a potent anti-HIV-1 activity which paves the way for new therapeutic avenues for the treatment of viral infections.

  17. Dendritic cells primed with a chimeric plasmid containing HIV-1-gag associated with lysosomal-associated protein-1 (LAMP/gag) is a potential therapeutic vaccine against HIV.

    Science.gov (United States)

    Lucas, Carolina G D O; Matassoli, Flavio L; Peçanha, Ligia M T; Santillo, Bruna Tereso; Oliveira, Luanda Mara da Silva; Oshiro, Telma Miyuki; Marques, Ernesto T D A; Oxenius, Annette; de Arruda, Luciana B

    2016-08-01

    The decline in number and function of T cells is a hallmark of HIV infection, and preservation or restoration of HIV-specific cellular immune response is a major goal of AIDS treatment. Dendritic cells (DCs) play a key role in the initiation and maintenance of the immune response, and their use as a vaccine vehicle is a promising strategy for enhancing vaccine efficacy. We evaluated the potential of DC-mediated immunization with a DNA vaccine consisting of HIV-1-p55gag (gag, group-specific antigen) associated to lysosomal associated protein (LAMP) sequence (LAMP/gag vaccine). Immunization of mice with mouse DCs transfected with LAMP/gag (Lg-mDCs) stimulated more potent B- and T-cell responses than naked DNA or DCs pulsed with inactivated HIV. Anti-Gag antibody levels were sustained for at least 3 mo after immunization, and recall T-cell responses were also strongly detected at this time point. Human DCs transfected with LAMP/gag (Lg-hDCs) were also activated and able to stimulate greater T-cell response than native gag-transfected DCs. Coculture between Lg-hDCs and T lymphocytes obtained from patients with HIV resulted in upregulation of CD38, CD69, HLA-DR, and granzyme B by CD4(+) and CD8(+) T cells, and increased IFN-γ and TNF-α production. These results indicate that the use of LAMP/gag-DC may be an efficient strategy for enhancing immune function in patients with HIV.-Lucas, C. G. D. O., Matassoli, F. L., Peçanha, L. M. T., Santillo, B. T., Oliveira, L. M. D. S., Oshiro, T. M., Marques, E. T. D. A., Jr., Oxenius, A., de Arruda, L. B. Dendritic cells primed with a chimeric plasmid containing HIV-1-gag associated with lysosomal-associated protein-1 (LAMP/gag) is a potential therapeutic vaccine against HIV.

  18. Extracellular Matrix Proteins Mediate HIV-1 gp120 Interactions with α4β7.

    Science.gov (United States)

    Plotnik, David; Guo, Wenjin; Cleveland, Brad; von Haller, Priska; Eng, Jimmy K; Guttman, Miklos; Lee, Kelly K; Arthos, James; Hu, Shiu-Lok

    2017-08-16

    Gut-homing α4β7(high) CD4(+) T lymphocytes have been shown to be preferentially targeted by Human Immunodeficiency Virus-1 (HIV), and are implicated in HIV pathogenesis. Previous studies demonstrated that HIV envelope protein gp120 binds and signals through α4β7, and that this likely contributes to the infection of α4β7(high) T cells and promotes cell-to-cell virus transmission. Structures within the second variable loop (V2) of gp120, including the tripeptide motif LDV/I, are thought to mediate gp120-α4β7 binding. However, lack of α4β7 binding has been reported in gp120 proteins containing LDV/I, and the precise determinants of gp120-α4β7 binding are not fully defined. In this work, we report the novel finding that fibronectins mediate indirect gp120-α4β7 interactions. We show that Chinese Hamster Ovary (CHO) cells used to express recombinant gp120 produced fibronectins and other extracellular matrix proteins that co-purified with gp120. CHO fibronectins were able to mediate the binding of a diverse panel of gp120 proteins to α4β7 in an in vitro cell binding assay. The V2 loop was not required for fibronectin-mediated binding of gp120 to α4β7, nor did V2-specific antibodies block this interaction. Removal of fibronectin through anion exchange chromatography abrogated V2-independent gp120-α4β7 binding. Additionally, we showed a recombinant human fibronectin fragment mediated gp120-α4β7 interactions in a similar manner to CHO fibronectin. These findings provide an explanation for the apparent contradictory observations regarding the gp120-α4β7 interaction and offer new insights into the potential role of fibronectin and other extracellular matrix proteins in HIV-1 biology.IMPORTANCE Immune tissues within the gut are severely damaged by HIV, and this plays an important role in the development of AIDS. Integrin α4β7 plays a major role in the trafficking of lymphocytes, including CD4(+) T cells, into gut lymphoid tissues. Previous reports

  19. IL-7 receptor recovery on CD8 T-cells isolated from HIV+ patients is inhibited by the HIV Tat protein.

    Directory of Open Access Journals (Sweden)

    Elliott M Faller

    Full Text Available Expression of the IL-7 receptor α-chain (CD127 is decreased on CD8 T-cells in HIV infected patients and partially recovers in those receiving antiretroviral therapy with sustained viral suppression. We have shown that soluble HIV Tat protein down regulates CD127 expression on CD8 T-cells isolated from healthy HIV-negative individuals. Tat is taken up by CD8 T-cells via endocytosis, exits the endosome and then translocates to the inner leaflet of the cell membrane where it binds to the cytoplasmic tail of CD127 inducing receptor internalization and degradation by the proteasome. This down regulation of CD127 by Tat results in impaired CD8 T-cell function. Interestingly, suppression of CD127 by Tat is reversible and requires the continual presence of Tat in the culture media. We thus questioned whether the low IL-7 receptor expression evident on CD8 T-cells in HIV+ patients was similarly reversible and if suppression of the receptor could be maintained ex vivo by Tat protein alone. We show here that when CD8 T-cells isolated from HIV+ patients are incubated alone in fresh medium, low CD127 expression on the cell surface recovers to normal levels. This recovery of CD127, however, is completely inhibited by the addition of HIV Tat protein to the culture media. This study then provides evidence that soluble factor(s are responsible for low CD127 expression on circulating CD8 T-cells in HIV+ individuals and further implicates Tat in suppressing this receptor essential to CD8 T-cell proliferation and function.

  20. IL-7 Receptor Recovery on CD8 T-Cells Isolated from HIV+ Patients Is Inhibited by the HIV Tat Protein

    Science.gov (United States)

    Faller, Elliott M.; McVey, Mark J.; MacPherson, Paul A.

    2014-01-01

    Expression of the IL-7 receptor α-chain (CD127) is decreased on CD8 T-cells in HIV infected patients and partially recovers in those receiving antiretroviral therapy with sustained viral suppression. We have shown that soluble HIV Tat protein down regulates CD127 expression on CD8 T-cells isolated from healthy HIV-negative individuals. Tat is taken up by CD8 T-cells via endocytosis, exits the endosome and then translocates to the inner leaflet of the cell membrane where it binds to the cytoplasmic tail of CD127 inducing receptor internalization and degradation by the proteasome. This down regulation of CD127 by Tat results in impaired CD8 T-cell function. Interestingly, suppression of CD127 by Tat is reversible and requires the continual presence of Tat in the culture media. We thus questioned whether the low IL-7 receptor expression evident on CD8 T-cells in HIV+ patients was similarly reversible and if suppression of the receptor could be maintained ex vivo by Tat protein alone. We show here that when CD8 T-cells isolated from HIV+ patients are incubated alone in fresh medium, low CD127 expression on the cell surface recovers to normal levels. This recovery of CD127, however, is completely inhibited by the addition of HIV Tat protein to the culture media. This study then provides evidence that soluble factor(s) are responsible for low CD127 expression on circulating CD8 T-cells in HIV+ individuals and further implicates Tat in suppressing this receptor essential to CD8 T-cell proliferation and function. PMID:25033393

  1. Antiretroviral treatment reduces increased CSF neurofilament protein (NFL) in HIV-1 infection.

    Science.gov (United States)

    Mellgren, A; Price, R W; Hagberg, L; Rosengren, L; Brew, B J; Gisslén, M

    2007-10-09

    Increased levels of the light-chain neurofilament protein (NFL) in CSF provide a marker of CNS injury in several neurodegenerative disorders and have been reported in the AIDS dementia complex (ADC). We examined the effects of highly active antiretroviral treatment (HAART) on CSF NFL in HIV-1-infected subjects with and without ADC who underwent repeated lumbar punctures (LPs). NFL was measured by ELISA (normal reference value NFL at baseline, with a median level of 780 ng/L and an intraquartile range (IQR) of 480 to 7300. After 3 months of treatment, NFL concentrations had fallen to normal in 48% (10/21), and the median decreased to 340 ng/L (IQR NFL levels. Thirty-two subjects had normal NFL at baseline, and all but one remained normal at follow-up. These effects on CSF NFL were seen in association with clinical improvement in ADC patients, decreases in plasma and CSF HIV-1 RNA and CSF neopterin, and increases in blood CD4 T cell counts. HAART seems to halt the neurodegenerative process(es) caused by HIV-1, as shown by the significant decrease in CSF NFL after treatment initiation. CSF NFL may serve as a useful marker in monitoring CNS injury in HIV-1 infection and in evaluating CNS efficacy of antiretroviral therapy.

  2. Virus-producing cells determine the host protein profiles of HIV-1 virion cores

    Directory of Open Access Journals (Sweden)

    Santos Steven

    2012-08-01

    Full Text Available Abstract Background Upon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs, which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized. Results We examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes, PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ, and non-activated THP1 cells (model of monocytes, mMN and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90 with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29, DNA binding (17, cytoskeleton (15, cytoskeleton regulation (21, chaperone (18, vesicular trafficking-associated (12 and ubiquitin-proteasome pathway-associated proteins (9 were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins

  3. Adhesion and fusion efficiencies of human immunodeficiency virus type 1 (HIV-1) surface proteins

    OpenAIRE

    2013-01-01

    In about half of patients infected with HIV-1 subtype B, viral populations shift from utilizing the transmembrane protein CCR5 to CXCR4, as well as or instead of CCR5, during late stage progression of the disease. How the relative adhesion efficiency and fusion competency of the viral Env proteins relate to infection during this transition is not well understood. Using a virus-cell fusion assay and live-cell single-molecule force spectroscopy, we compare the entry competency of viral clones t...

  4. Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: Implications for glycosylation and CD4 binding

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, C.I.; Lennick, M.; Lehar, S.M.; Beltz, G.A.; Young, E. (Cambridge Bioscience Corporation, Worcester, MA (USA))

    1990-10-01

    Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4.

  5. Labeling of multiple HIV-1 proteins with the biarsenical-tetracysteine system.

    Directory of Open Access Journals (Sweden)

    Cândida F Pereira

    Full Text Available Due to its small size and versatility, the biarsenical-tetracysteine system is an attractive way to label viral proteins for live cell imaging. This study describes the genetic labeling of the human immunodeficiency virus type 1 (HIV-1 structural proteins (matrix, capsid and nucleocapsid, enzymes (protease, reverse transcriptase, RNAse H and integrase and envelope glycoprotein 120 with a tetracysteine tag in the context of a full-length virus. We measure the impact of these modifications on the natural virus infection and, most importantly, present the first infectious HIV-1 construct containing a fluorescently-labeled nucleocapsid protein. Furthermore, due to the high background levels normally associated with the labeling of tetracysteine-tagged proteins we have also optimized a metabolic labeling system that produces infectious virus containing the natural envelope glycoproteins and specifically labeled tetracysteine-tagged proteins that can easily be detected after virus infection of T-lymphocytes. This approach can be adapted to other viral systems for the visualization of the interplay between virus and host cell during infection.

  6. Interferons and interferon (IFN)-inducible protein 10 during highly active anti-retroviral therapy (HAART)-possible immunosuppressive role of IFN-alpha in HIV infection

    DEFF Research Database (Denmark)

    Stylianou, E; Aukrust, P; Bendtzen, K;

    2000-01-01

    Interferons play an important, but incompletely understood role in HIV-related disease. We investigated the effect of HAART on plasma levels of IFN-alpha, IFN-gamma, neopterin and interferon-inducible protein 10 (IP-10) in 41 HIV-infected patients during 78 weeks of therapy. At baseline HIV-infec...

  7. Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses

    Directory of Open Access Journals (Sweden)

    Clavel François

    2008-02-01

    Full Text Available Abstract Background Numerous studies have shown that viral quasi-species with genetically diverse envelope proteins (Env replicate simultaneously in patients infected with the human immunodeficiency virus type 1 (HIV-1. Less information is available concerning the extent that envelope sequence diversity translates into a diversity of phenotypic properties, including infectivity and resistance to entry inhibitors. Methods To study these questions, we isolated genetically distinct contemporaneous clonal viral populations from the plasma of 5 HIV-1 infected individuals (n = 70, and evaluated the infectivity of recombinant viruses expressing Env proteins from the clonal viruses in several target cells. The sensitivity to entry inhibitors (enfuvirtide, TAK-799, soluble CD4 and monoclonal antibodies (2G12, 48d, 2F5 was also evaluated for a subset of the recombinant viruses (n = 20. Results Even when comparisons were restricted to viruses with similar tropism, the infectivity for a given target cell of viruses carrying different Env proteins from the same patient varied over an approximately 10-fold range, and differences in their relative ability to infect different target cells were also observed. Variable region haplotypes associated with high and low infectivity could be identified for one patient. In addition, clones carrying unique mutations in V3 often displayed low infectivity. No correlation was observed between viral infectivity and sensitivity to inhibition by any of the six entry inhibitors evaluated, indicating that these properties can be dissociated. Significant inter-patient differences, independent of infectivity, were observed for the sensitivity of Env proteins to several entry inhibitors and their ability to infect different target cells. Conclusion These findings demonstrate the marked functional heterogeneity of HIV-1 Env proteins expressed by contemporaneous circulating viruses, and underscore the advantage of clonal analyses in

  8. Enhanced HIV-1 neutralization by a CD4-VH3-IgG1 fusion protein

    Energy Technology Data Exchange (ETDEWEB)

    Meyuhas, Ronit; Noy, Hava; Fishman, Sigal [Laboratory of Immunology, MIGAL, P.O. Box 831, Kiryat Shmona 11016 (Israel); Margalit, Alon [Laboratory of Immunology, MIGAL, P.O. Box 831, Kiryat Shmona 11016 (Israel); Department of Biotechnology, Tel-Hai Academic College, Upper Galilee 12210 (Israel); Montefiori, David C. [Department of Surgery, Duke University Medical Center, Durham, NC 27710 (United States); Gross, Gideon, E-mail: gidi@migal.org.il [Laboratory of Immunology, MIGAL, P.O. Box 831, Kiryat Shmona 11016 (Israel); Department of Biotechnology, Tel-Hai Academic College, Upper Galilee 12210 (Israel)

    2009-08-21

    HIV-1 gp120 is an alleged B cell superantigen, binding certain VH3+ human antibodies. We reasoned that a CD4-VH3 fusion protein could possess higher affinity for gp120 and improved HIV-1 inhibitory capacity. To test this we produced several human IgG1 immunoligands harboring VH3. Unlike VH3-IgG1 or VH3-CD4-IgG1, CD4-VH3-IgG1 bound gp120 considerably stronger than CD4-IgG1. CD4-VH3-IgG1 exhibited {approx}1.5-2.5-fold increase in neutralization of two T-cell laboratory-adapted strains when compared to CD4-IgG1. CD4-VH3-IgG1 improved neutralization of 7/10 clade B primary isolates or pseudoviruses, exceeding 20-fold for JR-FL and 13-fold for Ba-L. It enhanced neutralization of 4/8 clade C viruses, and had negligible effect on 1/4 clade A pseudoviruses. We attribute this improvement to possible pairing of VH3 with CD4 D1 and stabilization of an Ig Fv-like structure, rather than to superantigen interactions. These novel findings support the current notion that CD4 fusion proteins can act as better HIV-1 entry inhibitors with potential clinical implications.

  9. HIV Tat protein and amyloid-β peptide form multifibrillar structures that cause neurotoxicity.

    Science.gov (United States)

    Hategan, Alina; Bianchet, Mario A; Steiner, Joseph; Karnaukhova, Elena; Masliah, Eliezer; Fields, Adam; Lee, Myoung-Hwa; Dickens, Alex M; Haughey, Norman; Dimitriadis, Emilios K; Nath, Avindra

    2017-02-20

    Deposition of amyloid-β plaques is increased in the brains of HIV-infected individuals, and the HIV transactivator of transcription (Tat) protein affects amyloidogenesis through several indirect mechanisms. Here, we investigated direct interactions between Tat and amyloid-β peptide. Our in vitro studies showed that in the presence of Tat, uniform amyloid fibrils become double twisted fibrils and further form populations of thick unstructured filaments and aggregates. Specifically, Tat binding to the exterior surfaces of the Aβ fibrils increases β-sheet formation and lateral aggregation into thick multifibrillar structures, thus producing fibers with increased rigidity and mechanical resistance. Furthermore, Tat and Aβ aggregates in complex synergistically induced neurotoxicity both in vitro and in animal models. Increased rigidity and mechanical resistance of the amyloid-β-Tat complexes coupled with stronger adhesion due to the presence of Tat in the fibrils may account for increased damage, potentially through pore formation in membranes.

  10. HIV-1 uncoating: connection to nuclear entry and regulation by host proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ambrose, Zandrea, E-mail: zaa4@pitt.edu [Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261 (United States); Aiken, Christopher [Department of Pathology, Microbiology and Immunology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States)

    2014-04-15

    The RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed by a capsid shell that dissociates within the cell in a multistep process known as uncoating, which influences completion of reverse transcription of the viral genome. Double-stranded viral DNA is imported into the nucleus for integration into the host genome, a hallmark of retroviral infection. Reverse transcription, nuclear entry, and integration are coordinated by a capsid uncoating process that is regulated by cellular proteins. Although uncoating is not well understood, recent studies have revealed insights into the process, particularly with respect to nuclear import pathways and protection of the viral genome from DNA sensors. Understanding uncoating will be valuable toward developing novel antiretroviral therapies for HIV-infected individuals.

  11. The histone chaperone protein Nucleosome Assembly Protein-1 (hNAP-1 binds HIV-1 Tat and promotes viral transcription

    Directory of Open Access Journals (Sweden)

    Marcello Alessandro

    2008-01-01

    Full Text Available Abstract Background Despite the large amount of data available on the molecular mechanisms that regulate HIV-1 transcription, crucial information is still lacking about the interplay between chromatin conformation and the events that regulate initiation and elongation of viral transcription. During transcriptional activation, histone acetyltransferases and ATP-dependent chromatin remodeling complexes cooperate with histone chaperones in altering chromatin structure. In particular, human Nucleosome Assembly Protein-1 (hNAP-1 is known to act as a histone chaperone that shuttles histones H2A/H2B into the nucleus, assembles nucleosomes and promotes chromatin fluidity, thereby affecting transcription of several cellular genes. Results Using a proteomic screening, we identified hNAP-1 as a novel cellular protein interacting with HIV-1 Tat. We observed that Tat specifically binds hNAP1, but not other members of the same family of factors. Binding between the two proteins required the integrity of the basic domain of Tat and of two separable domains of hNAP-1 (aa 162–290 and 290–391. Overexpression of hNAP-1 significantly enhanced Tat-mediated activation of the LTR. Conversely, silencing of the protein decreased viral promoter activity. To explore the effects of hNAP-1 on viral infection, a reporter HIV-1 virus was used to infect cells in which hNAP-1 had been either overexpressed or knocked-down. Consistent with the gene expression results, these two treatments were found to increase and inhibit viral infection, respectively. Finally, we also observed that the overexpression of p300, a known co-activator of both Tat and hNAP-1, enhanced hNAP-1-mediated transcriptional activation as well as its interaction with Tat. Conclusion Our study reveals that HIV-1 Tat binds the histone chaperone hNAP-1 both in vitro and in vivo and shows that this interaction participates in the regulation of Tat-mediated activation of viral gene expression.

  12. HIV-1 Nef binds with human GCC185 protein and regulates mannose 6 phosphate receptor recycling

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manjeet; Kaur, Supinder; Nazir, Aamir; Tripathi, Raj Kamal, E-mail: rajkamalcdri@gmail.com

    2016-05-20

    HIV-1 Nef modulates cellular function that enhances viral replication in vivo which culminate into AIDS pathogenesis. With no enzymatic activity, Nef regulates cellular function through host protein interaction. Interestingly, trans-cellular introduction of recombinant Nef protein in Caenorhabditis elegans results in AIDS like pathogenesis which might share common pathophysiology because the gene sequence of C. elegans and humans share considerable homology. Therefore employing C. elegans based initial screen complemented with sequence based homology search we identified GCC185 as novel host protein interacting with HIV-1 Nef. The detailed molecular characterization revealed N-terminal EEEE{sub 65} acidic domain of Nef as key region for interaction. GCC185 is a tethering protein that binds with Rab9 transport vesicles. Our results show that Nef-GCC185 interaction disrupts Rab9 interaction resulting in delocalization of CI-MPR (cation independent Mannose 6 phosphate receptor) resulting in elevated secretion of hexosaminidase. In agreement with this, our studies identified novel host GCC185 protein that interacts with Nef EEEE65 acidic domain interfering GCC185-Rab9 vesicle membrane fusion responsible for retrograde vesicular transport of CI-MPR from late endosomes to TGN. In light of existing report suggesting critical role of Nef-GCC185 interaction reveals valuable mechanistic insights affecting specific protein transport pathway in docking of late endosome derived Rab9 bearing transport vesicle at TGN elucidating role of Nef during viral pathogenesis. -- Highlights: •Nef, an accessory protein of HIV-1 interacts with host factor and culminates into AIDS pathogenesis. •Using Caenorhabditis elegans based screen system, novel Nef interacting cellular protein GCC185 was identified. •Molecular characterization of Nef and human protein GCC185 revealed Nef EEEE{sub 65} key region interacted with full length GCC185. •Nef impeded the GCC185-Rab 9 interaction and

  13. A multi-scale mathematical modeling framework to investigate anti-viral therapeutic opportunities in targeting HIV-1 accessory proteins.

    Science.gov (United States)

    Suryawanshi, Gajendra W; Hoffmann, Alexander

    2015-12-07

    Human immunodeficiency virus-1 (HIV-1) employs accessory proteins to evade innate immune responses by neutralizing the anti-viral activity of host restriction factors. Apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, A3G) and bone marrow stromal cell antigen 2 (BST2) are host resistance factors that potentially inhibit HIV-1 infection. BST2 reduces viral production by tethering budding HIV-1 particles to virus producing cells, while A3G inhibits the reverse transcription (RT) process and induces viral genome hypermutation through cytidine deamination, generating fewer replication competent progeny virus. Two HIV-1 proteins counter these cellular restriction factors: Vpu, which reduces surface BST2, and Vif, which degrades cellular A3G. The contest between these host and viral proteins influences whether HIV-1 infection is established and progresses towards AIDS. In this work, we present an age-structured multi-scale viral dynamics model of in vivo HIV-1 infection. We integrated the intracellular dynamics of anti-viral activity of the host factors and their neutralization by HIV-1 accessory proteins into the virus/cell population dynamics model. We calculate the basic reproductive ratio (Ro) as a function of host-viral protein interaction coefficients, and numerically simulated the multi-scale model to understand HIV-1 dynamics following host factor-induced perturbations. We found that reducing the influence of Vpu triggers a drop in Ro, revealing the impact of BST2 on viral infection control. Reducing Vif׳s effect reveals the restrictive efficacy of A3G in blocking RT and in inducing lethal hypermutations, however, neither of these factors alone is sufficient to fully restrict HIV-1 infection. Interestingly, our model further predicts that BST2 and A3G function synergistically, and delineates their relative contribution in limiting HIV-1 infection and disease progression. We provide a robust modeling framework for devising novel combination therapies that target

  14. Epstein Barr Virus detection and latent membrane protein 1 in oral hairy leukoplakia in HIV+ Venezuelan patients.

    Science.gov (United States)

    González, Xiomara; Correnti, María; Rivera, Helen; Perrone, Marianella

    2010-03-01

    To determine the prevalence of Epstein Barr virus (EBV) in oral hairy leukoplakia lesions (OHL) in HIV+ Venezuelan patients. In this case study, we evaluated 21 HIV+ adult patients with clinically present OHL lesions, 11 who were undergoing antiretroviral therapy, 10 who were not undergoing therapy and 10 HIV-negative adult patients with hyperkeratotic oral mucosal lesions. All of the subjects were assessed at the Infectious Disease Center, Faculty of Dentistry, Central University of Venezuela, and were clinically examined to detect oral mucosal lesions with the confirmed histopathologic diagnosis. Nested-PCR was used to determine the EBV infection and the latent membrane protein-1 (LMP-1) expression by immunohistochemistry. Of the subjects, 16/21 (76%) of the HIV+/AIDS patients tested positive for EBV, whereas 5/10 (50%) of the HIV-negative subjects tested positive for EBV. In the present study, a higher EBV prevalence was observed in HIV-positive patients when compared to HIV-negative patients without oral hairy leukoplakia, confirming the etiologic role in this entity. The LMP-1 in OHL patients who were both HIV+ and EBV+ was highly expressed (60%) at the epithelial basal cells. No association between the alcohol and tobacco consumption was observed among the EBV-positive cases.

  15. Human Antibodies that Recognize Novel Immunodominant Quaternary Epitopes on the HIV-1 Env Protein

    Science.gov (United States)

    Hicar, Mark D.; Chen, Xuemin; Sulli, Chidananda; Barnes, Trevor; Goodman, Jason; Sojar, Hakimuddin; Briney, Bryan; Willis, Jordan; Chukwuma, Valentine U.; Kalams, Spyros A.; Doranz, Benjamin J.; Spearman, Paul; Crowe, James E.

    2016-01-01

    Numerous broadly neutralizing antibodies (Abs) target epitopes that are formed or enhanced during mature HIV envelope formation (i.e. quaternary epitopes). Generally, it is thought that Env epitopes that induce broadly neutralizing Abs are difficult to access and poorly immunogenic because of the characteristic oligomerization, conformational flexibility, sequence diversity and extensive glycosylation of Env protein. To enhance for isolation of quaternary epitope-targeting Abs (QtAbs), we previously used HIV virus-like particles (VLPs) to bind B cells from long-term non-progressor subjects to identify a panel of monoclonal Abs. When expressed as recombinant full-length Abs, a subset of these novel Abs exhibited the binding profiles of QtAbs, as they either failed to bind to monomeric Env protein or showed much higher affinity for Env trimers and VLPs. These QtAbs represented a significant proportion of the B-cell response identified with VLPs. The Ab genes of these clones were highly mutated, but they did not neutralize common HIV strains. We sought to further define the epitopes targeted by these QtAbs. Competition-binding and mapping studies revealed these Abs targeted four separate epitopes; they also failed to compete for binding by Abs to known major neutralizing epitopes. Detailed epitope mapping studies revealed that two of the four epitopes were located in the gp41 subunit of Env. These QtAbs bound pre-fusion forms of antigen and showed differential binding kinetics depending on whether oligomers were produced as recombinant gp140 trimers or as full-length Env incorporated into VLPs. Antigenic regions within gp41 present unexpectedly diverse structural epitopes, including these QtAb epitopes, which may be targeted by the naturally occurring Ab response to HIV infection. PMID:27411063

  16. Human Antibodies that Recognize Novel Immunodominant Quaternary Epitopes on the HIV-1 Env Protein.

    Directory of Open Access Journals (Sweden)

    Mark D Hicar

    Full Text Available Numerous broadly neutralizing antibodies (Abs target epitopes that are formed or enhanced during mature HIV envelope formation (i.e. quaternary epitopes. Generally, it is thought that Env epitopes that induce broadly neutralizing Abs are difficult to access and poorly immunogenic because of the characteristic oligomerization, conformational flexibility, sequence diversity and extensive glycosylation of Env protein. To enhance for isolation of quaternary epitope-targeting Abs (QtAbs, we previously used HIV virus-like particles (VLPs to bind B cells from long-term non-progressor subjects to identify a panel of monoclonal Abs. When expressed as recombinant full-length Abs, a subset of these novel Abs exhibited the binding profiles of QtAbs, as they either failed to bind to monomeric Env protein or showed much higher affinity for Env trimers and VLPs. These QtAbs represented a significant proportion of the B-cell response identified with VLPs. The Ab genes of these clones were highly mutated, but they did not neutralize common HIV strains. We sought to further define the epitopes targeted by these QtAbs. Competition-binding and mapping studies revealed these Abs targeted four separate epitopes; they also failed to compete for binding by Abs to known major neutralizing epitopes. Detailed epitope mapping studies revealed that two of the four epitopes were located in the gp41 subunit of Env. These QtAbs bound pre-fusion forms of antigen and showed differential binding kinetics depending on whether oligomers were produced as recombinant gp140 trimers or as full-length Env incorporated into VLPs. Antigenic regions within gp41 present unexpectedly diverse structural epitopes, including these QtAb epitopes, which may be targeted by the naturally occurring Ab response to HIV infection.

  17. Adhesion and fusion efficiencies of human immunodeficiency virus type 1 (HIV-1) surface proteins

    Science.gov (United States)

    Dobrowsky, Terrence M.; Rabi, S. Alireza; Nedellec, Rebecca; Daniels, Brian R.; Mullins, James I.; Mosier, Donald E.; Siliciano, Robert F.; Wirtz, Denis

    2013-10-01

    In about half of patients infected with HIV-1 subtype B, viral populations shift from utilizing the transmembrane protein CCR5 to CXCR4, as well as or instead of CCR5, during late stage progression of the disease. How the relative adhesion efficiency and fusion competency of the viral Env proteins relate to infection during this transition is not well understood. Using a virus-cell fusion assay and live-cell single-molecule force spectroscopy, we compare the entry competency of viral clones to tensile strengths of the individual Env-receptor bonds of Env proteins obtained from a HIV-1 infected patient prior to and during coreceptor switching. The results suggest that the genetic determinants of viral entry were predominantly enriched in the C3, HR1 and CD regions rather than V3. Env proteins can better mediate entry into cells after coreceptor switch; this effective entry capacity does not correlate with the bond strengths between viral Env and cellular receptors.

  18. Adhesion and fusion efficiencies of human immunodeficiency virus type 1 (HIV-1) surface proteins.

    Science.gov (United States)

    Dobrowsky, Terrence M; Rabi, S Alireza; Nedellec, Rebecca; Daniels, Brian R; Mullins, James I; Mosier, Donald E; Siliciano, Robert F; Wirtz, Denis

    2013-10-22

    In about half of patients infected with HIV-1 subtype B, viral populations shift from utilizing the transmembrane protein CCR5 to CXCR4, as well as or instead of CCR5, during late stage progression of the disease. How the relative adhesion efficiency and fusion competency of the viral Env proteins relate to infection during this transition is not well understood. Using a virus-cell fusion assay and live-cell single-molecule force spectroscopy, we compare the entry competency of viral clones to tensile strengths of the individual Env-receptor bonds of Env proteins obtained from a HIV-1 infected patient prior to and during coreceptor switching. The results suggest that the genetic determinants of viral entry were predominantly enriched in the C3, HR1 and CD regions rather than V3. Env proteins can better mediate entry into cells after coreceptor switch; this effective entry capacity does not correlate with the bond strengths between viral Env and cellular receptors.

  19. FACT Proteins, SUPT16H and SSRP1, Are Transcriptional Suppressors of HIV-1 and HTLV-1 That Facilitate Viral Latency*

    Science.gov (United States)

    Huang, Huachao; Santoso, Netty; Power, Derek; Simpson, Sydney; Dieringer, Michael; Miao, Hongyu; Gurova, Katerina; Giam, Chou-Zen; Elledge, Stephen J.; Zhu, Jian

    2015-01-01

    Our functional genomic RNAi screens have identified the protein components of the FACT (facilitates chromatin transcription) complex, SUPT16H and SSRP1, as top host factors that negatively regulate HIV-1 replication. FACT interacts specifically with histones H2A/H2B to affect assembly and disassembly of nucleosomes, as well as transcription elongation. We further investigated the suppressive role of FACT proteins in HIV-1 transcription. First, depletion of SUPT16H or SSRP1 protein enhances Tat-mediated HIV-1 LTR (long terminal repeat) promoter activity. Second, HIV-1 Tat interacts with SUPT16H but not SSRP1 protein. However, both SUPT16H and SSRP1 are recruited to LTR promoter. Third, the presence of SUPT16H interferes with the association of Cyclin T1 (CCNT1), a subunit of P-TEFb, with the Tat-LTR axis. Removing inhibitory mechanisms to permit HIV-1 transcription is an initial and key regulatory step to reverse post-integrated latent HIV-1 proviruses for purging of reservoir cells. We therefore evaluated the role of FACT proteins in HIV-1 latency and reactivation. Depletion of SUPT16H or SSRP1 protein affects both HIV-1 transcriptional initiation and elongation and spontaneously reverses latent HIV-1 in U1/HIV and J-LAT cells. Similar effects were observed with a primary CD4+ T cell model of HIV-1 latency. FACT proteins also interfere with HTLV-1 Tax-LTR-mediated transcription and viral latency, indicating that they may act as general transcriptional suppressors for retroviruses. We conclude that FACT proteins SUPT16H and SSRP1 play a key role in suppressing HIV-1 transcription and promoting viral latency, which may serve as promising gene targets for developing novel HIV-1 latency-reversing agents. PMID:26378236

  20. Cerebral infarction in an HIV-infected patient with combined protein S and C deficiency and a patent foramen ovale.

    Science.gov (United States)

    Tomomasa, Ran; Yamashiro, Kazuo; Tanaka, Ryota; Hattori, Nobutaka

    2013-11-01

    A 41-year-old male with a history of human immunodeficiency virus (HIV) infection developed motor aphasia, dysarthria, and right hemiparesis. A magnetic resonance imaging scan of the brain revealed a cerebral infarction in the territory of the left middle cerebral artery. The laboratory data showed decreased levels of protein S and protein C. Transesophageal contrast-enhanced echocardiography revealed a patent foramen ovale (PFO). Prothrombotic states, such as protein S and C deficiency, have been reported in HIV-infected patients. In addition, previous studies have reported prothrombotic states to be risk factors for PFO-related cerebral infarction. An association between combined protein S and C deficiency caused by HIV infection and PFO-related cerebral infarction was suggested in our patient.

  1. The study of interaction of HIV-1 surface GP120 protein with CD4 cell receptor by EXAFS spectroscopy

    Science.gov (United States)

    Lukashev, Vitaly Alexeevich; Bausk, Nikolay Vladimirovich; Mazalov, Lev Nikolaevich; Kaurov, Oleg Alexeevich; Kolobov, Alexandr Alexandrovich; Naumochkin, Andrey Nikolaevich; Kulichkov, Vladimir Anatolevich

    1995-02-01

    This work is aimed at the study of the interaction between gp120 protein (HIV-1) and peptides, which are similar to CD4 protein receptors from T4 lymphocytes. The preliminary results demonstrated that peptide structural information could be obtained from fluorescent EXAFS.

  2. Active cAMP-dependent protein kinase incorporated within highly purified HIV-1 particles is required for viral infectivity and interacts with viral capsid protein.

    Science.gov (United States)

    Cartier, Christine; Hemonnot, Bénédicte; Gay, Bernard; Bardy, Martine; Sanchiz, Céline; Devaux, Christian; Briant, Laurence

    2003-09-12

    Host cell components, including protein kinases such as ERK-2/mitogen-activated protein kinase, incorporated within human immunodeficiency virus type 1 (HIV-1) virions play a pivotal role in the ability of HIV to infect and replicate in permissive cells. The present work provides evidence that the catalytic subunit of cAMP-dependent protein kinase (C-PKA) is packaged within HIV-1 virions as demonstrated using purified subtilisin-digested viral particles. Virus-associated C-PKA was shown to be enzymatically active and able to phosphorylate synthetic substrate in vitro. Suppression of virion-associated C-PKA activity by specific synthetic inhibitor had no apparent effect on viral precursor maturation and virus assembly. However, virus-associated C-PKA activity was demonstrated to regulate HIV-1 infectivity as assessed by single round infection assays performed by using viruses produced from cells expressing an inactive form of C-PKA. In addition, virus-associated C-PKA was found to co-precipitate with and to phosphorylate the CAp24gag protein. Altogether our results indicate that virus-associated C-PKA regulates HIV-1 infectivity, possibly by catalyzing phosphorylation of the viral CAp24gag protein.

  3. Simulations of HIV capsid protein dimerization reveal the effect of chemistry and topography on the mechanism of hydrophobic protein association

    CERN Document Server

    Yu, Naiyin

    2015-01-01

    Recent work has shown that the hydrophobic protein surfaces in aqueous solution sit near a drying transition. The tendency for these surfaces to expel water from their vicinity leads to self assembly of macromolecular complexes. In this article we show with a realistic model for a biologically pertinent system how this phenomenon appears at the molecular level. We focus on the association of the C-terminal domain (CA-C) of the human immunodeficiency virus (HIV) capsid protein. By combining all-atom simulations with specialized sampling techniques we measure the water density distribution during the approach of two CA-C proteins as a function of separation and amino acid sequence in the interfacial region. The simulations demonstrate that CA-C protein-protein interactions sit at the edge of a dewetting transition and that this mesoscopic manifestation of the underlying liquid-vapor phase transition can be readily manipulated by biology or protein engineering to significantly affect association behavior. While ...

  4. Generation and Characterization of a Bivalent HIV-1 Subtype C gp120 Protein Boost for Proof-of-Concept HIV Vaccine Efficacy Trials in Southern Africa.

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    Carlo Zambonelli

    Full Text Available The viral envelope glycoprotein (Env is the major target for antibody (Ab-mediated vaccine development against the Human Immunodeficiency Virus type 1 (HIV-1. Although several recombinant Env antigens have been evaluated in clinical trials, only the surface glycoprotein, gp120, (from HIV-1 subtype B, MN, and subtype CRF_01AE, A244 used in the ALVAC prime-AIDSVAX gp120 boost RV144 Phase III HIV vaccine trial was shown to contribute to protective efficacy, although modest and short-lived. Hence, for clinical trials in southern Africa, a bivalent protein boost of HIV-1 subtype C gp120 antigens composed of two complementary gp120s, from the TV1.C (chronic and 1086.C (transmitted founder HIV-1 strains, was selected. Stable Chinese Hamster Cell (CHO cell lines expressing these gp120s were generated, scalable purification methods were developed, and a detailed analytical analysis of the purified proteins was conducted that showed differences and complementarity in the antigenicity, glycan occupancy, and glycan content of the two gp120 molecules. Moreover, mass spectrometry revealed some disulfide heterogeneity in the expressed proteins, particularly in V1V2-C1 region and most prominently in the TV1 gp120 dimers. These dimers not only lacked binding to certain key CD4 binding site (CD4bs and V1V2 epitope-directed ligands but also elicited reduced Ab responses directed to those epitopes, in contrast to monomeric gp120, following immunization of rabbits. Both monomeric and dimeric gp120s elicited similarly high titer Tier 1 neutralizing Abs as measured in standard virus neutralization assays. These results provide support for clinical evaluations of bivalent preparations of purified monomeric TV1.C and 1086.C gp120 proteins.

  5. The use of Nanotrap particles technology in capturing HIV-1 virions and viral proteins from infected cells.

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    Elizabeth Jaworski

    Full Text Available HIV-1 infection results in a chronic but incurable illness since long-term HAART can keep the virus to an undetectable level. However, discontinuation of therapy rapidly increases viral burden. Moreover, patients under HAART frequently develop various metabolic disorders and HIV-associated neuronal disease. Today, the main challenge of HIV-1 research is the elimination of the residual virus in infected individuals. The current HIV-1 diagnostics are largely comprised of serological and nucleic acid based technologies. Our goal is to integrate the nanotrap technology into a standard research tool that will allow sensitive detection of HIV-1 infection. This study demonstrates that majority of HIV-1 virions in culture supernatants and Tat/Nef proteins spiked in culture medium can be captured by nanotrap particles. To determine the binding affinities of different baits, we incubated target molecules with nanotrap particles at room temperature. After short sequestration, materials were either eluted or remained attached to nanotrap particles prior to analysis. The unique affinity baits of nanotrap particles preferentially bound HIV-1 materials while excluded albumin. A high level capture of Tat or Tat peptide by NT082 and NT084 particles was measured by western blot (WB. Intracellular Nef protein was captured by NT080, while membrane-associated Nef was captured by NT086 and also detected by WB. Selective capture of HIV-1 particles by NT073 and NT086 was measured by reverse transcriptase assay, while capture of infectious HIV-1 by these nanoparticles was demonstrated by functional transactivation in TZM-bl cells. We also demonstrated specific capture of HIV-1 particles and exosomes-containing TAR-RNA in patients' serum by NT086 and NT082 particles, respectively, using specific qRT-PCR. Collectively, our data indicate that certain types of nanotrap particles selectively capture specific HIV-1 molecules, and we propose to use this technology as a

  6. The Use of Nanotrap Particles Technology in Capturing HIV-1 Virions and Viral Proteins from Infected Cells

    Science.gov (United States)

    Sampey, Gavin; Shafagati, Nazly; Van Duyne, Rachel; Iordanskiy, Sergey; Kehn-Hall, Kylene; Liotta, Lance; Petricoin, Emanuel; Young, Mary; Lepene, Benjamin; Kashanchi, Fatah

    2014-01-01

    HIV-1 infection results in a chronic but incurable illness since long-term HAART can keep the virus to an undetectable level. However, discontinuation of therapy rapidly increases viral burden. Moreover, patients under HAART frequently develop various metabolic disorders and HIV-associated neuronal disease. Today, the main challenge of HIV-1 research is the elimination of the residual virus in infected individuals. The current HIV-1 diagnostics are largely comprised of serological and nucleic acid based technologies. Our goal is to integrate the nanotrap technology into a standard research tool that will allow sensitive detection of HIV-1 infection. This study demonstrates that majority of HIV-1 virions in culture supernatants and Tat/Nef proteins spiked in culture medium can be captured by nanotrap particles. To determine the binding affinities of different baits, we incubated target molecules with nanotrap particles at room temperature. After short sequestration, materials were either eluted or remained attached to nanotrap particles prior to analysis. The unique affinity baits of nanotrap particles preferentially bound HIV-1 materials while excluded albumin. A high level capture of Tat or Tat peptide by NT082 and NT084 particles was measured by western blot (WB). Intracellular Nef protein was captured by NT080, while membrane-associated Nef was captured by NT086 and also detected by WB. Selective capture of HIV-1 particles by NT073 and NT086 was measured by reverse transcriptase assay, while capture of infectious HIV-1 by these nanoparticles was demonstrated by functional transactivation in TZM-bl cells. We also demonstrated specific capture of HIV-1 particles and exosomes-containing TAR-RNA in patients' serum by NT086 and NT082 particles, respectively, using specific qRT-PCR. Collectively, our data indicate that certain types of nanotrap particles selectively capture specific HIV-1 molecules, and we propose to use this technology as a platform to enhance HIV-1

  7. Identification of a human protein-derived HIV-1 fusion inhibitor targeting the gp41 fusion core structure.

    Directory of Open Access Journals (Sweden)

    Lijun Chao

    Full Text Available The HIV-1 envelope glycoprotein (Env gp41 plays a crucial role in the viral fusion process. The peptides derived from the C-terminal heptad repeat (CHR of gp41 are potent HIV fusion inhibitors. However, the activity of these anti-HIV-1 peptides in vivo may be attenuated by their induction of anti-gp41 antibodies. Thus, it is essential to identify antiviral peptides or proteins with low, or no, immunogenicity to humans. Here, we found that the C-terminal fragment (aa 462-521 of the human POB1 (the partner of RalBP1, designated C60, is an HIV-1 fusion inhibitor. It bound to N36, the peptide derived from the N-terminal heptad repeat (NHR of gp41, and to the six-helix bundle (6-HB formed by N36 and C34, a CHR-peptide, but it did not bind to C34. Unlike the CHR-peptides, C60 did not block gp41 6-HB formation. Rather, results suggest that C60 inhibits HIV-1 fusion by binding to the 6-HB, in particular, the residues in the gp41 NHR domain that are exposed on the surface of 6-HB. Since 6-HB plays a crucial role in the late stage of fusion between the viral envelope and endosomal membrane during the endocytic process of HIV-1, C60 may serve as a host restriction factor to suppress HIV-1 entry into CD4+ T lymphocytes. Taken together, it can be concluded from these results that C60 can be used as a lead for the development of anti-HIV-1 therapeutics or microbicides for the treatment and prevention of HIV-1 infection, as well as a molecular probe to study the fusogenic mechanism of HIV-1.

  8. CSF neurofilament protein (NFL) -- a marker of active HIV-related neurodegeneration.

    Science.gov (United States)

    Abdulle, Sahra; Mellgren, Asa; Brew, Bruce J; Cinque, Paola; Hagberg, Lars; Price, Richard W; Rosengren, Lars; Gisslén, Magnus

    2007-08-01

    The light subunit of the neurofilament protein (NFL), a major structural component of myelinated axons, is a sensitive indicator of axonal injury in the central nervous system (CNS) in a variety of neurodegenerative disorders. Cerebrospinal fluid (CSF) NFL concentrations were measured by ELISA (normal NFL concentrations were significantly higher in patients with ADC (median 2590 ng/l, IQR 780-7360) and CNS OIs (2315 ng/l, 985-7390 ng/l) than in neuroasymptomatic patients (NFL declined during HAART to the limit of detection in parallel with virological response and neurological improvement in ADC.CSF NFL concentrations were higher in neuroasymptomatic patients with lower CD4-cell strata than higher, p or =200/microl. The findings of this study support the value of CSF NFL as a useful marker of ongoing CNS damage in HIV infection. Markedly elevated CSF NFL concentrations in patients without CNS OIs are associated with ADC, follow the grade of severity, and decrease after initiation of effective antiretroviral treatment. Nearly all previously suggested CSF markers of ADC relate to immune activation or HIV viral load that do not directly indicate brain injury. By contrast NFL is a sensitive marker of such injury, and should prove useful in evaluating the presence and activity of ongoing CNS injury in HIV infection.

  9. Down-regulation of CTLA-4 by HIV-1 Nef protein.

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    Mohamed El-Far

    Full Text Available HIV-1 Nef protein down-regulates several cell surface receptors through its interference with the cell sorting and trafficking machinery. Here we demonstrate for the first time the ability of Nef to down-regulate cell surface expression of the negative immune modulator CTLA-4. Down-regulation of CTLA-4 required the Nef motifs DD175, EE155 and LL165, all known to be involved in vesicle trafficking. Disruption of the lysosomal functions by pH-neutralizing agents prevented CTLA-4 down-regulation by Nef, demonstrating the implication of the endosomal/lysosomal compartments in this process. Confocal microscopy experiments visualized the co-localization between Nef and CTLA-4 in the early and recycling endosomes but not at the cell surface. Overall, our results provide a novel mechanism by which HIV-1 Nef interferes with the surface expression of the negative regulator of T cell activation CTLA-4. Down-regulation of CTLA-4 may contribute to the mechanisms by which HIV-1 sustains T cell activation, a critical step in viral replication and dissemination.

  10. Distinct determinants in HIV-1 Vif and human APOBEC3 proteins are required for the suppression of diverse host anti-viral proteins.

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    Wenyan Zhang

    Full Text Available BACKGROUND: APOBEC3G (A3G and related cytidine deaminases of the APOBEC3 family of proteins are potent inhibitors of many retroviruses, including HIV-1. Formation of infectious HIV-1 requires the suppression of multiple cytidine deaminases by Vif. HIV-1 Vif suppresses various APOBEC3 proteins through the common mechanism of recruiting the Cullin5-ElonginB-ElonginC E3 ubiquitin ligase to induce target protein polyubiquitination and proteasome-mediated degradation. The domains in Vif and various APOBEC3 proteins required for APOBEC3 recognition and degradation have not been fully characterized. METHODS AND FINDINGS: In the present study, we have demonstrated that the regions of APOBEC3F (A3F that are required for its HIV-1-mediated binding and degradation are distinct from those reported for A3G. We found that the C-terminal cytidine deaminase domain (C-CDD of A3F alone is sufficient for its interaction with HIV-1 Vif and its Vif-mediated degradation. We also observed that the domains of HIV-1 Vif that are uniquely required for its functional interaction with full-length A3F are also required for the degradation of the C-CDD of A3F; in contrast, those Vif domains that are uniquely required for functional interaction with A3G are not required for the degradation of the C-CDD of A3F. Interestingly, the HIV-1 Vif domains required for the degradation of A3F are also required for the degradation of A3C and A3DE. On the other hand, the Vif domains uniquely required for the degradation of A3G are dispensable for the degradation of cytidine deaminases A3C and A3DE. CONCLUSIONS: Our data suggest that distinct regions of A3F and A3G are targeted by HIV-1 Vif molecules. However, HIV-1 Vif suppresses A3F, A3C, and A3DE through similar recognition determinants, which are conserved among Vif molecules from diverse HIV-1 strains. Mapping these determinants may be useful for the design of novel anti-HIV inhibitors.

  11. Urinary protein-to-creatinine ratio versus 24-h proteinuria in the screening for nephropathy in HIV patients.

    Science.gov (United States)

    Antonello, Vicente Sperb; Poli-De-Figueiredo, Carlos Eduardo; Antonello, Ivan Carlos Ferreira; Tovo, Cristiane Valle

    2015-06-01

    To determine the correlation between protein-to-creatinine ratio and 24-h urinary protein, proteinuria was measured in 45 patients attending a public HIV clinic in Porto Alegre, Brazil, using 24-h urinary protein excretion (24hUP) and urinary protein-to-creatinine ratio. Spearman's correlation test was done to evaluate the association between spot protein-to-creatinine ratio and 24hUP. The limits of agreement between the two methods were analysed by the Bland-Altman method. For protein excretion creatinine ratio and 24hUP were +0.112 and -0.097 g/day. A strong correlation (r = 0.957) was found between protein-to-creatinine ratio and 24hUP excretion. The conclusion is that the protein-to-creatinine ratio in spot urine specimens is an accurate, convenient and reliable screening method to estimate the urinary protein excretion in HIV patients to detect abnormal urinary protein loss. Further studies are required to evaluate renal disease in HIV patients with chronic renal disease and higher urinary protein excretion.

  12. The HIV-1 Vpu protein induces apoptosis in Drosophila via activation of JNK signaling.

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    Christelle Marchal

    Full Text Available The genome of the human immunodeficiency virus type 1 (HIV-1 encodes the canonical retroviral proteins, as well as additional accessory proteins that enhance the expression of viral genes, the infectivity of the virus and the production of virions. The accessory Viral Protein U (Vpu, in particular, enhances viral particle production, while also promoting apoptosis of HIV-infected human T lymphocytes. Some Vpu effects rely on its interaction with the ubiquitin-proteasome protein degradation system, but the mechanisms responsible for its pro-apoptotic effects in vivo are complex and remain largely to be elucidated.We took advantage of the Drosophila model to study the effects of Vpu activity in vivo. Expression of Vpu in the developing Drosophila wing provoked tissue loss due to caspase-dependent apoptosis. Moreover, Vpu induced expression of the pro-apoptotic gene reaper, known to down-regulate Inhibitor of Apoptosis Proteins (IAPs which are caspase-antagonizing E3 ubiquitin ligases. Indeed, Vpu also reduced accumulation of Drosophila IAP1 (DIAP1. Though our results demonstrate a physical interaction between Vpu and the proteasome-addressing SLIMB/β-TrCP protein, as in mammals, both SLIMB/βTrCP-dependent and -independent Vpu effects were observed in the Drosophila wing. Lastly, the pro-apoptotic effect of Vpu in this tissue was abrogated upon inactivation of the c-Jun N-terminal Kinase (JNK pathway. Our results in the fly thus provide the first functional evidence linking Vpu pro-apoptotic effects to activation of the conserved JNK pathway.

  13. The HIV-1 Vpu protein induces apoptosis in Drosophila via activation of JNK signaling.

    Science.gov (United States)

    Marchal, Christelle; Vinatier, Gérald; Sanial, Matthieu; Plessis, Anne; Pret, Anne-Marie; Limbourg-Bouchon, Bernadette; Théodore, Laurent; Netter, Sophie

    2012-01-01

    The genome of the human immunodeficiency virus type 1 (HIV-1) encodes the canonical retroviral proteins, as well as additional accessory proteins that enhance the expression of viral genes, the infectivity of the virus and the production of virions. The accessory Viral Protein U (Vpu), in particular, enhances viral particle production, while also promoting apoptosis of HIV-infected human T lymphocytes. Some Vpu effects rely on its interaction with the ubiquitin-proteasome protein degradation system, but the mechanisms responsible for its pro-apoptotic effects in vivo are complex and remain largely to be elucidated.We took advantage of the Drosophila model to study the effects of Vpu activity in vivo. Expression of Vpu in the developing Drosophila wing provoked tissue loss due to caspase-dependent apoptosis. Moreover, Vpu induced expression of the pro-apoptotic gene reaper, known to down-regulate Inhibitor of Apoptosis Proteins (IAPs) which are caspase-antagonizing E3 ubiquitin ligases. Indeed, Vpu also reduced accumulation of Drosophila IAP1 (DIAP1). Though our results demonstrate a physical interaction between Vpu and the proteasome-addressing SLIMB/β-TrCP protein, as in mammals, both SLIMB/βTrCP-dependent and -independent Vpu effects were observed in the Drosophila wing. Lastly, the pro-apoptotic effect of Vpu in this tissue was abrogated upon inactivation of the c-Jun N-terminal Kinase (JNK) pathway. Our results in the fly thus provide the first functional evidence linking Vpu pro-apoptotic effects to activation of the conserved JNK pathway.

  14. Structural basis of the association of HIV-1 matrix protein with DNA.

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    Mengli Cai

    Full Text Available HIV-1 matrix (MA is a multifunctional protein that is synthesized as a polyprotein that is cleaved by protease during viral maturation. MA contains a cluster of basic residues whose role is controversial. Proposed functions include membrane anchoring, facilitating viral assembly, and directing nuclear import of the viral DNA. Since MA has been reported to be a component of the preintegration complex (PIC, we have used NMR to probe its interaction with other PIC components. We show that MA interacts with DNA and this is likely sufficient to account for its association with the PIC.

  15. Levels of procalcitonin, C-reactive protein and neopterin in patients with advanced HIV-1 infection

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    P Bipath

    2012-06-01

    Full Text Available Objectives. To compare the value of procalcitonin, C-reactive protein (CRP and neopterin as indicators of immune deficiency, co-infection, efficacy of treatment, and disease progression, in patients with advanced HIV-1 infection. Design. Cross-sectional, investigating baseline blood measurements and clinical observations in 82 HIV-positive patients divided into an antiretroviral treatment (ART group and an ART-naïve group. Setting. Secondary general hospital in Pretoria. Results. Procalcitonin and CRP levels showed no significant differences between the ART and ART-naïve groups, and no correlations with CD4 counts or viral loads. CRP levels were significantly higher with TB co-infection (p<0.05. Neopterin levels were raised above normal in 92% of the ART-naïve group and in 75% of the ART group. The levels were significantly higher (p<0.05 in the ART- naïve group. Negative correlations were found between neopterin and CD4 counts for the total patient group (r=-0.482; p<0.001. Neopterin was significantly (p<0.05 higher in the HIV/TB co-infection group than in those without TB. Higher neopterin levels at baseline were associated with a decline in CD4 counts over the ensuing 6-month period, and patients with higher baseline neopterin levels developed more complications over the 6-month period. Conclusions. Compared with procalcitonin and CRP, neopterin appears to be associated with the degree of immunodeficiency and of co-infection with TB. Neopterin levels may be investigated further as a measure of disease progression or treatment response. S Afr J HIV Med 2012;13(2:78-82.

  16. Proteochemometric modeling of the bioactivity spectra of HIV-1 protease inhibitors by introducing protein-ligand interaction fingerprint.

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    Qi Huang

    Full Text Available HIV-1 protease is one of the main therapeutic targets in HIV. However, a major problem in treatment of HIV is the rapid emergence of drug-resistant strains. It should be particularly helpful to clinical therapy of AIDS if one method can be used to predict antivirus capability of compounds for different variants. In our study, proteochemometric (PCM models were created to study the bioactivity spectra of 92 chemical compounds with 47 unique HIV-1 protease variants. In contrast to other PCM models, which used Multiplication of Ligands and Proteins Descriptors (MLPD as cross-term, one new cross-term, i.e. Protein-Ligand Interaction Fingerprint (PLIF was introduced in our modeling. With different combinations of ligand descriptors, protein descriptors and cross-terms, nine PCM models were obtained, and six of them achieved good predictive abilities (Q(2(test>0.7. These results showed that the performance of PCM models could be improved when ligand and protein descriptors were complemented by the newly introduced cross-term PLIF. Compared with the conventional cross-term MLPD, the newly introduced PLIF had a better predictive ability. Furthermore, our best model (GD & P & PLIF: Q(2(test = 0.8271 could select out those inhibitors which have a broad antiviral activity. As a conclusion, our study indicates that proteochemometric modeling with PLIF as cross-term is a potential useful way to solve the HIV-1 drug-resistant problem.

  17. The utility of protein structure as a predictor of site-wise dN/dS varies widely among HIV-1 proteins.

    Science.gov (United States)

    Meyer, Austin G; Wilke, Claus O

    2015-10-06

    Protein structure acts as a general constraint on the evolution of viral proteins. One widely recognized structural constraint explaining evolutionary variation among sites is the relative solvent accessibility (RSA) of residues in the folded protein. In influenza virus, the distance from functional sites has been found to explain an additional portion of the evolutionary variation in the external antigenic proteins. However, to what extent RSA and distance from a reference site in the protein can be used more generally to explain protein adaptation in other viruses and in the different proteins of any given virus remains an open question. To address this question, we have carried out an analysis of the distribution and structural predictors of site-wise dN/dS in HIV-1. Our results indicate that the distribution of dN/dS in HIV follows a smooth gamma distribution, with no special enrichment or depletion of sites with dN/dS at or above one. The variation in dN/dS can be partially explained by RSA and distance from a reference site in the protein, but these structural constraints do not act uniformly among the different HIV-1 proteins. Structural constraints are highly predictive in just one of the three enzymes and one of three structural proteins in HIV-1. For these two proteins, the protease enzyme and the gp120 structural protein, structure explains between 30 and 40% of the variation in dN/dS. Finally, for the gp120 protein of the receptor-binding complex, we also find that glycosylation sites explain just 2% of the variation in dN/dS and do not explain gp120 evolution independently of either RSA or distance from the apical surface. © 2015 The Author(s).

  18. Alteration of Methamphetamine-induced stereotypic behaviour in transgenic mice expressing HIV-1 envelope protein gp120.

    Science.gov (United States)

    Roberts, Amanda J; Maung, Ricky; Sejbuk, Natalia E; Ake, Christopher; Kaul, Marcus

    2010-02-15

    The use of drugs for recreational purposes, in particular Methamphetamine, is associated with an increased risk of infection with human immunodeficiency virus (HIV)-1. HIV-1 infection in turn can lead to HIV-associated neurological disorders (HAND) that range from mild cognitive and motor impairment to HIV-associated dementia (HAD). Interestingly, post mortem brain specimens from HAD patients and transgenic (tg) mice expressing the viral envelope protein gp120 in the central nervous system display similar neuropathological signs. In HIV patients, the use of Methamphetamine appears to aggravate neurocognitive alterations. In the present study, we injected HIV/gp120tg mice and non-transgenic littermate control animals with Methamphetamine dissolved in Saline or Saline vehicle and assessed locomotion and stereotyped behaviour. We found that HIVgp120-transgenic mice differ significantly from non-transgenic controls in certain domains of their behavioural response to Methamphetamine. Thus this experimental model system may be useful to further study the mechanistic interaction of both the viral envelope protein and the psychostimulant drug in behavioural alterations and neurodegenerative disease.

  19. Alphavirus replicon DNA expressing HIV antigens is an excellent prime for boosting with recombinant modified vaccinia Ankara (MVA or with HIV gp140 protein antigen.

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    Maria L Knudsen

    Full Text Available Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 μg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 μg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 μg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose.

  20. Alphavirus replicon DNA expressing HIV antigens is an excellent prime for boosting with recombinant modified vaccinia Ankara (MVA) or with HIV gp140 protein antigen.

    Science.gov (United States)

    Knudsen, Maria L; Ljungberg, Karl; Tatoud, Roger; Weber, Jonathan; Esteban, Mariano; Liljeström, Peter

    2015-01-01

    Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP) vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA) and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF) adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 μg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 μg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 μg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose.

  1. Trans-cellular introduction of HIV-1 protein Nef induces pathogenic response in Caenorhabditis elegans.

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    Aamir Nazir

    Full Text Available BACKGROUND: Caenorhabditis elegans has emerged as a very powerful model for studying the host pathogen interactions. Despite the absence of a naturally occurring viral infection for C. elegans, the model is now being exploited experimentally to study the basic aspects of virus-host interplay. The data generated from recent studies suggests that the virus that infects mammalian cells does infect, replicate and accumulate in C. elegans. METHODOLOGY/PRINCIPAL FINDINGS: We took advantage of the easy-to-achieve protein introduction in C. elegans and employing the methodology, we administered HIV-1 protein Nef into live worms. Nef is known to be an important protein for exacerbating HIV-1 pathogenesis in host by enhancing viral replication. The deletion of nef from the viral genome has been reported to inhibit its replication in the host, thereby leading to delayed pathogenesis. Our studies, employing Nef introduction into C. elegans, led to creation of an in-vivo model that allowed us to study, whether or not, the protein induces effect in the whole organism. We observed a marked lipodystrophy, effect on neuromuscular function, impaired fertility and reduced longevity in the worms exposed to Nef. The observed effects resemble to those observed in Nef transgenic mice and most interestingly the effects also relate to some of the pathogenic aspects exhibited by human AIDS patients. CONCLUSIONS/SIGNIFICANCE: Our studies underline the importance of this in vivo model for studying the interactions of Nef with host proteins, which could further be used for identifying possible inhibitors of such interactions.

  2. Semisynthetic analogues of PSC-RANTES, a potent anti-HIV protein.

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    Gaertner, Hubert; Offord, Robin; Botti, Paolo; Kuenzi, Gabriel; Hartley, Oliver

    2008-02-01

    New HIV prevention methods are needed, and among those currently being explored are "microbicides", substances applied topically to prevent HIV acquisition during sexual intercourse. The chemokine analogue PSC-RANTES (N(alpha)(n-nonanoyl)-des-Ser(1)-[ L-thioprolyl(2), L-cyclohexylglycyl(3)]-RANTES(4-68)) is a highly potent HIV entry inhibitor which has shown promising efficacy in its initial evaluation as a candidate microbicide. However, a way must be found to produce the molecule by cheaper means than total chemical synthesis. Since the only noncoded structures are located at the N-terminus, a possible solution would be to produce a protein fragment representing all but the N-terminal region using low-cost recombinant production methods and then to attach, site specifically, a short synthetic fragment containing the noncoded N-terminal structures. Here, we describe the evaluation of a range of different conjugation chemistries in order to identify those with potential for development as economical routes to production of a PSC-RANTES analogue with antiviral activity as close as possible to that of the parent protein. The strategies tested involved linkage through oxime, hydrazone/hydrazide, and Psi[CH2-NH] bonds, as well as through a peptide bond obtained either by a thiazolidine rearrangement or by direct alpha-amino acylation of a protein fragment in which 4 of the 5 lysine residues of the native sequence were replaced by arginine (the fifth lysine is essential for activity). Where conjugation involved replacement of one or more residues with a linker moiety, the point in the main chain at which the linker was introduced was varied. The resulting panel of 22 PSC-RANTES analogues was evaluated for anti-HIV activity in an entry inhibition assay. The [Arg (25,45,56,57)] PSC-RANTES analogue has comparable potency to PSC-RANTES, and one of the oxime linked analogues, 4L-57, has potency only 5-fold lower, with scope for improvement. Both represent promising leads for

  3. Hyperdopaminergic tone in HIV-1 protein treated rats and cocaine sensitization

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    Ferris, Mark J.; Frederick-Duus, Danielle; Fadel, Jim; Mactutus, Charles F; Booze, Rosemarie M.

    2013-01-01

    In the United States, one-third of infected individuals contracted Human Immunodeficiency Virus-1 (HIV-1) via injecting drugs with contaminated needles or through risky behaviors associated with drug use. Research demonstrates concomitant administration of psychostimulants and HIV-1-proteins damage neurons to a greater extent than viral proteins or the drug alone. To model the onset of HIV-1-infection in relation to a history of drug use, the current research compared behavior and extracellular dopamine and metabolite levels following Tat1–86 infusions in animals with and without a history of cocaine (Coc) experience (10 mg/kg; i.p.; 1 injection/day × 9 days). Animals receiving a behaviorally sensitizing regimen of Coc demonstrated a decrease in extracellular dopamine concentration in the nucleus accumbens, consistent with evidence describing up-regulation of dopamine transporter uptake. Contrary to this effect, Tat1–86 microinfusion into the nucleus accumbens following the sensitizing regimen of Coc caused a significant increase in extracellular dopamine levels (nM) within 48 h with no difference in percent of baseline response to Coc. After 72 h, Tat + Coc treated animals demonstrated a blunted effect on potassium-stimulated extracellular dopamine release (percent of baseline) with a corresponding decrease in expression of behavioral sensitization to Coc challenge. A persistent decrease in extracellular dopamine metabolite levels was found across all time-points in Tat-treated animals, regardless of experience with Coc. The current study provides evidence for divergent neurochemical and behavioral outcomes following Tat-treatment; contingent upon experience with Coc. PMID:20796175

  4. APOBEC3G impairs the multimerization of the HIV-1 Vif protein in living cells.

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    Batisse, Julien; Guerrero, Santiago Xavier; Bernacchi, Serena; Richert, Ludovic; Godet, Julien; Goldschmidt, Valérie; Mély, Yves; Marquet, Roland; de Rocquigny, Hugues; Paillart, Jean-Christophe

    2013-06-01

    The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in nonpermissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G [A3G]) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55(Gag). Recently, we showed that oligomerization of Vif into high-molecular-mass complexes induces Vif folding and influences its binding to high-affinity RNA binding sites present in the HIV genomic RNA. To get further insight into the role of Vif multimerization in viral assembly and A3G repression, we used fluorescence lifetime imaging microscopy (FLIM)- and fluorescence resonance energy transfer (FRET)-based assays to investigate Vif-Vif interactions in living cells. By using two N-terminally tagged Vif proteins, we show that Vif-Vif interactions occur in living cells. This oligomerization is strongly reduced when the putative Vif multimerization domain ((161)PPLP(164)) is mutated, indicating that this domain is crucial, but that regions outside this motif also participate in Vif oligomerization. When coexpressed together with Pr55(Gag), Vif is largely relocated to the cell membrane, where Vif oligomerization also occurs. Interestingly, wild-type A3G strongly interferes with Vif multimerization, contrary to an A3G mutant that does not bind to Vif. These findings confirm that Vif oligomerization occurs in living cells partly through its C-terminal motif and suggest that A3G may target and perturb the Vif oligomerization state to limit its functions in the cell.

  5. Neurotoxic Activity of the HIV-1 Envelope Glycoprotein: Activation of Protein Kinase C in Rat Astrocytes

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    Isaac Adebayo

    2002-11-01

    Full Text Available Abstract: Envelope glycoprotein (gp120 of the human immunodeficiency virus type one (HIV-1, has adverse effects on glial cells and neurons. This study reports on the direct effect of recombinant gp120 (r-gp120 produced from different expression systems on protein kinase C, as a measure of relative neurotoxicity. Brain cells were grown in vitro from explants of the cerebral cortex of newborn rats, and recombinant gp120 preparations expressed in mammalian cell/vaccinia virus and insect cell/baculovirus systems were applied to astrocyte-enriched cultures. The gp120 preparations activated protein kinase C (PKC to similar levels in these cells. Mutant recombinant gp120 lacking the amino-terminal 29 amino acids produced from the mammalian and insect cells also activated PKC to similar levels as did the full-length protein. The recombinant proteins specifically activated PKC β and ζ, suggesting that they are able to induce both Ca2+-dependent and Ca2+-independent isoforms of this enzyme. Alteration of PKC activity in astrocytes by gp120 indicates its ability to modulate gene expression, which is associated with the neurotoxicity of this protein. Furthermore, the results suggest that the deletion of the first 29 residues of NH2-terminal end of the gp120 does not affect the functional activity of this protein with regard to modulation of signal transduction in astrocytes.

  6. STRUCTURAL VIROLOGY. X-ray crystal structures of native HIV-1 capsid protein reveal conformational variability.

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    Gres, Anna T; Kirby, Karen A; KewalRamani, Vineet N; Tanner, John J; Pornillos, Owen; Sarafianos, Stefan G

    2015-07-03

    The detailed molecular interactions between native HIV-1 capsid protein (CA) hexamers that shield the viral genome and proteins have been elusive. We report crystal structures describing interactions between CA monomers related by sixfold symmetry within hexamers (intrahexamer) and threefold and twofold symmetry between neighboring hexamers (interhexamer). The structures describe how CA builds hexagonal lattices, the foundation of mature capsids. Lattice structure depends on an adaptable hydration layer modulating interactions among CA molecules. Disruption of this layer alters interhexamer interfaces, highlighting an inherent structural variability. A CA-targeting antiviral affects capsid stability by binding across CA molecules and subtly altering interhexamer interfaces remote to the ligand-binding site. Inherent structural plasticity, hydration layer rearrangement, and effector binding affect capsid stability and have functional implications for the retroviral life cycle. Copyright © 2015, American Association for the Advancement of Science.

  7. NMR insights into the conformational properties of Man-9 and its recognition by two HIV binding proteins.

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    Shahzad-Ul-Hussan, Syed; Sastry, Mallika; Lemmin, Thomas; Soto, Cinque; Loesgen, Sandra; Scott, Danielle A; Davison, Jack Robert; O'Connor, Robert; Kwong, Peter D; Bewley, Carole A

    2017-02-06

    Man9GlcNAc2 (Man-9) present at the surface of HIV constitutes the binding sites of several HIV neutralizing agents and mammalian lectin DC-SIGN that is involved in cellular immunity and trans-infections. We describe the conformational properties of Man-9 in free state and when bound by an HIV entry inhibitor protein and define the minimum epitopes of two HIV binding proteins using NMR spectroscopy. In this regard we developed a robust expression system for production of 13C,15N-labeled glycans in mammalian cells that facilitated the implementation of 3D 13C-edited spectra to deconvolute spectral overlap allowing for the solution structure determination of Man-9. The studies reveal that Man-9 interacts with HIV-binding proteins via distinct binding epitopes and adopts divergent conformations in the bound state. In combination with molecular dynamics we find that these receptor-bound conformations are sampled by Man-9 in the free state suggesting a mechanism for diverse recognition.

  8. Circulating heat shock protein 60 levels are elevated in HIV patients and are reduced by anti-retroviral therapy.

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    Itaru Anraku

    Full Text Available Circulating heat shock protein 60 (Hsp60 and heat shock protein 10 (Hsp10 have been associated with pro- and anti-inflammatory activity, respectively. To determine whether these heat shock proteins might be associated with the immune activation seen in HIV-infected patients, the plasma levels of Hsp60 and Hsp10 were determined in a cohort of 20 HIV-infected patients before and after effective combination anti-retroviral therapy (cART. We show for the first time that circulating Hsp60 levels are elevated in HIV-infected patients, with levels significantly reduced after cART, but still higher than those in HIV-negative individuals. Hsp60 levels correlated significantly with viral load, CD4 counts, and circulating soluble CD14 and lipopolysaccharide levels. No differences or correlations were seen for Hsp10 levels. Elevated circulating Hsp60 may contribute to the immune dysfunction and non-AIDS clinical events seen in HIV-infected patients.

  9. Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

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    St-Laurent Georges

    2006-11-01

    Full Text Available Abstract Background The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. Results Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. Conclusion The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.

  10. Comparing Models of Intelligence in Project TALENT: The VPR Model Fits Better than the CHC and Extended Gf-Gc Models

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    Major, Jason T.; Johnson, Wendy; Deary, Ian J.

    2012-01-01

    Three prominent theories of intelligence, the Cattell-Horn-Carroll (CHC), extended fluid-crystallized (Gf-Gc) and verbal-perceptual-image rotation (VPR) theories, provide differing descriptions of the structure of intelligence (McGrew, 2009; Horn & Blankson, 2005; Johnson & Bouchard, 2005b). To compare these theories, models representing them were…

  11. HIV-1 structural proteins serve as PAMPs for TLR2 heterodimers significantly increasing infection and innate immune activation

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    Kenneth Lee Rosenthal

    2015-08-01

    Full Text Available Immune activation is critical to HIV infection and pathogenesis; however, our understanding of HIV innate immune activation remains incomplete. Recently we demonstrated that soluble TLR2 (sTLR2 physically inhibited HIV-induced, NFκB activation and inflammation, as well as HIV-1 infection. In light of these findings, we hypothesized that HIV-1 structural proteins may serve as pathogen-associated molecular patterns (PAMPs for cellular TLR2 heterodimers. These studies made use of primary human T cells and TZMbl cells stably transformed to express TLR2 (TZMbl-2. Our results demonstrated that cells expressing TLR2 showed significantly increased proviral DNA compared to cells lacking TLR2, and mechanistically this may be due to a TLR2-mediated increased CCR5 expression. Importantly, we show that HIV-1 structural proteins, p17, p24, and gp41 act as viral PAMPs signalling through TLR2 and its heterodimers leading to significantly increased immune activation via the NFκB signaling pathway. Using co-immunoprecipitation and a dot blot method, we demonstrated direct protein interactions between these viral PAMPs and TLR2, while only p17 and gp41 bound to TLR1. Specifically, TLR2/1 heterodimer recognized p17 and gp41, while p24 lead to immune activation through TLR2/6. These results were confirmed using TLR2/1 siRNA knock down assays which ablated p17 and gp41-induced cellular activation and through studies of HEK293 cells expressing selected TLRs. Interestingly, our results show in the absence of TLR6, p24 bound to TLR2 and blocked p17 and gp41-induced activation, thus providing a novel mechanism by which HIV-1 can manipulate innate sensing. Taken together, our results identified, for the first time, novel HIV-1 PAMPs that play a role in TLR2-mediated cellular activation and increased proviral DNA. These findings have important implications for our fundamental understanding of HIV-1 immune activation and pathogenesis, as well as HIV-1 vaccine development.

  12. Probability weighted ensemble transfer learning for predicting interactions between HIV-1 and human proteins.

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    Suyu Mei

    Full Text Available Reconstruction of host-pathogen protein interaction networks is of great significance to reveal the underlying microbic pathogenesis. However, the current experimentally-derived networks are generally small and should be augmented by computational methods for less-biased biological inference. From the point of view of computational modelling, data scarcity, data unavailability and negative data sampling are the three major problems for host-pathogen protein interaction networks reconstruction. In this work, we are motivated to address the three concerns and propose a probability weighted ensemble transfer learning model for HIV-human protein interaction prediction (PWEN-TLM, where support vector machine (SVM is adopted as the individual classifier of the ensemble model. In the model, data scarcity and data unavailability are tackled by homolog knowledge transfer. The importance of homolog knowledge is measured by the ROC-AUC metric of the individual classifiers, whose outputs are probability weighted to yield the final decision. In addition, we further validate the assumption that only the homolog knowledge is sufficient to train a satisfactory model for host-pathogen protein interaction prediction. Thus the model is more robust against data unavailability with less demanding data constraint. As regards with negative data construction, experiments show that exclusiveness of subcellular co-localized proteins is unbiased and more reliable than random sampling. Last, we conduct analysis of overlapped predictions between our model and the existing models, and apply the model to novel host-pathogen PPIs recognition for further biological research.

  13. Determining confidence of predicted interactions between HIV-1 and human proteins using conformal method.

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    Nouretdinov, Ilia; Gammerman, Alex; Qi, Yanjun; Klein-Seetharaman, Judith

    2012-01-01

    Identifying protein-protein interactions (PPI's) is critical for understanding virtually all cellular molecular mechanisms. Previously, predicting PPI's was treated as a binary classification task and has commonly been solved in a supervised setting which requires a positive labeled set of known PPI's and a negative labeled set of non-interacting protein pairs. In those methods, the learner provides the likelihood of the predicted interaction, but without a confidence level associated with each prediction. Here, we apply a conformal prediction framework to make predictions and estimate confidence of the predictions. The conformal predictor uses a function measuring relative 'strangeness' interacting pairs to check whether prediction of a new example added to the sequence of already known PPI's would conform to the 'exchangeability' assumption: distribution of interacting pairs is invariant with any permutations of the pairs. In fact, this is the only assumption we make about the data. Another advantage is that the user can control a number of errors by providing a desirable confidence level. This feature of CP is very useful for a ranking list of possible interactive pairs. In this paper, the conformal method has been developed to deal with just one class - class interactive proteins - while there is not clearly defined of 'non-interactive'pairs. The confidence level helps the biologist in the interpretation of the results, and better assists the choices of pairs for experimental validation. We apply the proposed conformal framework to improve the identification of interacting pairs between HIV-1 and human proteins.

  14. Efficient trapping of HIV-1 envelope protein by hetero-oligomerization with an N-helix chimera

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    Silver Jonathan

    2005-08-01

    Full Text Available Abstract Background The N-heptad repeat region of the HIV-1 Transmembrane Envelope protein is a trimerization domain that forms part of a "six helix bundle" crucial to Envelope-mediated membrane fusion. N-heptad repeat peptides have been used as extracellular reagents to inhibit virus fusion. Results When expressed intracellularly with wild-type HIV-1 Envelope protein, the N-heptad repeat domain efficiently hetero-oligomerized with Envelope and trapped it in the endoplasmic reticulum or early Golgi, as indicated by lack of transport to the cell surface, absent proteolytic processing, and aberrant glycosylation. Conclusion Post-translational processing of HIV Envelope is very sensitive to an agent that binds to the N-heptad repeat during synthesis, suggesting that it might be possible to modify drugs that bind to this region to have transport-blocking properties.

  15. A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice

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    Tomusange, Khamis; Wijesundara, Danushka; Gummow, Jason; Garrod, Tamsin; Li, Yanrui; Gray, Lachlan; Churchill, Melissa; Grubor-Bauk, Branka; Gowans, Eric J.

    2016-01-01

    DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAX-sTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans. PMID:27358023

  16. Differentially co-expressed interacting protein pairs discriminate samples under distinct stages of HIV type 1 infection.

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    Yoon, Dukyong; Kim, Hyosil; Suh-Kim, Haeyoung; Park, Rae Woong; Lee, KiYoung

    2011-01-01

    Microarray analyses based on differentially expressed genes (DEGs) have been widely used to distinguish samples across different cellular conditions. However, studies based on DEGs have not been able to clearly determine significant differences between samples of pathophysiologically similar HIV-1 stages, e.g., between acute and chronic progressive (or AIDS) or between uninfected and clinically latent stages. We here suggest a novel approach to allow such discrimination based on stage-specific genetic features of HIV-1 infection. Our approach is based on co-expression changes of genes known to interact. The method can identify a genetic signature for a single sample as contrasted with existing protein-protein-based analyses with correlational designs. Our approach distinguishes each sample using differentially co-expressed interacting protein pairs (DEPs) based on co-expression scores of individual interacting pairs within a sample. The co-expression score has positive value if two genes in a sample are simultaneously up-regulated or down-regulated. And the score has higher absolute value if expression-changing ratios are similar between the two genes. We compared characteristics of DEPs with that of DEGs by evaluating their usefulness in separation of HIV-1 stage. And we identified DEP-based network-modules and their gene-ontology enrichment to find out the HIV-1 stage-specific gene signature. Based on the DEP approach, we observed clear separation among samples from distinct HIV-1 stages using clustering and principal component analyses. Moreover, the discrimination power of DEPs on the samples (70-100% accuracy) was much higher than that of DEGs (35-45%) using several well-known classifiers. DEP-based network analysis also revealed the HIV-1 stage-specific network modules; the main biological processes were related to "translation," "RNA splicing," "mRNA, RNA, and nucleic acid transport," and "DNA metabolism." Through the HIV-1 stage-related modules, changing

  17. Systematic Protein-Protein Docking and Molecular Dynamics Studies of HIV-1 gp120 and CD4: Insights for New Drug Development

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    M. Rizman-Idid

    2011-12-01

    Full Text Available Background and the purpose of the study: The interactions between HIV-1 gp120 and mutated CD4 proteins were investigated in order to identify a lead structure for therapy based on competitive blocking of the HIV binding receptor for human T-cells. Crystal structures of HIV gp120-CD4 complexes reveal a close interaction of the virus receptor with CD4 Phe43, which is embedded in a pocket of the virus protein.Methods: This study applies computer simulations to determine the best binding of amino acid 43 CD4 mutants to HIV gp120. Besides natural CD4, three mutants carrying alternate aromatic residues His, Trp and Tyr at position 43 were investigated. Several docking programs were applied on isolated proteins based on selected crystal structures of gp120-CD4 complexes, as well as a 5 ns molecular dynamics study on the protein complexes. The initial structures were minimized in Gromacs to avoid crystal packing effects, and then subjected to docking experiments using AutoDock4, FireDock, ClusPro and ZDock. In molecular dynamics, the Gibbs free binding energy was calculated for the gp120-CD4 complexes. The docking outputs were analyzed on energy within the respective docking software.Results and conclusion: Visualization and hydrogen bonding analysis were performed using the Swiss-PdbViewer. Strong binding to HIV gp120 can be achieved with an extended aromatic group (Trp. However, the sterical demand of the interaction affects the binding kinetics. In conclusion, a ligand for an efficient blocking of HIV gp120 should involve an extended but conformational flexible aromatic group, i.e. a biphenyl. A docking study on biphenylalanine-43 confirms this expectation

  18. Three-Dimensional Visualizations in Teaching Genomics and Bioinformatics: Mutations in HIV Envelope Proteins and Their Consequences for Vaccine Design

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    Kathy Takayama

    2009-11-01

    Full Text Available This project addresses the need to provide a visual context to teach the practical applications of genome sequencing and bioinformatics. Present-day research relies on indirect visualization techniques (e.g., fluorescence-labeling of DNA in sequencing reactions and sophisticated computer analysis. Such methods are impractical and prohibitively expensive for laboratory classes. More importantly, there is a need for curriculum resources that visually demonstrate the application of genome sequence information rather than the DNA sequencing methodology itself. This project is a computer-based lesson plan that engages students in collaborative, problem-based learning. The specific example focuses on approaches to Human Immunodeficiency Virus-1 (HIV-1 vaccine design based on HIV-1 genome sequences using a case study. Students performed comparative alignments of variant HIV-1 sequences available from a public database. Students then examined the consequences of HIV-1 mutations by applying the alignments to three-dimensional images of the HIV-1 envelope protein structure, thus visualizing the implications for applications such as vaccine design. The lesson enhances problem solving through the application of one type of information (genomic or protein sequence into concrete visual conceptualizations. Assessment of student comprehension and problem-solving ability revealed marked improvement after the computer tutorial. Furthermore, contextual presentation of these concepts within a case study resulted in student responses that demonstrated higher levels of cognitive ability than was expected by the instructor.

  19. Kinetic analysis of the effect of HIV nucleocapsid protein (NCp) on internal strand transfer reactions.

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    Raja, A; DeStefano, J J

    1999-04-20

    The mechanism of HIV reverse transcriptase (RT) catalyzed strand transfer synthesis (i.e., switching of the primer to a new template) from internal regions on RNA templates in the presence and absence of HIV nucleocapsid protein (NCp) was investigated. Two different systems each consisting of DNA-primed RNA donor (on which primer extension initiated) and acceptor (to which DNAs initiated on the donor could transfer) templates were used to determine kinetic parameters of strand transfer. The donor and acceptor shared an internal region of homology where homologous strand transfer could occur. The rate of strand transfer at various acceptor concentrations was determined by monitoring the production of transfer products over time. These rates were used to construct Lineweaver-Burk plots. In each system, NCp increased the Vmax about 3-fold while the Km for acceptor template was decreased severalfold. NCp's effects on RT extension ranged from no effect to inhibition depending on the primer-template used. The lowered Km shows that NCp increases the affinity of the acceptor template for the transferring DNA. Vmax increases despite the inhibition of RT extension. The increased Vmax implies a stimulatory mechanism that cannot be mimicked by high acceptor concentrations. Therefore, NCp does not act by merely increasing the effective concentration of nucleic acids.

  20. Primate TRIM5 proteins form hexagonal nets on HIV-1 capsids

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    Li, Yen-Li; Chandrasekaran, Viswanathan; Carter, Stephen D; Woodward, Cora L; Christensen, Devin E; Dryden, Kelly A; Pornillos, Owen; Yeager, Mark; Ganser-Pornillos, Barbie K; Jensen, Grant J; Sundquist, Wesley I

    2016-01-01

    TRIM5 proteins are restriction factors that block retroviral infections by binding viral capsids and preventing reverse transcription. Capsid recognition is mediated by C-terminal domains on TRIM5α (SPRY) or TRIMCyp (cyclophilin A), which interact weakly with capsids. Efficient capsid recognition also requires the conserved N-terminal tripartite motifs (TRIM), which mediate oligomerization and create avidity effects. To characterize how TRIM5 proteins recognize viral capsids, we developed methods for isolating native recombinant TRIM5 proteins and purifying stable HIV-1 capsids. Biochemical and EM analyses revealed that TRIM5 proteins assembled into hexagonal nets, both alone and on capsid surfaces. These nets comprised open hexameric rings, with the SPRY domains centered on the edges and the B-box and RING domains at the vertices. Thus, the principles of hexagonal TRIM5 assembly and capsid pattern recognition are conserved across primates, allowing TRIM5 assemblies to maintain the conformational plasticity necessary to recognize divergent and pleomorphic retroviral capsids. DOI: http://dx.doi.org/10.7554/eLife.16269.001 PMID:27253068

  1. The role of G protein gene GNB3 C825T Polymorphism in HIV-1 acquisition, progression and immune activation

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    Juno Jennifer

    2012-01-01

    Full Text Available Abstract Background The GNB3 C825T polymorphism is associated with increased G protein-mediated signal transduction, SDF-1α-mediated lymphocyte chemotaxis, accelerated HIV-1 progression, and altered responses to antiretroviral therapy among Caucasian subjects. The GNB3 825T allele is highly prevalent in African populations, and as such any impact on HIV-1 acquisition or progression rates could have a dramatic impact. This study examines the association of the 825T polymorphism with HIV-1 acquisition, disease progression and immune activation in two African cohorts. GNB3 825 genotyping was performed for enrolees in both a commercial sex worker cohort and a perinatal HIV transmission (PHT cohort in Nairobi, Kenya. Ex vivo immune activation was quantified by flow cytometry, and plasma chemokine levels were assessed by cytokine bead array. Results GNB3 genotype was not associated with sexual or vertical HIV-1 acquisition within these cohorts. Within the Pumwani cohort, GNB3 genotype did not affect HIV-1 disease progression among seroconverters or among HIV-1-positive individuals after adjustment for baseline CD4 count. Maternal CD4 decline and viral load increase in the PHT cohort did not differ between genotypes. Multi-parametric flow cytometry assessment of T cell activation (CD69, HLA-DR, CD38 and Treg frequency (CD25+FOXP3+ found no differences between genotype groups. Plasma SDF-1α, MIP-1β and TRAIL levels quantified by cytokine bead array were also similar between groups. Conclusions In contrast to previous reports, we were unable to provide evidence to suggest that the GNB3 C825T polymorphism affects HIV-1 acquisition or disease progression within African populations. Ex vivo immune activation and plasma chemokine levels were similarly unaffected by GNB3 genotype in both HIV-1-negative and HIV-1-positive individuals. The paucity of studies investigating the impact of GNB3 polymorphism among African populations and the lack of mechanistic

  2. Construction of doxycyline-dependent mini-HIV-1 variants for the development of a virotherapy against leukemias

    Directory of Open Access Journals (Sweden)

    Berkhout Ben

    2006-09-01

    Full Text Available Abstract T-cell acute lymphoblastic leukemia (T-ALL is a high-risk type of blood-cell cancer. We describe the improvement of a candidate therapeutic virus for virotherapy of leukemic cells. Virotherapy is based on the exclusive replication of a virus in leukemic cells, leading to the selective removal of these malignant cells. To improve the safety of such a virus, we constructed an HIV-1 variant that replicates exclusively in the presence of the nontoxic effector doxycycline (dox. This was achieved by replacement of the viral TAR-Tat system for transcriptional activation by the Escherichia coli-derived Tet system for inducible gene expression. This HIV-rtTA virus replicates in a strictly dox-dependent manner. In this virus, additional deletions and/or inactivating mutations were introduced in the genes for accessory proteins. These proteins are essential for virus replication in untransformed cells, but dispensable in leukemic T cells. These minimized HIV-rtTA variants contain up to 7 deletions/inactivating mutations (TAR, Tat, vif, vpR, vpU, nef and U3 and replicate efficiently in the leukemic SupT1 T cell line, but do not replicate in normal peripheral blood mononuclear cells. These virus variants are also able to efficiently remove leukemic cells from a mixed culture with untransformed cells. The therapeutic viruses use CD4 and CXCR4 for cell entry and could potentially be used against CXCR4 expressing malignancies such as T-lymphoblastic leukemia/lymphoma, NK leukemia and some myeloid leukemias.

  3. Boosting with Subtype C CN54rgp140 Protein Adjuvanted with Glucopyranosyl Lipid Adjuvant after Priming with HIV-DNA and HIV-MVA Is Safe and Enhances Immune Responses: A Phase I Trial.

    Directory of Open Access Journals (Sweden)

    Agricola Joachim

    Full Text Available A vaccine against HIV is widely considered the most effective and sustainable way of reducing new infections. We evaluated the safety and impact of boosting with subtype C CN54rgp140 envelope protein adjuvanted in glucopyranosyl lipid adjuvant (GLA-AF in Tanzanian volunteers previously given three immunizations with HIV-DNA followed by two immunizations with recombinant modified vaccinia virus Ankara (HIV-MVA.Forty volunteers (35 vaccinees and five placebo recipients were given two CN54rgp140/GLA-AF immunizations 30-71 weeks after the last HIV-MVA vaccination. These immunizations were delivered intramuscularly four weeks apart.The vaccine was safe and well tolerated except for one episode of asymptomatic hypoglycaemia that was classified as severe adverse event. Two weeks after the second HIV-MVA vaccination 34 (97% of the 35 previously vaccinated developed Env-specific binding antibodies, and 79% and 84% displayed IFN-γ ELISpot responses to Gag and Env, respectively. Binding antibodies to subtype C Env (included in HIV-DNA and protein boost, subtype B Env (included only in HIV-DNA and CRF01_AE Env (included only in HIV-MVA were significantly boosted by the CN54rgp140/GLA-AF immunizations. Functional antibodies detected using an infectious molecular clone virus/peripheral blood mononuclear cell neutralization assay, a pseudovirus/TZM-bl neutralization assay or by assays for antibody-dependent cellular cytotoxicity (ADCC were not significantly boosted. In contrast, T-cell proliferative responses to subtype B MN antigen and IFN-γ ELISpot responses to Env peptides were significantly enhanced. Four volunteers not primed with HIV-DNA and HIV-MVA before the CN54rgp140/GLA-AF immunizations mounted an antibody response, while cell-mediated responses were rare. After the two Env subtype C protein immunizations, a trend towards higher median subtype C Env binding antibody titers was found in vaccinees who had received HIV-DNA and HIV-MVA prior to the

  4. Viral replication is enhanced by an HIV-1 intersubtype recombination-derived Vpu protein

    Directory of Open Access Journals (Sweden)

    Salomón Horacio

    2010-10-01

    Full Text Available Abstract Background Multiple HIV-1 intersubtype recombinants have been identified in human populations. Previous studies from our lab group have shown that the epidemic in Argentina is characterized by the high prevalence of a circulating recombinant form, CRF12_BF, and many related BF recombinant forms. In these genomic structures a recombination breakpoint frequently involved the vpu coding region. Due to the scarce knowledge of Vpu participation in the virion release process and its impact on pathogenesis and of the functional capacities of intersubtype recombinant Vpu proteins, the aim of this work was to perform a comparative analysis on virion release capacity and relative replication capacity among viral variants harboring either a BF recombinant Vpu or a subtype B Vpu. Results Our results showed that BF recombinant Vpu was associated to an increased viral particles production when compared to WT B variant in tetherin-expressing cell lines. This observation was tested in the context of a competition assay between the above mentioned variants. The results showed that the replication of the BF Vpu-harboring variant was more efficient in cell cultures than subtype B, reaching a higher frequency in the viral population in a short period of time. Conclusion This study showed that as a result of intersubtype recombination, a structurally re-organized HIV-1 Vpu has an improved in vitro capacity of enhancing viral replication, and provides evidence of the changes occurring in this protein function that could play an important role in the successful spread of intersubtype recombinant variants.

  5. Structural basis for the potent inhibition of the HIV integrase-LEDGF/p75 protein-protein interaction.

    Science.gov (United States)

    Ribone, Sergio R; Quevedo, Mario A

    2017-08-01

    Integrase (IN) constitutes one of the key enzymes involved in the lifecycle of the Human Immunodeficiency Virus (HIV), the etiological agent of AIDS. The biological role of IN strongly depends on the recognition and binding of cellular cofactors belonging to the infected host cell. Thus, the inhibition of the protein-protein interaction (PPI) between IN and cellular cofactors has been envisioned as a promising therapeutic target. In the present work we explore a structure-activity relationship for a set of 14 compounds reported as inhibitors of the PPI between IN and the lens epithelium-derived growth factor (LEDGF/p75). Our results demonstrate that the possibility to adopt the bioactive conformation capable of interacting with the hotspots IN-LEDGF/p75 hotspots residues constitutes a critical feature to obtain a potent inhibition. A ligand efficiency (|Lig-Eff|) quantitative descriptor combining both interaction energetics and conformational requirements was developed and correlated with the reported biological activity. Our results contribute to the rational development of IN-LEDGF/p75 interaction inhibitors providing a solid quantitative structure-activity relationship aimed for the screening of new IN-LEDGF/p75 interaction inhibitors. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. The protein tyrosine kinase Fyn activates transcription from the HIV promoter via activation of NF kappa B-like DNA-binding proteins.

    Science.gov (United States)

    Hohashi, N; Hayashi, T; Fusaki, N; Takeuchi, M; Higurashi, M; Okamoto, T; Semba, K; Yamamoto, T

    1995-11-01

    Protein tyrosine kinase p59fyn (Fyn) associates with the TCR-CD3 complex, which suggests that Fyn plays a significant role in the signal transduction involving TCR complex. In addition to cellular genes, viral promoters such as the HIV long terminal repeat (LTR) are also activated upon T cell activation. To elucidate the functional significance of Fyn in the expression of viral promoters, we transfected a Fyn-expression vector together with a reporter plasmid containing the chloramphenicol acetyltransferase gene driven by HIV LTR into a human T cell line, Jurkat. In this assay, Fyn stimulated the promoter in HIV LTR when the transfected cells were treated with both concanavalin A and PMA as an antigen-mimic stimulation. This activation required the intact SH2 domain of Fyn. Mutational analysis of HIV LTR showed that the NF kappa B binding sites were responsible for this effect. Electrophoretic mobility shift assays and UV cross-linking experiments showed that activation of T cells by anti-CD3 antibody induced four kappa B-binding proteins (50, 60, 65 and 100 kDa) in Fyn-overexpressing cells more efficiently than in the parental cells. Our results suggested that Fyn was able to regulate expression of a subset of genes via kappa B-binding proteins upon T cell activation.

  7. HIV-1 Vpu protein mediates the transport of potassium in Saccharomyces cerevisiae.

    Science.gov (United States)

    Herrero, Laura; Monroy, Noemí; González, María Eugenia

    2013-01-01

    Human immunodeficiency virus type 1 (HIV-1) Vpu is an integral membrane protein that belongs to the viroporin family. Viroporins interact with cell membranes, triggering membrane permeabilization and promoting release of viral particles. In vitro electrophysiological methods have revealed changes in membrane ion currents when Vpu is present; however, in vivo the molecular mechanism of Vpu at the plasma membrane is still uncertain. We used the yeast Saccharomyces cerevisiae as a genetic model system to analyze how Vpu ion channel impacts cellular homeostasis. Inducible expression of Vpu impaired cell growth, suggesting that this viral protein is toxic to yeast cultures. This toxicity decreased with extracellular acidic pH. Also, Vpu toxicity diminished as the extracellular K(+) concentration was increased. However, expression of the Vpu protein suppresses the growth defect of K(+) uptake-deficient yeast (Δtrk1,2). The phenotype rescue of these highly hyperpolarized cells was almost total when they were grown in medium supplemented with high concentrations of KCl (100 mM) at pH 7.0 but was significantly reduced when the extracellular K(+) concentration or pH was decreased. These results indicate that Vpu has the ability to modify K(+) transport in both yeast strains. Here, we show also that Vpu confers tolerance to the aminoglycoside antibiotic hygromycin B in Δtrk1,2 yeast. Our results suggest that Vpu interferes with cell growth of wild-type yeast but improves proliferation of the hyperpolarized trk1,2 mutant by inducing plasma membrane depolarization. Furthermore, evaluation of the ion channel activity of the Vpu protein in Δtrk1,2 yeast could aid in the development of a high-throughput screening assay for molecules that target the retroviral protein.

  8. Immune evasion activities of accessory proteins Vpu, Nef and Vif are conserved in acute and chronic HIV-1 infection.

    Science.gov (United States)

    Mlcochova, Petra; Apolonia, Luis; Kluge, Silvia F; Sridharan, Aishwarya; Kirchhoff, Frank; Malim, Michael H; Sauter, Daniel; Gupta, Ravindra K

    2015-08-01

    Heterosexual HIV-1 transmission has been identified as a genetic bottleneck and a single transmitted/founder (T/F) variant with reduced sensitivity to type I interferon initiates productive infection in most cases. We hypothesized that particularly active accessory protein(s) may confer T/F viruses with a selective advantage in establishing HIV infection. Thus, we tested vpu, vif and nef alleles from six T/F and six chronic (CC) viruses in assays for 9 immune evasion activities involving the counteraction of interferon-stimulated genes and modulation of ligands known to activate innate immune cells. All functions were highly conserved with no significant differences between T/F and CC viruses, suggesting that these accessory protein functions are important throughout the course of infection.

  9. Protein–Protein Interaction between Surfactant Protein D and DC-SIGN via C-Type Lectin Domain Can Suppress HIV-1 Transfer

    Directory of Open Access Journals (Sweden)

    Eswari Dodagatta-Marri

    2017-07-01

    Full Text Available Surfactant protein D (SP-D is a soluble C-type lectin, belonging to the collectin (collagen-containing calcium-dependent lectin family, which acts as an innate immune pattern recognition molecule in the lungs at other mucosal surfaces. Immune regulation and surfactant homeostasis are salient functions of SP-D. SP-D can bind to a range of viral, bacterial, and fungal pathogens and trigger clearance mechanisms. SP-D binds to gp120, the envelope protein expressed on HIV-1, through its C-type lectin or carbohydrate recognition domain. This is of importance since SP-D is secreted by human mucosal epithelial cells and is present in the female reproductive tract, including vagina. Another C-type lectin, dendritic cell (DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN, present on the surface of the DCs, also binds to HIV-1 gp120 and facilitates viral transfer to the lymphoid tissues. DCs are also present at the site of HIV-1 entry, embedded in vaginal or rectal mucosa. In the present study, we report a direct protein–protein interaction between recombinant forms of SP-D (rfhSP-D and DC-SIGN via their C-type lectin domains. Both SP-D and DC-SIGN competed for binding to immobilized HIV-1 gp120. Pre-incubation of human embryonic kidney cells expressing surface DC-SIGN with rfhSP-D significantly inhibited the HIV-1 transfer to activated peripheral blood mononuclear cells. In silico analysis revealed that SP-D and gp120 may occupy same sites on DC-SIGN, which may explain the reduced transfer of HIV-1. In summary, we demonstrate, for the first time, that DC-SIGN is a novel binding partner of SP-D, and this interaction can modulate HIV-1 capture and transfer to CD4+ T cells. In addition, the present study also reveals a novel and distinct mechanism of host defense by SP-D against HIV-1.

  10. The HIV matrix protein p17 subverts nuclear receptors expression and induces a STAT1-dependent proinflammatory phenotype in monocytes.

    Directory of Open Access Journals (Sweden)

    Barbara Renga

    Full Text Available BACKGROUND: Long-term remission of HIV-1 disease can be readily achieved by combinations of highly effective antiretroviral therapy (HAART. However, a residual persistent immune activation caused by circulating non infectious particles or viral proteins is observed under HAART and might contribute to an higher risk of non-AIDS pathologies and death in HIV infected persons. A sustained immune activation supports lipid dysmetabolism and increased risk for development of accelerated atehrosclerosis and ischemic complication in virologically suppressed HIV-infected persons receiving HAART. AIM: While several HIV proteins have been identified and characterized for their ability to maintain immune activation, the role of HIV-p17, a matrix protein involved in the viral replication, is still undefined. RESULTS: Here, we report that exposure of macrophages to recombinant human p17 induces the expression of proinflammatory and proatherogenic genes (MCP-1, ICAM-1, CD40, CD86 and CD36 while downregulating the expression of nuclear receptors (FXR and PPARγ that counter-regulate the proinflammatory response and modulate lipid metabolism in these cells. Exposure of macrophage cell lines to p17 activates a signaling pathway mediated by Rack-1/Jak-1/STAT-1 and causes a promoter-dependent regulation of STAT-1 target genes. These effects are abrogated by sera obtained from HIV-infected persons vaccinated with a p17 peptide. Ligands for FXR and PPARγ counteract the effects of p17. CONCLUSIONS: The results of this study show that HIV p17 highjacks a Rack-1/Jak-1/STAT-1 pathway in macrophages, and that the activation of this pathway leads to a simultaneous dysregulation of immune and metabolic functions. The binding of STAT-1 to specific responsive elements in the promoter of PPARγ and FXR and MCP-1 shifts macrophages toward a pro-atherogenetic phenotype characterized by high levels of expression of the scavenger receptor CD36. The present work identifies p17 as a

  11. Interferons and interferon (IFN)-inducible protein 10 during highly active anti-retroviral therapy (HAART)-possible immunosuppressive role of IFN-alpha in HIV infection

    DEFF Research Database (Denmark)

    Stylianou, E; Aukrust, P; Bendtzen, K;

    2000-01-01

    Interferons play an important, but incompletely understood role in HIV-related disease. We investigated the effect of HAART on plasma levels of IFN-alpha, IFN-gamma, neopterin and interferon-inducible protein 10 (IP-10) in 41 HIV-infected patients during 78 weeks of therapy. At baseline HIV...... seemed not to involve enhanced lymphocyte apoptosis. Our findings suggest a pathogenic role of IFN-alpha in HIV infection, which may be a potential target for immunomodulating therapy in combination with HAART....

  12. Patterns of recombination in HIV-1M are influenced by selection disfavouring the survival of recombinants with disrupted genomic RNA and protein structures.

    Directory of Open Access Journals (Sweden)

    Michael Golden

    Full Text Available Genetic recombination is a major contributor to the ongoing diversification of HIV. It is clearly apparent that across the HIV-genome there are defined recombination hot and cold spots which tend to co-localise both with genomic secondary structures and with either inter-gene boundaries or intra-gene domain boundaries. There is also good evidence that most recombination breakpoints that are detectable within the genes of natural HIV recombinants are likely to be minimally disruptive of intra-protein amino acid contacts and that these breakpoints should therefore have little impact on protein folding. Here we further investigate the impact on patterns of genetic recombination in HIV of selection favouring the maintenance of functional RNA and protein structures. We confirm that chimaeric Gag p24, reverse transcriptase, integrase, gp120 and Nef proteins that are expressed by natural HIV-1 recombinants have significantly lower degrees of predicted folding disruption than randomly generated recombinants. Similarly, we use a novel single-stranded RNA folding disruption test to show that there is significant, albeit weak, evidence that natural HIV recombinants tend to have genomic secondary structures that more closely resemble parental structures than do randomly generated recombinants. These results are consistent with the hypothesis that natural selection has acted both in the short term to purge recombinants with disrupted RNA and protein folds, and in the longer term to modify the genome architecture of HIV to ensure that recombination prone sites correspond with those where recombination will be minimally deleterious.

  13. Potent in vitro antiviral activity of Cistus incanus extract against HIV and Filoviruses targets viral envelope proteins.

    Science.gov (United States)

    Rebensburg, Stephanie; Helfer, Markus; Schneider, Martha; Koppensteiner, Herwig; Eberle, Josef; Schindler, Michael; Gürtler, Lutz; Brack-Werner, Ruth

    2016-02-02

    Novel therapeutic options are urgently needed to improve global treatment of virus infections. Herbal products with confirmed clinical safety features are attractive starting material for the identification of new antiviral activities. Here we demonstrate that Cistus incanus (Ci) herbal products inhibit human immunodeficiency virus (HIV) infections in vitro. Ci extract inhibited clinical HIV-1 and HIV-2 isolates, and, importantly, a virus isolate with multiple drug resistances, confirming broad anti-HIV activity. Antiviral activity was highly selective for virus particles, preventing primary attachment of the virus to the cell surface and viral envelope proteins from binding to heparin. Bioassay-guided fractionation indicated that Ci extract contains numerous antiviral compounds and therefore has favorably low propensity to induce virus resistance. Indeed, no resistant viruses emerged during 24 weeks of continuous propagation of the virus in the presence of Ci extracts. Finally, Ci extracts also inhibited infection by virus particles pseudotyped with Ebola and Marburg virus envelope proteins, indicating that antiviral activity of Ci extract extends to emerging viral pathogens. These results demonstrate that Ci extracts show potent and broad in vitro antiviral activity against viruses that cause life-threatening diseases in humans and are promising sources of agents that target virus particles.

  14. Activation of the HIV-1 enhancer by the LEF-1 HMG protein on nucleosome-assembled DNA in vitro.

    Science.gov (United States)

    Sheridan, P L; Sheline, C T; Cannon, K; Voz, M L; Pazin, M J; Kadonaga, J T; Jones, K A

    1995-09-01

    Lymphoid enhancer-binding factor 1 (LEF-1) is a regulatory high mobility group (HMG) protein that activates the T cell receptor alpha (TCR alpha) enhancer in a context-restricted manner in T cells. In this paper we demonstrate that the distal region of the human immunodeficiency virus-1 (HIV-1) enhancer, which contains DNA-binding sites for LEF-1 and Ets-1, also provides a functional context for activation by LEF-1. First, we show that mutations in the LEF-1-binding site inhibit the activity of multimerized copies of the HIV-1 enhancer in Jurkat T cells, and that LEF-1/GAL4 can activate a GAL4-substituted HIV-1 enhancer 80- to 100-fold in vivo. Second, recombinant LEF-1 is shown to activate HIV-1 transcription on chromatin-assembled DNA in vitro. By using a nucleosome-assembly system derived from Drosophila embryos, we find that the packaging of DNA into chromatin in vitro strongly represses HIV-1 transcription and that repression can be counteracted efficiently by preincubation of the DNA with LEF-1 (or LEF-1 and Ets-1) supplemented with fractions containing the promoter-binding protein, Sp1. Addition of TFE-3, which binds to an E-box motif upstream of the LEF-1 and Ets-1 sites, further augments transcription in this system. Individually or collectively, none of the three enhancer-binding proteins (LEF-1, Ets-1, and TFE-3) could activate transcription in the absence of Sp1. A truncation mutant of LEF-1 (HMG-88), which contains the HMG box but lacks the trans-activation domain, did not activate transcription from nucleosomal DNA, indicating that bending of DNA by the HMG domain is not sufficient to activate transcription in vitro. We conclude that transcription activation by LEF-1 in vitro is a chromatin-dependent process that requires a functional trans-activation domain in addition to the HMG domain.

  15. LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein

    Directory of Open Access Journals (Sweden)

    Marker Daniel F

    2012-11-01

    Full Text Available Abstract Background Human Immunodeficiency Virus-1 (HIV-1 associated neurocognitive disorders (HANDs are accompanied by significant morbidity, which persists despite the use of combined antiretroviral therapy (cART. While activated microglia play a role in pathogenesis, changes in their immune effector functions, including phagocytosis and proinflammatory signaling pathways, are not well understood. We have identified leucine-rich repeat kinase 2 (LRRK2 as a novel regulator of microglial phagocytosis and activation in an in vitro model of HANDs, and hypothesize that LRRK2 kinase inhibition will attenuate microglial activation during HANDs. Methods We treated BV-2 immortalized mouse microglia cells with the HIV-1 trans activator of transcription (Tat protein in the absence or presence of LRRK2 kinase inhibitor (LRRK2i. We used Western blot, qRT-PCR, immunocytochemistry and latex bead engulfment assays to analyze LRRK2 protein levels, proinflammatory cytokine and phagocytosis receptor expression, LRRK2 cellular distribution and phagocytosis, respectively. Finally, we utilized ex vivo microfluidic chambers containing primary hippocampal neurons and BV-2 microglia cells to investigate microglial phagocytosis of neuronal axons. Results We found that Tat-treatment of BV-2 cells induced kinase activity associated phosphorylation of serine 935 on LRRK2 and caused the formation of cytoplasmic LRRK2 inclusions. LRRK2i decreased Tat-induced phosphorylation of serine 935 on LRRK2 and inhibited the formation of Tat-induced cytoplasmic LRRK2 inclusions. LRRK2i also decreased Tat-induced process extension in BV-2 cells. Furthermore, LRRK2i attenuated Tat-induced cytokine expression and latex bead engulfment. We examined relevant cellular targets in microfluidic chambers and found that Tat-treated BV-2 microglia cells cleared axonal arbor and engulfed neuronal elements, whereas saline treated controls did not. LRRK2i was found to protect axons in the presence

  16. Responsiveness of muscle protein synthesis to growth hormone administration in HIV-infected individuals declines with severity of disease.

    OpenAIRE

    McNurlan, M A; Garlick, P J; Steigbigel, R T; DeCristofaro, K A; Frost, R A; Lang, C H; Johnson, R. W.; Santasier, A M; Cabahug, C J; Fuhrer, J; Gelato, M C

    1997-01-01

    This study was undertaken to determine if human recombinant growth hormone (hrGH, 6 mg/d for 2 wk) would stimulate muscle protein synthesis in AIDS wasting. Healthy controls were compared with patients who were HIV+, had AIDS without weight loss, and had AIDS with > 10% weight loss. Before hrGH, rates of skeletal muscle protein synthesis, measured with l-[2H5]phenylalanine, were the same in controls and in all stages of disease. Rates of myofibrillar protein degradation, however, assessed fro...

  17. Bloch spin waves and emergent structure in protein folding with HIV envelope glycoprotein as an example

    Science.gov (United States)

    Dai, Jin; Niemi, Antti J.; He, Jianfeng; Sieradzan, Adam; Ilieva, Nevena

    2016-03-01

    We inquire how structure emerges during the process of protein folding. For this we scrutinize collective many-atom motions during all-atom molecular dynamics simulations. We introduce, develop, and employ various topological techniques, in combination with analytic tools that we deduce from the concept of integrable models and structure of discrete nonlinear Schrödinger equation. The example we consider is an α -helical subunit of the HIV envelope glycoprotein gp41. The helical structure is stable when the subunit is part of the biological oligomer. But in isolation, the helix becomes unstable, and the monomer starts deforming. We follow the process computationally. We interpret the evolving structure both in terms of a backbone based Heisenberg spin chain and in terms of a side chain based XY spin chain. We find that in both cases the formation of protein supersecondary structure is akin the formation of a topological Bloch domain wall along a spin chain. During the process we identify three individual Bloch walls and we show that each of them can be modelled with a precision of tenths to several angstroms in terms of a soliton solution to a discrete nonlinear Schrödinger equation.

  18. X-Ray Structures of Native HIV-1 Capsid Protein Reveal Conformational Variability

    Science.gov (United States)

    Gres, Anna T.; Kirby, Karen A.; KewalRamani, Vineet N.; Tanner, John J.; Pornillos, Owen; Sarafianos, Stefan G.

    2015-01-01

    The detailed molecular interactions between Human Immunodeficiency Virus type 1 (HIV-1) capsid protein (CA) hexamers have been elusive in the context of a native protein. We report crystal structures describing novel interactions between CA monomers related by 6-fold symmetry within a hexamer (intra-hexamer) and by 3-fold and 2-fold symmetry between neighboring hexamers (inter-hexamer). These structures help elucidate how CA builds a hexagonal lattice, the foundation of the mature capsid. Lattice structure depends on an adaptable hydration layer that modulates interactions among CA molecules. Disruption of this layer by crystal dehydration treatment alters inter-hexamer interfaces and condenses CA packing, highlighting an inherent structural variability. Capsid stability changes imparted by high concentrations of CA-targeting antiviral PF74 can be explained by variations at inter-hexamer interfaces remote to the ligand binding site. Inherent structural plasticity, hydration layer rearrangement, and effector molecule binding may perturb capsid uncoating or assembly and have functional implications for the retroviral life cycle. PMID:26044298

  19. Preparation and Characterization of Three Monoclonal Antibodies against HIV-1 p24 Capsid Protein

    Institute of Scientific and Technical Information of China (English)

    Guangjie Liu; Jianping Wang; Jianchun Xiao; Zhiwei Zhao; Yongtang Zheng

    2007-01-01

    HIV-1 p24 detection provides a means to aid the early diagnosis of HIV-1 infection, track the progression of disease and assess the efficacy of antiretroviral therapy. In the present study, three monoclonal antibodies (mAbs) p3JB9,p5F1 and p6F4 against HIV-1 p24 were generated. All mAbs could detect p24 of HIV-1ⅢB, HIV-1Ada-M, HIV-174v mAbs p5F1 and p6F4 could detect HIV-1KM018, while p3JB9 could not. Three mAbs did not react with HIV-2ROD,HIV-2CBL-20 and SIVagmTyo-1. The recognized epitope of p5F1 was located on the Gag amino acid region DCKTILKALGPAATLEEMMTAC. The p5F1 was used to establish a modified sandwich ELISA with rabbit anti-p24 serum and showed good specificity and high sensitivity, which has been used to measure HIV-1 p24 antigen levels in research.

  20. Potent Suppression of Viral Infectivity by the Peptides That Inhibit Multimerization of Human Immunodeficiency Virus Type 1 (HIV-1) Vif Proteins*

    OpenAIRE

    YANG Bin; Gao, Ling; Lin LI; Lu, Zhixian; Fan, Xuejun; Patel, Charvi A.; Pomerantz, Roger J.; DuBois, Garrett C.; Zhang, Hui

    2002-01-01

    Virion infectivity factor (Vif) is essential for the replication of human immunodeficiency virus type 1 (HIV-1) in vivo, but its function remains uncertain. Recently, we have shown that Vif proteins are able to form multimers, including dimers, trimers, or tetramers. Because the multimerization of Vif proteins is required for Vif function in the viral life cycle, we propose that it could be a novel target for anti-HIV-1 therapeutics. Through a phage peptide display method, we have identified ...

  1. A Novel HIV-1 Therapeutic Target:Tat Transactivator protein%一个新的HIV-1治疗靶--Tat转录激活蛋白

    Institute of Scientific and Technical Information of China (English)

    梁伟; 王亚芹; DAVALIAN DARIUSH

    2004-01-01

    Tat is a HIV-1 transaction transcriptional activator protein and plays a pivotal role in viral replication and in several AIDS-associated pathologies. Its biological properties and functions make it as a good candidate for the development of an anti-AIDS vaccine and/or drug. Strategies designing vaccines and drugs for anti-AIDS include vaccines derived from Tat, extracellular Tat-binding antagonists, inhibitors of Tat-activated intracellular second messengers, anti-Tat antisense,anti-TAR antisense, decoy and antagonists, anti-Tat intracellular intrabody, inhibitors of intracellular Tat cofactors and so on. An effective anti-AIDS therapy will require a multi-targeted approach in which classic antiviral drugs and protease inhibitors are combined with novel extracellular and intracellular Tat antagonists. This approach could prevent the development of drug-resistant HIV strains and decrease the dosage and related toxicity of each single drug and lead to a cure for AIDS-associated pathologies.%Tat是HIV-1病毒进行转录和复制的一个十分重要的蛋白质,同时,Tat也与HIV-1感染引起的严重病理学程度密切相关.Tat的生物学性质和功能决定了其是一个理想的开发抗AIDS疫苗和药物的靶蛋白.基于Tat自身及其作用的TARRNA,可以设计Tat疫苗、细胞外结合Tat的拮抗剂、抗Tat的反义核酸、抗TAR的反义核酸、抗Tat的细胞内抗体和细胞内Tat协同因子的抑制剂等.传统的抗病毒药物及蛋白酶抑制剂与新的细胞内和细胞外Tat拮抗剂联合使用,多靶点地抑制HIV-1的复制将是一个有效的抗AIDS的治疗方案.这一治疗方案能够防止HIV病毒耐药株的产生,减少单一作用靶点药物的用药剂量和降低相应的毒性,最终治愈AIDS相关的病理学变化.

  2. Development of a robust cell-based high-throughput screening assay to identify targets of HIV-1 viral protein R dimerization

    NARCIS (Netherlands)

    Zych, Courtney; Dömling, A.; Ayyavoo, Velpandi

    2013-01-01

    Targeting protein-protein interactions (PPI) is an emerging field in drug discovery. Dimerization and PPI are essential properties of human immunodeficiency virus (HIV)-1 proteins, their mediated functions, and virus biology. Additionally, dimerization is required for the functional interaction of H

  3. High-affinity binding of southern African HIV type 1 subtype C envelope protein, gp120, to the CCR5 coreceptor.

    Science.gov (United States)

    Fromme, Bernhard J; Coetsee, Marla; Van Der Watt, Pauline; Chan, Mei-Chi; Sperling, Karin M; Katz, Arieh A; Flanagan, Colleen A

    2008-12-01

    HIV-1 subtype C is the fastest spreading subtype worldwide and predominantly uses the CCR5 coreceptor, showing minimal transition to the X4 phenotype. This raises the possibility that envelope proteins of HIV-1 subtype C have structural features that favor interaction with CCR5. Preference for CCR5 could arise from enhanced affinity of HIV-1 subtype C for CCR5. To test this, we have characterized the interaction of gp120 envelope proteins from HIV-1 subtype C clones with CD4 and CCR5. Recombinant gp120 proteins from isolates of HIV-1 subtypes B and C were expressed, purified, and assessed in a CD4 binding assay and a CCR5 chemokine competition binding assay. All gp120 proteins bound to CD4-expressing cells, except one, 97ZA347ts, which had Arg substituted for the Cys239 in the conserved C2 loop. Reconstitution of Cys239, using site-directed mutagenesis, restored CD4 binding, while introducing Arg or Ser into position 239 of the functional Du151 gp120 protein abrogated CD4 binding. This shows that the Cys228-Cys239 disulfide bond of gp120 is required for high-affinity binding to CD4. Recombinant gp120 proteins from two HIV-1 subtype B clones bound CCR5 in the presence of CD4, while gp120 from the X4-tropic, HxB2, clone did not bind CCR5. gp120 from two functional HIV-1 subtype C clones, Du151 and MOLE1, bound CCR5 with high affinity in the presence of CD4 and Du151 showed significant CCR5 binding in the absence of CD4. A gp120 from a nonfunctional subtype C clone had lower affinity for CCR5. These results indicate that HIV-1 subtype C proteins have high affinity for CCR5 with variable dependence on CD4.

  4. Quantification of IgA and IgG and specificities of antibodies to viral proteins in parotid saliva at different stages of HIV-1 infection

    Science.gov (United States)

    CARTRY, O; MOJA, P; QUESNEL, A; POZZETTO, B; LUCHT, F R; GENIN, C

    1997-01-01

    Paired sera and parotid saliva from 75 HIV-1-infected patients, divided in three equal groups with CD4+ cell counts > 500, 200–500 and < 200/mm3, respectively, were analysed for IgG, IgA and secretory IgA (sIgA) concentrations and for IgG and IgA antibody directed to HIV-1. Twenty-nine age-matched HIV− subjects were used as controls. In serum the concentrations of immunoglobulins were significantly increased in HIV-infected subjects compared with controls, and a progressive increase of IgA and sIgA was noticed while the CD4+ cell count decreased. In contrast, concentrations of IgA and sIgA were not different in parotid saliva between the four subject groups. By an ELISA test directed towards HIV-1 proteins, 73 of the 75 serum specimens from the HIV-infected subjects (97%) and 43 of the corresponding saliva (57%) were found positive for specific IgA antibodies to HIV-1, with an even distribution among the three groups of patients. By Western blotting multiple specificities of IgA to HIV-1 proteins were not frequently found in patients. By contrast, in spite of an IgG concentration in saliva about 100 times lower than that of IgA, reactivities were significantly higher for IgG than for IgA antibodies, especially to env and to pol HIV-1 products. Altogether, these data suggest that the regulation of IgA production in HIV-infected subjects is independent in serum and in parotid saliva. This imbalance of IgA/IgG antibodies to HIV-1 at the mucosal level appears to be a specific feature of HIV-1 infection, and may raise important issues in terms of local protection after immunization. PMID:9218823

  5. HIV-infected patients show functionally defective high-density lipoprotein (HDL paralleled with changes in HDL-associated proteins

    Directory of Open Access Journals (Sweden)

    V Estrada

    2012-11-01

    Full Text Available Purpose of the study Epidemiological studies have consistently demonstrated an inverse association between plasma HDL concentrations and cardiovascular risk. Although this cardioprotective role has been mainly attributed to its role in promoting cellular cholesterol efflux, there is an emerging interest in the anti-inflammatory and antioxidant properties of HDL. The aim of the study is to investigate the anti-inflammatory properties of HDL isolated from HIV-infected patients. Methods Cross-sectional study of 113 HIV-infected patients and 70 non-infected control subjects without evident CVD. From each subject, HDL was isolated by ultracentrifugation and its anti-inflammatory status was tested as its ability to inhibit MCP-1-induced migration of the monocytic cell line THP-1 using transwell cell culture chamber inserts with micropore filters of 5 microns pore size. HDL-associated proteins were measured by commercial ELISAs. Results Twenty-three HIV-infected patients were ART-naïve (32±15 years, 66.7% male and ninety were currently on ART (46±11 years, 78.9 % male. Most patients on ART (91.1% had undetectable viral load (<50 copies/mL. When compared to healthy subjects, both naïve and treated HIV-infected patients had lower plasma HDL-cholesterol levels (naïve: 45±12, ART: 50±10, controls: 55±10 mg/dL, p<0.05. HDL isolated from HIV naïve patients showed a significantly reduced anti-inflammatory activity (THP-1 monocyte migration capacity was 203% times higher in HIV patients than in control subjects. The anti-inflammatory activity of HDL from ART-treated HIV-infected patients was significantly improved when compared to naïve patients, although it remained significantly lower than controls (130% THP-1 monocyte migration vs controls. HDL from HIV-infected patients had a decreased concentration of the anti-inflammatory proteins Apo A1 (controls: 2.1±0.3, naïve: 1.4±0.2, ART: 1.6±0.1 mg/ml and LCAT (controls: 0.53±0.09, naïve: 0.34±0

  6. Unique and differential protein signatures within the mononuclear cells of HIV-1 and HCV mono-infected and co-infected patients.

    Science.gov (United States)

    Boukli, Nawal M; Shetty, Vivekananda; Cubano, Luis; Ricaurte, Martha; Coelho-Dos-Reis, Jordana; Nickens, Zacharie; Shah, Punit; Talal, Andrew H; Philip, Ramila; Jain, Pooja

    2012-09-07

    Pathogenesis of liver damage in patients with HIV and HCV co-infection is complex and multifactorial. Although global awareness regarding HIV-1/HCV co-infection is increasing little is known about the pathophysiology that mediates the rapid progression to hepatic disease in the co-infected individuals. In this study, we investigated the proteome profiles of peripheral blood mononuclear cells from HIV-1 mono-, HCV mono-, and HIV-1/HCV co-infected patients. The results of high-resolution 2D gel electrophoresis and PD quest software quantitative analysis revealed that several proteins were differentially expressed in HIV-1, HCV, and HIV-1/HCV co-infection. Liquid chromatography-mass spectrometry and Mascot database matching (LC-MS/MS analysis) successfully identified 29 unique and differentially expressed proteins. These included cytoskeletal proteins (tropomyosin, gelsolin, DYPLSL3, DYPLSL4 and profilin-1), chaperones and co-chaperones (HSP90-beta and stress-induced phosphoprotein), metabolic and pre-apoptotic proteins (guanosine triphosphate [GTP]-binding nuclear protein Ran, the detoxifying enzyme glutathione S-transferase (GST) and Rho GDP-dissociation inhibitor (Rho-GDI), proteins involved in cell prosurvival mechanism, and those involved in matrix synthesis (collagen binding protein 2 [CBP2]). The six most significant and relevant proteins were further validated in a group of mono- and co-infected patients (n = 20) at the transcriptional levels. The specific pro- and anti- apoptotic protein signatures revealed in this study could facilitate the understanding of apoptotic and protective immune-mediated mechanisms underlying HIV-1 and HCV co-infection and their implications on liver disease progression in co-infected patients.

  7. Unique and differential protein signatures within the mononuclear cells of HIV-1 and HCV mono-infected and co-infected patients

    Directory of Open Access Journals (Sweden)

    Boukli Nawal M

    2012-09-01

    Full Text Available Abstract Background Pathogenesis of liver damage in patients with HIV and HCV co-infection is complex and multifactorial. Although global awareness regarding HIV-1/HCV co-infection is increasing little is known about the pathophysiology that mediates the rapid progression to hepatic disease in the co-infected individuals. Results In this study, we investigated the proteome profiles of peripheral blood mononuclear cells from HIV-1 mono-, HCV mono-, and HIV-1/HCV co-infected patients. The results of high-resolution 2D gel electrophoresis and PD quest software quantitative analysis revealed that several proteins were differentially expressed in HIV-1, HCV, and HIV-1/HCV co-infection. Liquid chromatography-mass spectrometry and Mascot database matching (LC-MS/MS analysis successfully identified 29 unique and differentially expressed proteins. These included cytoskeletal proteins (tropomyosin, gelsolin, DYPLSL3, DYPLSL4 and profilin-1, chaperones and co-chaperones (HSP90-beta and stress-induced phosphoprotein, metabolic and pre-apoptotic proteins (guanosine triphosphate [GTP]-binding nuclear protein Ran, the detoxifying enzyme glutathione S-transferase (GST and Rho GDP-dissociation inhibitor (Rho-GDI, proteins involved in cell prosurvival mechanism, and those involved in matrix synthesis (collagen binding protein 2 [CBP2]. The six most significant and relevant proteins were further validated in a group of mono- and co-infected patients (n = 20 at the transcriptional levels. Conclusions The specific pro- and anti- apoptotic protein signatures revealed in this study could facilitate the understanding of apoptotic and protective immune-mediated mechanisms underlying HIV-1 and HCV co-infection and their implications on liver disease progression in co-infected patients.

  8. Proteomic profiling of SupT1 cells reveal modulation of host proteins by HIV-1 Nef variants.

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    Reshu Saxena

    Full Text Available Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1 through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01 in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1, VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 (55AAAAAAA61 and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein

  9. Purified protein derivative skin testing on HIV/AIDS patients and logistic regression analysis of its risk factors

    Institute of Scientific and Technical Information of China (English)

    Fengren Liu; Jianjun Ye; Changfu Xiong; Jiguo Yin; Weihua He; Gaobo Li; Dingyuan Zhao; Linxiang Ye

    2007-01-01

    Objective: To understand the reactivity of purified protein derivative skin test(PPD test) in HIV-infected persons and to determine the influential factors associated with PPD. Methods: 174 HIV/AIDS patients registered in the local center for disease control and prevention(CDC) participated this study from April to June in 2006. Questionnaire, CD4 count and thoracic roentgenogram were performed for all participants. Results: In this study, response rate of questionnaires was 83.65%. The majority of these participants had a different degree of immunodeficiency that accounted for 93.64%. Female patients had a higher CD4 count than that of males. The total positive rate of PPD was 38.15%. Analysis of single factor in our study indicated that CD4 count, previous tuberculosis history, tuberculosis contact history and thoracic roentgenogram manifestation of patients were related to their PPD diameters. Further analysis of multiple factors also supports the previous conclusion that CD4 count and previous tuberculosis history of patients were risk factors in the PPD test. Conclusion: The PPD test of HIV/AIDS patients could be affected by several factors. For persons infected with HIV, the confirmation of latent tuberculosis infection (LTBI) should be considered the combination effect of previous MTB infection and body cellular immune function.

  10. HIV-1 Tat activates neuronal ryanodine receptors with rapid induction of the unfolded protein response and mitochondrial hyperpolarization.

    Directory of Open Access Journals (Sweden)

    John P Norman

    Full Text Available Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1 is ultimately refractory to highly active antiretroviral therapy (HAART because of failure of complete virus eradication in the central nervous system (CNS, and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR, followed by the unfolded protein response (UPR and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS.

  11. Boosting of HIV-1 neutralizing antibody responses by a distally related retroviral envelope protein

    DEFF Research Database (Denmark)

    Uchtenhagen, Hannes; Schiffner, Torben; Bowles, Emma

    2014-01-01

    Our knowledge of the binding sites for neutralizing Abs (NAb) that recognize a broad range of HIV-1 strains (bNAb) has substantially increased in recent years. However, gaps remain in our understanding of how to focus B cell responses to vulnerable conserved sites within the HIV-1 envelope glycop...

  12. Inhibition of HIV-1 Maturation via Small-Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein.

    Science.gov (United States)

    Wang, Weifeng; Zhou, Jing; Halambage, Upul D; Jurado, Kellie A; Jamin, Augusta V; Wang, Yujie; Engelman, Alan N; Aiken, Christopher

    2017-05-01

    The human immunodeficiency virus type 1 (HIV-1) capsid protein is an attractive therapeutic target, owing to its multifunctionality in virus replication and the high fitness cost of amino acid substitutions in capsids to HIV-1 infectivity. To date, small-molecule inhibitors have been identified that inhibit HIV-1 capsid assembly and/or impair its function in target cells. Here, we describe the mechanism of action of the previously reported capsid-targeting HIV-1 inhibitor, Boehringer-Ingelheim compound 1 (C1). We show that C1 acts during HIV-1 maturation to prevent assembly of a mature viral capsid. However, unlike the maturation inhibitor bevirimat, C1 did not significantly affect the kinetics or fidelity of Gag processing. HIV-1 particles produced in the presence of C1 contained unstable capsids that lacked associated electron density and exhibited impairments in early postentry stages of infection, most notably reverse transcription. C1 inhibited assembly of recombinant HIV-1 CA in vitro and induced aberrant cross-links in mutant HIV-1 particles capable of spontaneous intersubunit disulfide bonds at the interhexamer interface in the capsid lattice. Resistance to C1 was conferred by a single amino acid substitution within the compound-binding site in the N-terminal domain of the CA protein. Our results demonstrate that the binding site for C1 represents a new pharmacological vulnerability in the capsid assembly stage of the HIV-1 life cycle.IMPORTANCE The HIV-1 capsid protein is an attractive but unexploited target for clinical drug development. Prior studies have identified HIV-1 capsid-targeting compounds that display different mechanisms of action, which in part reflects the requirement for capsid function at both the efferent and afferent phases of viral replication. Here, we show that one such compound, compound 1, interferes with assembly of the conical viral capsid during virion maturation and results in perturbations at a specific protein-protein

  13. Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion.

    Directory of Open Access Journals (Sweden)

    Zhen Gong

    Full Text Available The membrane proximal region (MPR, residues 649-683 and transmembrane domain (TMD, residues 684-705 of the gp41 subunit of HIV-1's envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662-683 of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649-705, in Escherichia coli as a fusion protein with maltose binding protein (MBP. MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM. Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.

  14. Bacterial expression, correct membrane targeting and functional folding of the HIV-1 membrane protein Vpu using a periplasmic signal peptide.

    Science.gov (United States)

    Deb, Arpan; Johnson, William A; Kline, Alexander P; Scott, Boston J; Meador, Lydia R; Srinivas, Dustin; Martin-Garcia, Jose M; Dörner, Katerina; Borges, Chad R; Misra, Rajeev; Hogue, Brenda G; Fromme, Petra; Mor, Tsafrir S

    2017-01-01

    Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.

  15. Bacterial expression, correct membrane targeting and functional folding of the HIV-1 membrane protein Vpu using a periplasmic signal peptide

    Science.gov (United States)

    Deb, Arpan; Johnson, William A.; Kline, Alexander P.; Scott, Boston J.; Meador, Lydia R.; Srinivas, Dustin; Martin-Garcia, Jose M.; Dörner, Katerina; Borges, Chad R.; Misra, Rajeev; Hogue, Brenda G.; Fromme, Petra

    2017-01-01

    Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models. PMID:28225803

  16. Structure-based design of ligands for protein basic domains: Application to the HIV-1 Tat protein

    Science.gov (United States)

    Filikov, Anton V.; James, Thomas L.

    1998-05-01

    A methodology has been developed for designing ligands to bind a flexible basic protein domain where the structure of the domain is essentially known. It is based on an empirical binding free energy function developed for highly charged complexes and on Monte Carlo simulations in internal coordinates with both the ligand and the receptor being flexible. HIV-1 encodes a transactivating regulatory protein called Tat. Binding of the basic domain of Tat to TAR RNA is required for efficient transcription of the viral genome. The structure of a biologically active peptide containing the Tat basic RNA-binding domain is available from NMR studies. The goal of the current project is to design a ligand which will bind to that basic domain and potentially inhibit the TAR-Tat interaction. The basic domain contains six arginine and two lysine residues. Our strategy was to design a ligand for arginine first and then a superligand for the basic domain by joining arginine ligands with a linker. Several possible arginine ligands were obtained by searching the Available Chemicals Directory with DOCK 3.5 software. Phytic acid, which can potentially bind multiple arginines, was chosen as a building block for the superligand. Calorimetric binding studies of several compounds to methylguanidine and Arg-/Lys-containing peptides were performed. The data were used to develop an empirical binding free energy function for prediction of affinity of the ligands for the Tat basic domain. Modeling of the conformations of the complexes with both the superligand and the basic domain being flexible has been carried out via Biased Probability Monte Carlo (BPMC) simulations in internal coordinates (ICM 2.6 suite of programs). The simulations used parameters to ensure correct folding, i.e., consistent with the experimental NMR structure of a 25-residue Tat peptide, from a random starting conformation. Superligands for the basic domain were designed by joining together two molecules of phytic acid with

  17. Quantitative and phenotypic analyses of lymphocyte-monocyte heterokaryons induced by the HIV envelope proteins: Significant loss of lymphoid markers.

    Science.gov (United States)

    Rivera-Toledo, Evelyn; Huerta, Leonor; Larralde, Carlos; Lamoyi, Edmundo

    2011-04-01

    Cells infected with the human immunodeficiency virus (HIV) can fuse with CD4(+) cells leading to the formation of multinucleated cells. The presence of multinucleated cells infected with HIV in tissues of patients has been documented, although their cellular composition and role in AIDS pathogenesis is still under study. Here, we present evidence of in vitro heterotypic lymphocyte-monocyte fusion in cocultures of lymphocytic Jurkat T cells expressing the HIV-1 gp120/gp41 glycoproteins (Env) and CD4(+) monocytic THP-1 cells. Using a previously characterized method that involves differential labeling of fusion partners with fluorescent probes and flow cytometry analysis after coculture, up to 20% of double fluorescent cells were detected in 48h. This double fluorescent cell population was produced by heterotypic lymphocyte-monocyte fusion as it was not observed when Jurkat T cells expressing a mutant non-fusogenic Env protein were used. Heterokaryon formation was inhibited by an anti-CD4 monoclonal antibody and the HIV-fusion inhibitor peptide T-20. About 68% of heterokaryons remained alive and non-apoptotic after 2days of coculture. In heterokaryons, CD4 was barely detectable and the expression of the CD3 and CD28 lymphoid markers was greatly reduced, whereas the expression of CD32 and the intracellular antigen CD68, both markers of monocytic cells, remained unchanged. In contrast with unfused T cells, heterokaryons only expressed very low levels of the lymphoid activation marker CD25 following treatment with PMA plus ionomycin. These studies point to the possible generation of lymphocyte-monocyte heterokaryons with a myeloid phenotype during HIV infection, with unknown consequences for AIDS pathogenesis. Copyright © 2010 Elsevier Inc. All rights reserved.

  18. HIV-1 protein induced modulation of primary human osteoblast differentiation and function via a Wnt/β-catenin-dependent mechanism.

    LENUS (Irish Health Repository)

    Butler, Joseph S

    2013-02-01

    HIV infection is associated with metabolic bone disease resulting in bone demineralization and reduced bone mass. The molecular mechanisms driving this disease process have yet to be elucidated. Wnt\\/β-catenin signaling plays a key role in bone development and remodeling. We attempted to determine the effects of the HIV-1 protein, gp120, on Wnt\\/β-catenin signaling at an intracellular and transcriptional level in primary human osteoblasts (HOBs). This work, inclusive of experimental controls, was part of a greater project assessing the effects of a variety of different agents on Wnt\\/β-catenin signaling (BMC Musculoskelet Disord 2010;11:210).We examined the phenotypic effects of silencing and overexpressing the Wnt antagonist, Dickkopf-1 (Dkk1) in HOBs treated with gp120. HOBs exposed to gp120 displayed a significant reduction in alkaline phosphatase activity (ALP) activity and cell proliferation and increased cellular apoptosis over a 48 h time course. Immunocytochemistry demonstrated a significant reduction in intracytosolic and intranuclear β-catenin in response to HIV-1 protein exposure. These changes were associated with a reduction of TCF\\/LEF-mediated transcription, the transcriptional outcome of canonical Wnt β-catenin signaling. Silencing Dkk1 expression in HOBs exposed to gp120 resulted in increased ALP activity and cell proliferation, and decreased cellular apoptosis relative to scrambled control. Dkk1 overexpression exacerbated the inhibitory effect of gp120 on HOB function, with decreases in ALP activity and cell proliferation and increased cellular apoptosis relative to vector control. Wnt\\/β-catenin signaling plays a key regulatory role in HIV-associated bone loss, with Dkk1, aputative central mediator in this degenerative process. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 218-226, 2013.

  19. Elevation of intact and proteolytic fragments of acute phase proteins constitutes the earliest systemic antiviral response in HIV-1 infection.

    Directory of Open Access Journals (Sweden)

    Holger B Kramer

    2010-05-01

    Full Text Available The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, beta-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA occurred as early as 5-7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha-1-antitrypsin (AAT, termed virus inhibitory peptide (VIRIP, was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies.

  20. Attenuating HIV Tat/TAR-mediated protein expression by exploring the side chain length of positively charged residues.

    Science.gov (United States)

    Wu, Cheng-Hsun; Chen, Yi-Ping; Liu, Shing-Lung; Chien, Fan-Ching; Mou, Chung-Yuan; Cheng, Richard P

    2015-12-07

    RNA is a drug target involved in diverse cellular functions and viral processes. Molecules that inhibit the HIV TAR RNA-Tat protein interaction may attenuate Tat/TAR-dependent protein expression and potentially serve as anti-HIV therapeutics. By incorporating positively charged residues with mixed side chain lengths, we designed peptides that bind TAR RNA with enhanced intracellular activity. Tat-derived peptides that were individually substituted with positively charged residues with varying side chain lengths were evaluated for TAR RNA binding. Positively charged residues with different side chain lengths were incorporated at each Arg and Lys position in the Tat-derived peptide to enhance TAR RNA binding. The resulting peptides showed enhanced TAR RNA binding affinity, cellular uptake, nuclear localization, proteolytic resistance, and inhibition of intracellular Tat/TAR-dependent protein expression compared to the parent Tat-derived peptide with no cytotoxicity. Apparently, the enhanced inhibition of protein expression by these peptides was not determined by RNA binding affinity, but by proteolytic resistance. Despite the high TAR binding affinity, a higher binding specificity would be necessary for practical purposes. Importantly, altering the positively charged residue side chain length should be a viable strategy to generate potentially useful RNA-targeting bioactive molecules.

  1. Rigid proteins and softening of biological membranes—with application to HIV-induced cell membrane softening

    Science.gov (United States)

    Agrawal, Himani; Zelisko, Matthew; Liu, Liping; Sharma, Pradeep

    2016-05-01

    A key step in the HIV-infection process is the fusion of the virion membrane with the target cell membrane and the concomitant transfer of the viral RNA. Experimental evidence suggests that the fusion is preceded by considerable elastic softening of the cell membranes due to the insertion of fusion peptide in the membrane. What are the mechanisms underpinning the elastic softening of the membrane upon peptide insertion? A broader question may be posed: insertion of rigid proteins in soft membranes ought to stiffen the membranes not soften them. However, experimental observations perplexingly appear to show that rigid proteins may either soften or harden membranes even though conventional wisdom only suggests stiffening. In this work, we argue that regarding proteins as merely non-specific rigid inclusions is flawed, and each protein has a unique mechanical signature dictated by its specific interfacial coupling to the surrounding membrane. Predicated on this hypothesis, we have carried out atomistic simulations to investigate peptide-membrane interactions. Together with a continuum model, we reconcile contrasting experimental data in the literature including the case of HIV-fusion peptide induced softening. We conclude that the structural rearrangements of the lipids around the inclusions cause the softening or stiffening of the biological membranes.

  2. Rigid proteins and softening of biological membranes-with application to HIV-induced cell membrane softening.

    Science.gov (United States)

    Agrawal, Himani; Zelisko, Matthew; Liu, Liping; Sharma, Pradeep

    2016-05-06

    A key step in the HIV-infection process is the fusion of the virion membrane with the target cell membrane and the concomitant transfer of the viral RNA. Experimental evidence suggests that the fusion is preceded by considerable elastic softening of the cell membranes due to the insertion of fusion peptide in the membrane. What are the mechanisms underpinning the elastic softening of the membrane upon peptide insertion? A broader question may be posed: insertion of rigid proteins in soft membranes ought to stiffen the membranes not soften them. However, experimental observations perplexingly appear to show that rigid proteins may either soften or harden membranes even though conventional wisdom only suggests stiffening. In this work, we argue that regarding proteins as merely non-specific rigid inclusions is flawed, and each protein has a unique mechanical signature dictated by its specific interfacial coupling to the surrounding membrane. Predicated on this hypothesis, we have carried out atomistic simulations to investigate peptide-membrane interactions. Together with a continuum model, we reconcile contrasting experimental data in the literature including the case of HIV-fusion peptide induced softening. We conclude that the structural rearrangements of the lipids around the inclusions cause the softening or stiffening of the biological membranes.

  3. Virus-Like Particles Derived from HIV-1 for Delivery of Nuclear Proteins: Improvement of Production and Activity by Protein Engineering.

    Science.gov (United States)

    Robert, Marc-André; Lytvyn, Viktoria; Deforet, Francis; Gilbert, Rénald; Gaillet, Bruno

    2017-01-01

    Virus-like particles (VLPs) derived from retroviruses and lentiviruses can be used to deliver recombinant proteins without the fear of causing insertional mutagenesis to the host cell genome. In this study we evaluate the potential of an inducible lentiviral vector packaging cell line for VLP production. The Gag gene from HIV-1 was fused to a gene encoding a selected protein and it was transfected into the packaging cells. Three proteins served as model: the green fluorescent protein and two transcription factors-the cumate transactivator (cTA) of the inducible CR5 promoter and the human Krüppel-like factor 4 (KLF4). The sizes of the VLPs were 120-150 nm in diameter and they were resistant to freeze/thaw cycles. Protein delivery by the VLPs reached up to 100% efficacy in human cells and was well tolerated. Gag-cTA triggered up to 1100-fold gene activation of the reporter gene in comparison to the negative control. Protein engineering was required to detect Gag-KLF4 activity. Thus, insertion of the VP16 transactivation domain increased the activity of the VLPs by eightfold. An additional 2.4-fold enhancement was obtained by inserting nuclear export signal. In conclusion, our platform produced VLPs capable of efficient protein transfer, and it was shown that protein engineering can be used to improve the activity of the delivered proteins as well as VLP production.

  4. HIV-1 and IL-1β regulate astrocytic CD38 through mitogen-activated protein kinases and nuclear factor-κB signaling mechanisms

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    Mamik Manmeet K

    2011-10-01

    Full Text Available Abstract Background Infection with human immunodeficiency virus type-1 (HIV-1 leads to some form of HIV-1-associated neurocognitive disorders (HAND in approximately half of the cases. The mechanisms by which astrocytes contribute to HIV-1-associated dementia (HAD, the most severe form of HAND, still remain unresolved. HIV-1-encephalitis (HIVE, a pathological correlate of HAD, affects an estimated 9-11% of the HIV-1-infected population. Our laboratory has previously demonstrated that HIVE brain tissues show significant upregulation of CD38, an enzyme involved in calcium signaling, in astrocytes. We also reported an increase in CD38 expression in interleukin (IL-1β-activated astrocytes. In the present investigation, we studied regulatory mechanisms of CD38 gene expression in astrocytes activated with HIV-1-relevant stimuli. We also investigated the role of mitogen-activated protein kinases (MAPKs and nuclear factor (NF-κB in astrocyte CD38 regulation. Methods Cultured human astrocytes were transfected with HIV-1YU-2 proviral clone and levels of CD38 mRNA and protein were measured by real-time PCR gene expression assay, western blot analysis and immunostaining. Astrocyte activation by viral transfection was determined by analyzing proinflammatory chemokine levels using ELISA. To evaluate the roles of MAPKs and NF-κB in CD38 regulation, astrocytes were treated with MAPK inhibitors (SB203580, SP600125, U0126, NF-κB interfering peptide (SN50 or transfected with dominant negative IκBα mutant (IκBαM prior to IL-1β activation. CD38 gene expression and CD38 ADP-ribosyl cyclase activity assays were performed to analyze alterations in CD38 levels and function, respectively. Results HIV-1YU-2-transfection significantly increased CD38 mRNA and protein expression in astrocytes (p YU-2-transfected astrocytes significantly increased HIV-1 gene expression (p Conclusion The present findings demonstrate a direct involvement of HIV-1 and virus

  5. Change in High-Sensitivity C-Reactive Protein Levels Following Initiation of Efavirenz-Based Antiretroviral Regimens in HIV-Infected Individuals

    OpenAIRE

    Shikuma, Cecilia M.; Ribaudo, Heather J.; Zheng, Yu; Gulick, Roy M.; Meyer, William A.; Tashima, Karen T.; Bastow, Barbara; Kuritzkes, Daniel R.; Glesby, Marshall J.

    2011-01-01

    Elevations in C-reactive protein (CRP) are associated with increased cardiovascular disease (CVD) risk, increased HIV disease progression, and death in HIV-infected patients. Use of abacavir has been reported to increase CVD risk. We assessed the effect of virologically suppressive efavirenz (EFV)-based antiretroviral therapy on high sensitivity CRP (hsCRP) levels over a 96-week period with particular attention to the effect of gender and abacavir use. Banked sera from entry and week 96 visit...

  6. Mutational Analysis and Allosteric Effects in the HIV-1 Capsid Protein Carboxyl-Terminal Dimerization Domain

    Science.gov (United States)

    2009-01-01

    The carboxyl-terminal domain (CTD, residues 146−231) of the HIV-1 capsid (CA) protein plays an important role in the CA−CA dimerization and viral assembly of the human immunodeficiency virus type 1. Disrupting the native conformation of the CA is essential for blocking viral capsid formation and viral replication. Thus, it is important to identify the exact nature of the structural changes and driving forces of the CTD dimerization that take place in mutant forms. Here, we compare the structural stability, conformational dynamics, and association force of the CTD dimers for both wild-type and mutated sequences using all-atom explicit-solvent molecular dynamics (MD). The simulations show that Q155N and E159D at the major homology region (MHR) and W184A and M185A at the helix 2 region are energetically less favorable than the wild-type, imposing profound negative effects on intermolecular CA−CA dimerization. Detailed structural analysis shows that three mutants (Q155N, E159D, and W184A) display much more flexible local structures and weaker CA−CA association than the wild-type, primarily due to the loss of interactions (hydrogen bonds, side chain hydrophobic contacts, and π-stacking) with their neighboring residues. Most interestingly, the MHR that is far from the interacting dimeric interface is more sensitive to the mutations than the helix 2 region that is located at the CA−CA dimeric interface, indicating that structural changes in the distinct motif of the CA could similarly allosterically prevent the CA capsid formation. In addition, the structural and free energy comparison of the five residues shorter CA (151−231) dimer with the CA (146−231) dimer further indicates that hydrophobic interactions, side chain packing, and hydrogen bonds are the major, dominant driving forces in stabilizing the CA interface. PMID:19199580

  7. The prevalence and predictive value of dipstick urine protein in HIV-positive persons in Europe

    Directory of Open Access Journals (Sweden)

    Amanda Mocroft

    2014-11-01

    Full Text Available Introduction: Proteinuria (PTU is an important marker for the development and progression of renal disease, cardiovascular disease and death, but there is limited information about the prevalence and factors associated with confirmed PTU in predominantly white European HIV+ persons, especially in those with an estimated glomerular filtration rate (eGFR of 60 mL/min/1.73 m2. Patients and methods: Baseline was defined as the first of two consecutive dipstick urine protein (DPU measurements during prospective follow-up >1/6/2011 (when systematic data collection began. PTU was defined as two consecutive DUP >1+ (>30 mg/dL >3 months apart; persons with eGFR 90 and those with prior abacavir use had lower odds of PTU (Figure 1. There was no significant association between past or current use of tenofovir, lopinavir, atazanvir (boosted or unboosted or any other boosted PI and PTU (p>0.2. During 688.2 person-years of follow up (PYFU, three persons developed chronic kidney disease (CKD; confirmed [>3 months apart] eGFR60 was almost 10 times higher than in those without baseline PTU and eGFR>60 (rate ratio 9.61; 95% CI 0.87–105.9, p=0.065. Conclusions: One in 25 persons with eGFR>60 had confirmed proteinuria at baseline. Factors associated with PTU were similar to those associated with CKD. The lack of association with antiretrovirals, particularly tenofovir, may be due to the cross-sectional design of this study, and additional follow-up is required to address progression to PTU in those without PTU at baseline. It may also suggest other markers are needed to capture the deteriorating renal function associated with antiretrovirals may be needed at higher eGFRs. Our findings suggest PTU is an early marker for impaired renal function.

  8. The 73 kDa subunit of the CPSF complex binds to the HIV-1 LTR promoter and functions as a negative regulatory factor that is inhibited by the HIV-1 Tat protein.

    Science.gov (United States)

    de la Vega, Laureano; Sánchez-Duffhues, Gonzalo; Fresno, Manuel; Schmitz, M Lienhard; Muñoz, Eduardo; Calzado, Marco A

    2007-09-14

    Gene expression in eukaryotes requires the post-transcriptional cleavage of mRNA precursors into mature mRNAs. The cleavage and polyadenylation specificity factor (CPSF) is critical for this process and its 73 kDa subunit (CPSF-73) mediates cleavage coupled to polyadenylation and histone pre-mRNA processing. Using CPSF-73 over-expression and siRNA-mediated knockdown experiments, this study identifies CPSF-73 as an important regulatory protein that represses the basal transcriptional activity of the HIV-1 LTR promoter. Similar results were found with over-expression of the CPSF-73 homologue RC-68, but not with CPSF 100 kDa subunit (CPSF-100) and RC-74. Chromatin immunoprecipitation assays revealed the physical interaction of CPSF-73 with the HIV-1 LTR promoter. Further experiments revealed indirect CPSF-73 binding to the region between -275 to -110 within the 5' upstream region. Functional assays revealed the importance for the 5' upstream region (-454 to -110) of the LTR for CPSF-73-mediated transcription repression. We also show that HIV-1 Tat protein interacts with CPSF-73 and counteracts its repressive activity on the HIV-1 LTR promoter. Our results clearly show a novel function for CPSF-73 and add another candidate protein for explaining the molecular mechanisms underlying HIV-1 latency.

  9. Mining the protein data bank to differentiate error from structural variation in clustered static structures: an examination of HIV protease.

    Science.gov (United States)

    Venkatakrishnan, Balasubramanian; Palii, Miorel-Lucian; Agbandje-McKenna, Mavis; McKenna, Robert

    2012-03-01

    The Protein Data Bank (PDB) contains over 71,000 structures. Extensively studied proteins have hundreds of submissions available, including mutations, different complexes, and space groups, allowing for application of data-mining algorithms to analyze an array of static structures and gain insight about a protein's structural variation and possibly its dynamics. This investigation is a case study of HIV protease (PR) using in-house algorithms for data mining and structure superposition through generalized formulæ that account for multiple conformations and fractional occupancies. Temperature factors (B-factors) are compared with spatial displacement from the mean structure over the entire study set and separately over bound and ligand-free structures, to assess the significance of structural deviation in a statistical context. Space group differences are also examined.

  10. [Interaction of human apolipoprotein AI and HIV-1 envelope proteins with the native and recombinant CD4 receptors].

    Science.gov (United States)

    Panin, L E; Kostina, N E

    2003-01-01

    The method of enzyme-linked immunosorbent assay (ELISA) was used to show an interaction of soluble recombinant CD4-receptor (rsCD4) with human apolipoprotein A-1. Competitive interactions between envelope proteins VIH-1 (gp120 and gp41), on the one hand, and human apolipoprotein A-1 with CD4 receptor, present in the cellular membranes of line MT4 human lymphocytes, were demonstrated by the method of flow cytofluorimetry. It was suggested that the competitive interactions between the above proteins could manifest in respect to the apolipoprotein A-1 receptor, which affects the involvement of the latter in the regulation of protein biosynthesis and which leads to a decrease in the body weight of HIV-infected patients.

  11. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. Importance of the C-terminal unstructured tail.

    Science.gov (United States)

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisne, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55(Gag), reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNA(Lys) 3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity.

  12. HIV and schistosomiasis in rural Zimbabwe: the association of Retinol-binding protein with disease progression, inflammation and mortality

    Directory of Open Access Journals (Sweden)

    Sebastian Ranzi Kotzé

    2015-04-01

    Conclusions: In HIV-infected individuals, RBP was negatively associated with levels of inflammatory markers, markers of HIV progression, infection with schistosomiasis and markers of schistosomal intensity.

  13. The membrane-spanning domain of gp41 plays a critical role in intracellular trafficking of the HIV envelope protein

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    Kondo Naoyuki

    2010-11-01

    Full Text Available Abstract Background The sequences of membrane-spanning domains (MSDs on the gp41 subunit are highly conserved among many isolates of HIV-1. The GXXXG motif, a potential helix-helix interaction motif, and an arginine residue (rare in hydrophobic MSDs are especially well conserved. These two conserved elements are expected to locate on the opposite sides of the MSD, if the MSD takes a α-helical secondary structure. A scanning alanine-insertion mutagenesis was performed to elucidate the structure-function relationship of gp41 MSD. Results A circular dichroism analysis of a synthetic gp41 MSD peptide determined that the secondary structure of the gp41 MSD was α-helical. We then performed a scanning alanine-insertion mutagenesis of the entire gp41 MSD, progressively shifting the relative positions of MSD segments around the helix axis. Altering the position of Gly694, the last residue of the GXXXG motif, relative to Arg696 (the number indicates the position of the amino acid residues in HXB2 Env around the axis resulted in defective fusion. These mutants showed impaired processing of the gp160 precursor into gp120 and gp41. Furthermore, these Env mutants manifested inefficient intracellular transport in the endoplasmic reticulum and Golgi regions. Indeed, a transplantation of the gp41 MSD portion into the transmembrane domain of another membrane protein, Tac, altered its intracellular distribution. Our data suggest that the intact MSD α-helix is critical in the intracellular trafficking of HIV-1 Env. Conclusions The relative position between the highly conserved GXXXG motif and an arginine residue around the gp41 MSD α-helix is critical for intracellular trafficking of HIV-1 Env. The gp41 MSD region not only modulates membrane fusion but also controls biosynthesis of HIV-1 Env.

  14. Effect of rosiglitazone on visfatin and retinol-binding protein-4 plasma concentrations in HIV-positive patients.

    Science.gov (United States)

    Haider, D G; Schindler, K; Mittermayer, F; Müller, M; Nowotny, P; Rieger, A; Luger, A; Ludvik, B; Wolzt, M

    2007-04-01

    Thiazolidinediones (TZD) may improve insulin resistance in patients with diabetes and HIV. The novel adipocytokines visfatin and retinol-binding protein-4 (RBP-4) have been proposed to influence the development of impaired glucose tolerance. The impact of TZD on these cytokines is yet unknown. In this randomized, double-blind, placebo-controlled parallel group study, 37 lean HIV-positive subjects aged 19-50 years were treated with 8 mg/day rosiglitazone (n=20) or placebo (n=17) for 6 months. Insulin sensitivity was estimated from the homeostasis model assessment (HOMA) index. Fasting visfatin, RBP-4, leptin, and adiponectin plasma concentrations were analyzed by immunoassays. Rosiglitazone had no effect on impaired insulin sensitivity, but increased median plasma visfatin from 6.2 ng/ml (95% CI: 5.9; 6.5) to 13.7 ng/ml (12.6; 19.1) (P<0.001) and adiponectin from 3.2 ng/ml (2.2; 4.0) to 4.0 ng/ml (3.3; 8.5; P<0.001). RBP-4 was lowered from 21.0 ng/ml (19.6; 23.1) to 16.3 ng/ml (15.2; 17.0; P<0.001), and leptin concentrations were unchanged. Adipocytokine concentrations were stable in subjects receiving placebo, where a deterioration in insulin sensitivity was detectable (P<0.05). Changes in visfatin and RBP-4 were correlated in subjects receiving rosiglitazone (r=-0.64, P<0.01) but not placebo (r=0.12, P=0.15). TZD treatment affects circulating adipocytokine concentrations in subjects with HIV. Reductions in RBP-4 and increases in visfatin may contribute to the pharmacodynamic action of TZD on glucose homeostasis. Quantification of adipocytokines might be useful to assess TZD treatment effectiveness in insulin-resistant subjects with HIV.

  15. Gold nanoparticles mediated colorimetric assay for HIV-Tat protein detection

    Science.gov (United States)

    Hashwan, Saeed S. Ba; Ruslinda, A. Rahim; Fatin, M. F.; Gopinath, Subash C. B.; Thivina, V.; Tony, V. C. S.; Arshad, M. K. Md.; Hashim, U.

    2016-07-01

    Gold-nanoparticle (AuNP) based colorimetric assays have been formulated for different biomolecular interactions. With this assay the probe such as antibody immobilized on the Au surface and in the presence of appropriate binding partner (antigen), will interact with each other on the Au surface. By following this strategy, herein we formulated a detection system with two anti-HIV-Tat antibodies, Mono (McAb) - and polyclonal (PcAb) by immobilizing them independently with different AuNPs. Under this condition, these two antibodies are under dispersed condition, and in the presence of HIV-Tat antigen, these molecules will be connected and forms the aggregation of AuNPs. This strategy yield rapid results, can be monitored by the spectral changes in UV-Vis spectrophotometry. Experiments were performed with two different methods using two anti-HIV-Tats monoclonal and one Polyclonal antibody against the antigen HIV-Tat. Between these methods conjugation of HIV-Tat and McAb on the AuNP followed by addition of PcAb yielded better results.

  16. Expression of a Recombinant Anti-HIV and Anti-Tumor Protein, MAP30, in Nicotiana tobacum Hairy Roots: A pH-Stable and Thermophilic Antimicrobial Protein.

    Science.gov (United States)

    Moghadam, Ali; Niazi, Ali; Afsharifar, Alireza; Taghavi, Seyed Mohsen

    2016-01-01

    In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents.

  17. Expression of a Recombinant Anti-HIV and Anti-Tumor Protein, MAP30, in Nicotiana tobacum Hairy Roots: A pH-Stable and Thermophilic Antimicrobial Protein.

    Directory of Open Access Journals (Sweden)

    Ali Moghadam

    Full Text Available In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents.

  18. Expression of a Recombinant Anti-HIV and Anti-Tumor Protein, MAP30, in Nicotiana tobacum Hairy Roots: A pH-Stable and Thermophilic Antimicrobial Protein

    Science.gov (United States)

    Moghadam, Ali; Niazi, Ali; Afsharifar, Alireza; Taghavi, Seyed Mohsen

    2016-01-01

    In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents. PMID:27459300

  19. In vitro inhibition of vesicular stomatitis virus replication by purified porcine Mx1 protein fused to HIV-1 Tat protein transduction domain (PTD).

    Science.gov (United States)

    Zhang, Xiao-min; He, Dan-Ni; Zhou, Bin; Pang, Ran; Liu, Ke; Zhao, Jin; Chen, Pu-yan

    2013-08-01

    Vesicular stomatitis virus (VSV) is the causative agent of Vesicular stomatitis (VS), a highly contagious fatal disease of human and pigs. Few effective antiviral drugs are currently available against VSV infection. Mx proteins are interferon (IFN)-induced dynamin-like GTPases present in all vertebrates with a range of antiviral activities. Previous studies have shown that the transfected cell lines expressing either porcine Mx1 or human MxA acquired a high degree of resistance to VSV. To explore the feasibility of taking porcine Mx1 protein expressed in Escherichia coli as an antiviral agent, we applied the pCold system to express this fusion protein (PTD-poMx1), which consisted of an N-terminal HIV-1 Tat protein transduction domain (PTD) and the full-length porcine Mx1, and investigated its effects on the replication of VSV in Vero cells. The results demonstrated that the purified PTD-poMx1 fusion proteins could transduct into cells after incubated for 5h and had no cytotoxic. Furthermore, plaque reduction assay, determination of TCID50, real-time PCR and Western blot analyses were carried out to confirm the antiviral activity of purified fusion proteins in VSV-infected Vero cells. Altogether, these data suggested that PTD-poMx1 fusion proteins might be applicable to inhibit VSV replication as a novel antiviral therapeutic agent.

  20. The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

    Science.gov (United States)

    Scalabrin, Matteo; Frasson, Ilaria; Ruggiero, Emanuela; Perrone, Rosalba; Tosoni, Elena; Lago, Sara; Tassinari, Martina; Palù, Giorgio; Richter, Sara N.

    2017-01-01

    G-quadruplexes are four-stranded conformations of nucleic acids that act as cellular epigenetic regulators. A dynamic G-quadruplex forming region in the HIV-1 LTR promoter represses HIV-1 transcription when in the folded conformation. This activity is enhanced by nucleolin, which induces and stabilizes the HIV-1 LTR G-quadruplexes. In this work by a combined pull-down/mass spectrometry approach, we consistently found hnRNP A2/B1 as an additional LTR-G-quadruplex interacting protein. Surface plasmon resonance confirmed G-quadruplex specificity over linear sequences and fluorescence resonance energy transfer analysis indicated that hnRNP A2/B1 is able to efficiently unfold the LTR G-quadruplexes. Evaluation of the thermal stability of the LTR G-quadruplexes in different-length oligonucleotides showed that the protein is fit to be most active in the LTR full-length environment. When hnRNP A2/B1 was silenced in cells, LTR activity decreased, indicating that the protein acts as a HIV-1 transcription activator. Our data highlight a tightly regulated control of transcription based on G-quadruplex folding/unfolding, which depends on interacting cellular proteins. These findings provide a deeper understanding of the viral transcription mechanism and may pave the way to the development of drugs effective against the integrated HIV-1, present both in actively and latently infected cells.

  1. Solution structure of the Equine Infectious Anemia Virus p9 protein: a rationalization of its different ALIX binding requirements compared to the analogous HIV-p6 protein

    Directory of Open Access Journals (Sweden)

    Henklein Peter

    2009-12-01

    Full Text Available Abstract Background The equine infection anemia virus (EIAV p9 Gag protein contains the late (L- domain required for efficient virus release of nascent virions from the cell membrane of infected cell. Results In the present study the p9 protein and N- and C-terminal fragments (residues 1-21 and 22-51, respectively were chemically synthesized and used for structural analyses. Circular dichroism and 1H-NMR spectroscopy provide the first molecular insight into the secondary structure and folding of this 51-amino acid protein under different solution conditions. Qualitative 1H-chemical shift and NOE data indicate that in a pure aqueous environment p9 favors an unstructured state. In its most structured state under hydrophobic conditions, p9 adopts a stable helical structure within the C-terminus. Quantitative NOE data further revealed that this α-helix extends from Ser-27 to Ser-48, while the N-terminal residues remain unstructured. The structural elements identified for p9 differ substantially from that of the functional homologous HIV-1 p6 protein. Conclusions These structural differences are discussed in the context of the different types of L-domains regulating distinct cellular pathways in virus budding. EIAV p9 mediates virus release by recruiting the ALG2-interacting protein X (ALIX via the YPDL-motif to the site of virus budding, the counterpart of the YPXnL-motif found in p6. However, p6 contains an additional PTAP L-domain that promotes HIV-1 release by binding to the tumor susceptibility gene 101 (Tsg101. The notion that structures found in p9 differ form that of p6 further support the idea that different mechanisms regulate binding of ALIX to primary versus secondary L-domains types.

  2. Identification of a nuclear transport inhibitory signal (NTIS) in the basic domain of HIV-1 Vif protein.

    Science.gov (United States)

    Friedler, A; Zakai, N; Karni, O; Friedler, D; Gilon, C; Loyter, A

    1999-06-11

    The HIV-1 auxiliary protein Vif contains a basic domain within its sequence. This basic region,90RKKR93, is similar to the prototypic nuclear localization signal (NLS). However, Vif is not a nuclear protein and does not function in the nucleus. Here we have studied the karyophilic properties of this basic region. We have synthesized peptides corresponding to this positively charged NLS-like region and observed that these peptides inhibited nuclear transport via the importin pathway in vitro with IC50values in the micromolar range. Inhibition was observed only with peptides derived from the positively charged region, but not from other regions of the Vif protein, showing sequence specificity. On the other hand, the Vif inhibitory peptide Vif88-98 did not confer karyophilic properties when conjugated to BSA. The inactive Vif conjugate and the active SV40-NLS-BSA conjugate both contained a similar number of peptides conjugated to each BSA molecule, as was determined by amino acid analysis of the peptide-BSA conjugates. Thus, the lack of nuclear import of the Vif peptide-BSA conjugate cannot be attributed to insufficient number of conjugated peptide molecules per BSA molecule. Our results suggest that the HIV-1 Vif protein carries an NLS-like sequence that inhibits, but does not mediate, nuclear import via the importin pathway. We have termed such signals as nuclear transport inhibitory signals (NTIS). The possible role of NTIS in controlling nuclear uptake, and specifically during virus infection, is discussed herein. Our results raise the possibility that NLS-like sequences of certain low molecular weight viral proteins may serve as regulators of nucleocytoplasmic trafficking and not neccessarily as mediators of nuclear import.

  3. GB Virus C Proteine interferieren mit der Replikation von HIV-1

    OpenAIRE

    2010-01-01

    GB Virus C (GBV-C) was discovered in 1995 within the search for new hepatitis viruses. The human apathogenic enveloped RNA virus belongs to the family of the Flaviviridae and replicates primarily in lymphocytes. Transmission occurs mainly by sexual or parenteral exposure. In developed countries up to 6% of the healthy population and up to 40% of multiply exposed individuals, such as HIV patients, are viremic for GBV-C. Since the late 1990s, GBV-C has been investigated in the context of HIV. U...

  4. Effect of medroxyprogesterone acetate on the efficiency of an oral protein-rich nutritional support in HIV-infected patients.

    Science.gov (United States)

    Rochon, Cécile; Prod'homme, Magali; Laurichesse, Henri; Tauveron, Igor; Balage, Michèle; Gourdon, Florence; Baud, Olivier; Jacomet, Christine; Jouvency, Sylvie; Bayle, Gérard; Champredon, Claude; Thieblot, Philippe; Beytout, Jean; Grizard, Jean

    2003-01-01

    We have examined the effect of a medroxyprogesterone therapy in HIV-infected patients under appropriate nutrition for anabolism. The experiments were performed on 12 men (mean age 40 y), HIV seropositive but free of any clinically active opportunistic infection for at least one month. The patients underwent a 2-week baseline diet period (1.2 g protein x kg(-1) body weight (BW) x d(-1)) and then a 5-week experimental period with again the baseline diet in conjunction with supplements including Tonexis HP (0.7 g protein x kg(-1) BW) x d(-1)), L-threonine (0.018 g x kg(-1) BW x d(-1)) and L-methionine (0.013 g x kg(-1) BW x d(-1)). Indeed HIV-infected patients showed deficiencies in these amino acids. They were randomly divided into groups I and II under double-blinded condition. Group II was given medroxyprogesterone acetate (0.4 g x d(-1)) during the last 3 weeks whereas group I received a placebo. All the patients significantly increased their body weight (P < 0.05) during the experimental periods. Those under medroxyprogesterone tended to show a higher but not significant weight gain (+3.1 +/- 1.0 kg in group II and +1.9 +/- 0.3 kg in group I). Blood free amino acids were used as rough indicators of amino acid utilization and were analyzed prior and during acute 150 min intravenous infusion of a complete glucose-amino acid mixture. This test was done before and at the end of the experimental periods. Basal essential blood free amino acids were similar in the two groups and did not change during the experimental period. Most essential amino acids increased following glucose-amino acid infusions. The incremental increase was of less magnitude after the experimental period than before when medroxyprogesterone was present (P < 0.05 for valine, leucine, lysine, threonine and methionine). This was not the case in the absence of the hormone. We concluded that medroxyprogesterone might improve the efficacy of an oral protein-rich nutritional support in HIV

  5. HIV-1 Protein Nef Inhibits Activity of ATP-binding Cassette Transporter A1 by Targeting Endoplasmic Reticulum Chaperone Calnexin*

    Science.gov (United States)

    Jennelle, Lucas; Hunegnaw, Ruth; Dubrovsky, Larisa; Pushkarsky, Tatiana; Fitzgerald, Michael L.; Sviridov, Dmitri; Popratiloff, Anastas; Brichacek, Beda; Bukrinsky, Michael

    2014-01-01

    HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered high density lipoprotein profile exacerbated by down-modulation and impairment of ATP-binding cassette transporter A1 (ABCA1) activity by the HIV-1 protein Nef. However, the mechanisms of this Nef effect remain unknown. Here, we show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which regulates folding and maturation of glycosylated proteins. Nef disrupted interaction between calnexin and ABCA1 but increased affinity and enhanced interaction of calnexin with HIV-1 gp160. The Nef mutant that did not bind to calnexin did not affect the calnexin-ABCA1 interaction. Interaction with calnexin was essential for functionality of ABCA1, as knockdown of calnexin blocked the ABCA1 exit from the endoplasmic reticulum, reduced ABCA1 abundance, and inhibited cholesterol efflux; the same effects were observed after Nef overexpression. However, the effects of calnexin knockdown and Nef on cholesterol efflux were not additive; in fact, the combined effect of these two factors together did not differ significantly from the effect of calnexin knockdown alone. Interestingly, gp160 and ABCA1 interacted with calnexin differently; although gp160 binding to calnexin was dependent on glycosylation, glycosylation was of little importance for the interaction between ABCA1 and calnexin. Thus, Nef regulates the activity of calnexin to stimulate its interaction with gp160 at the expense of ABCA1. This study identifies a mechanism for Nef-dependent inactivation of ABCA1 and dysregulation of cholesterol metabolism. PMID:25170080

  6. Role of the HIV-1 Matrix Protein in Gag Intracellular Trafficking and Targeting to the Plasma Membrane for Virus Assembly

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    Ruba H Ghanam

    2012-02-01

    Full Text Available Human immunodeficiency virus type-1 (HIV-1 encodes a polypeptide called Gag that is able to form virus-like particles (VLPs in vitro in the absence of any cellular or viral constituents. During the late phase of the HIV-1 infection, Gag polyproteins are transported to the plasma membrane (PM for assembly. In the past two decades, in vivo, in vitro and structural studies have shown that Gag trafficking and targeting to the PM are orchestrated events that are dependent on multiple factors including cellular proteins and specific membrane lipids. The matrix (MA domain of Gag has been the focus of these studies as it appears to be engaged in multiple intracellular interactions that are suggested to be critical for virus assembly and replication. The interaction between Gag and the PM is perhaps the most understood. It is now established that the ultimate localization of Gag on punctate sites on the PM is mediated by specific interactions between the MA domain of Gag and phosphatidylinositol-4,5-bisphosphate (PI(4,5P2, a minor lipid localized on the inner leaflet of the PM. Structure-based studies revealed that binding of PI(4,5P2 to MA induces minor conformational changes, leading to exposure of the myristyl (myr group. Exposure of the myr group is also triggered by binding of calmodulin, enhanced by factors that promote protein self-association like the capsid domain of Gag, and is modulated by pH. Despite the steady progress in defining both the viral and cellular determinants of retroviral assembly and release, Gag’s intracellular interactions and trafficking to its assembly sites in the infected cell are poorly understood. In this review, we summarize the current understanding of the structural and functional role of MA in HIV replication.

  7. Highly conserved serine residue 40 in HIV-1 p6 regulates capsid processing and virus core assembly

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    Solbak Sara MØ

    2011-02-01

    Full Text Available Abstract Background The HIV-1 p6 Gag protein regulates the final abscission step of nascent virions from the cell membrane by the action of two late assembly (L- domains. Although p6 is located within one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds at least to two cellular budding factors (Tsg101 and ALIX, is a substrate for phosphorylation, ubiquitination, and sumoylation, and mediates the incorporation of the HIV-1 accessory protein Vpr into viral particles. As expected, known functional domains mostly overlap with several conserved residues in p6. In this study, we investigated the importance of the highly conserved serine residue at position 40, which until now has not been assigned to any known function of p6. Results Consistently with previous data, we found that mutation of Ser-40 has no effect on ALIX mediated rescue of HIV-1 L-domain mutants. However, the only feasible S40F mutation that preserves the overlapping pol open reading frame (ORF reduces virus replication in T-cell lines and in human lymphocyte tissue cultivated ex vivo. Most intriguingly, L-domain mediated virus release is not dependent on the integrity of Ser-40. However, the S40F mutation significantly reduces the specific infectivity of released virions. Further, it was observed that mutation of Ser-40 selectively interferes with the cleavage between capsid (CA and the spacer peptide SP1 in Gag, without affecting cleavage of other Gag products. This deficiency in processing of CA, in consequence, led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Moreover, the defects induced by the S40F mutation in p6 can be rescued by the A1V mutation in SP1 that generally enhances processing of the CA-SP1 cleavage site. Conclusions Overall, these data support a so far unrecognized function of p6 mediated by Ser-40 that occurs independently of the L-domain function, but selectively

  8. Speciifc T-cell Responses to CFP10, an Secreted Antigens of Mycobacterium Tuberculosis Protein, in Chinese hIV Positive Individuals

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

    Objective To construct prokaryotic expression vector of CFP-10 gene, and obtain recombinant protein, and the recombinant CFP-10 protein was taken as stimulus to detect speciifc T cell responses, to set up a method to faciliate to detect potential TB infection in China. Methods CFP-10 was cloned into inducible prokaryotic expression vector pET-32a (+) and transfected into E. coli BL21 (DE3). After IPTG induction, the product were veriifed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot hybridization were carried out to verify the antigenicity;the recombinant CFP-10 protein was taken as stimulus to detect speciifc T cell responses in HIV (+) persons with or without clinical manifestation of TB diseases, and HIV (-) controls with or without TB diseases. Results The CFP-10 recombinant protein exsited in the form of inclusion body and accounted for 94%in total bacterial protein of E. coli and the molecular weight is 31 kD;Western blot conifrmed the recombinant proteins had high antigenicity;our in-house ELISpot-IFN-γassay with recombinant antigen derived from CFP-10 proteins showed significant higher frequencies in TB patients with or without HIV infection than that in the healthy controls and only HIV (+) group. Conclusions The recombinant CFP-10 genes can be expressed successfully in prokaryotic expression system of E. coli and recombinant proteins with high antigenicity were obtained, which will set foundation for further study on their immunogenicity and bioinformatics. Our results proved that it is indeed true that some HIV positive patient have high frequencies of TB specific T cell responses, which maybe a clue to find latent TB infection in this population.

  9. Pokeweed antiviral protein restores levels of cellular APOBEC3G during HIV-1 infection by depurinating Vif mRNA.

    Science.gov (United States)

    Krivdova, Gabriela; Hudak, Katalin A

    2015-10-01

    Pokeweed antiviral protein (PAP) is an RNA glycosidase that inhibits production of human immunodeficiency virus type 1 (HIV-1) when expressed in human culture cells. Previously, we showed that the expression of PAP reduced the levels of several viral proteins, including virion infectivity factor (Vif). However, the mechanism causing Vif reduction and the consequences of the inhibition were not determined. Here we show that the Vif mRNA is directly depurinated by PAP. Because of depurination at two specific sites within the Vif ORF, Vif levels decrease during infections and the progeny viruses that are generated are ∼ 10-fold less infectious and compromised for proviral integration. These results are consistent with PAP activity inhibiting translation of Vif, which in turn reduces the effect of Vif to inactivate the host restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like editing complex 3G). Our findings identify Vif mRNA as a new substrate for PAP and demonstrate that derepression of innate immunity against HIV-1 contributes to its antiviral activity.

  10. HDAC inhibition induces HIV-1 protein and enables immune-based clearance following latency reversal

    DEFF Research Database (Denmark)

    Wu, Guoxin; Swanson, Michael; Talla, Aarthi

    2017-01-01

    Promising therapeutic approaches for eradicating HIV include transcriptional activation of provirus from latently infected cells using latency-reversing agents (LRAs) and immune-mediated clearance to purge reservoirs. Accurate detection of cells capable of producing viral antigens and virions, an...

  11. A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication.

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    Anna Garbelli

    Full Text Available DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

  12. A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication.

    Science.gov (United States)

    Garbelli, Anna; Beermann, Sandra; Di Cicco, Giulia; Dietrich, Ursula; Maga, Giovanni

    2011-05-12

    DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

  13. Lipid and acute-phase protein alterations in HIV-1 infected patients in the early stages of infection: correlation with CD4+ lymphocytes

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    Treitinger Arício

    2001-01-01

    Full Text Available Lipid and acute-phase protein alterations have been described in various infection diseases, and they have been recorded during the early stages of HIV infection. Lipid and acute-phase protein profiles also have been correlated with cellular immunological abnormalities. To document these correlations during HIV infection, we studied 75 HIV-infected patients and 26 HIV-negative controls. Patients were classified according to the criteria proposed by the Walter Reed Army Institute: as WR-1 (CD4 lymphocytes, 1154 ± 268/mm³, WR-2 (CD4, 793 ± 348/mm³ and WR3/4 (CD4, 287±75 mm³. Triglycerides, total cholesterol and HDL-cholesterol concentrations were measured by enzymatic methods. Immunoglobulins (IgA and IgG and acute-phase proteins (haptoglobin, a1-acid glycoprotein, C-reactive protein and transferrin were determined by immunonephelometry. Haptoglobin levels were significantly increased in HIV-positive patients and correlated with the progression of HIV-infection (controlHIV-negative controls. Elevated triglyceride levels (1.51±0.75 mmol/L were found only in WR3/4 patients, when compared to the control individuals (1.05±0.04 mmol/L. No differences were found in transferrin and C-reactive protein concentrations among the studied groups. CD4+ lymphocyte counts were inversely correlated with triglycerides, IgA, a1-acid glycoprotein and haptoglobin, and they were positively correlated with albumin, total cholesterol and HDL-cholesterol. Multiple linear regression analysis showed that increased haptoglobin and IgA levels were the best predictive variables of a decreasing CD4+ lymphocyte count. In conclusion, our data showed that: 1 a decrease in total cholesterol, HDL-cholesterol and albumin levels occurred earlier than hypertriglyceridemia in the course of

  14. Lipid and acute-phase protein alterations in HIV-1 infected patients in the early stages of infection: correlation with CD4+ lymphocytes.

    Science.gov (United States)

    Treitinger, A; Spada, C; da Silva, L M; Hermes, E M; Amaral, J A; Abdalla, D S

    2001-08-01

    Lipid and acute-phase protein alterations have been described in various infection diseases, and they have been recorded during the early stages of HIV infection. Lipid and acute-phase protein profiles also have been correlated with cellular immunological abnormalities. To document these correlations during HIV infection, we studied 75 HIV-infected patients and 26 HIV-negative controls. Patients were classified according to the criteria proposed by the Walter Reed Army Institute: as WR-1 (CD4 lymphocytes, 1154 +/- 268/mm3), WR-2 (CD4, 793 +/- 348/mm3) and WR3/4 (CD4, 287+/-75 mm3). Triglycerides, total cholesterol and HDL-cholesterol concentrations were measured by enzymatic methods. Immunoglobulins (IgA and IgG) and acute-phase proteins (haptoglobin, alpha1-acid glycoprotein, C-reactive protein and transferrin) were determined by immunonephelometry. Haptoglobin levels were significantly increased in HIV-positive patients and correlated with the progression of HIV-infection (controlHIV-negative controls. Elevated triglyceride levels (1.51+/-0.75 mmol/L) were found only in WR3/4 patients, when compared to the control individuals (1.05+/-0.04 mmol/L). No differences were found in transferrin and C-reactive protein concentrations among the studied groups. CD4+ lymphocyte counts were inversely correlated with triglycerides, IgA, alpha1-acid glycoprotein and haptoglobin, and they were positively correlated with albumin, total cholesterol and HDL-cholesterol. Multiple linear regression analysis showed that increased haptoglobin and IgA levels were the best predictive variables of a decreasing CD4+ lymphocyte count. In conclusion, our data showed that: 1) a decrease in total cholesterol, HDL-cholesterol and albumin levels occurred earlier than hypertriglyceridemia in the course of

  15. Lipid and acute-phase protein alterations in HIV-1 infected patients in the early stages of infection: correlation with CD4+ lymphocytes

    Directory of Open Access Journals (Sweden)

    Arício Treitinger

    Full Text Available Lipid and acute-phase protein alterations have been described in various infection diseases, and they have been recorded during the early stages of HIV infection. Lipid and acute-phase protein profiles also have been correlated with cellular immunological abnormalities. To document these correlations during HIV infection, we studied 75 HIV-infected patients and 26 HIV-negative controls. Patients were classified according to the criteria proposed by the Walter Reed Army Institute: as WR-1 (CD4 lymphocytes, 1154 ± 268/mm³, WR-2 (CD4, 793 ± 348/mm³ and WR3/4 (CD4, 287±75 mm³. Triglycerides, total cholesterol and HDL-cholesterol concentrations were measured by enzymatic methods. Immunoglobulins (IgA and IgG and acute-phase proteins (haptoglobin, a1-acid glycoprotein, C-reactive protein and transferrin were determined by immunonephelometry. Haptoglobin levels were significantly increased in HIV-positive patients and correlated with the progression of HIV-infection (controlHIV-negative controls. Elevated triglyceride levels (1.51±0.75 mmol/L were found only in WR3/4 patients, when compared to the control individuals (1.05±0.04 mmol/L. No differences were found in transferrin and C-reactive protein concentrations among the studied groups. CD4+ lymphocyte counts were inversely correlated with triglycerides, IgA, a1-acid glycoprotein and haptoglobin, and they were positively correlated with albumin, total cholesterol and HDL-cholesterol. Multiple linear regression analysis showed that increased haptoglobin and IgA levels were the best predictive variables of a decreasing CD4+ lymphocyte count. In conclusion, our data showed that: 1 a decrease in total cholesterol, HDL-cholesterol and albumin levels occurred earlier than hypertriglyceridemia in the course of

  16. [A novel immunization strategy to induce strong humoral responses against HIV-1 using combined DNA, recombinant vaccinia virus and protein vaccines].

    Science.gov (United States)

    Liu, Chang; Wang, Shu-hui; Ren, Li; Hao, Yan-ling; Zhang, Qi-cheng; Liu, Ying

    2014-11-01

    To optimize the immunization strategy against HIV-1, a DNA vaccine was combined with a recombinant vaccinia virus (rTV) vaccine and a protein vaccine. Immune responses against HIV-1 were detected in 30 female guinea pigs divided into six groups. Three groups of guinea pigs were primed with HIV-1 DNA vaccine three times, boosted with rTV at week 14, and then boosted with gp140 protein at intervals of 4, 8 or 12 weeks. Simultaneously, the other three groups of animals were primed with rTV vaccine once, and then boosted with gp140 after 4, 8 or 12 weeks. The HIV-1 specific binding antibody and neutralizing antibody, in addition to the relative affinity of these antibodies, were detected at different time points after the final administration of vaccine in each group. The DNA-rTV-gp140 immune regimen induced higher titers and affinity levels of HIV-1 gp120/gp140 antibodies and stronger V1V2-gp70 antibodies than the rTV-gp140 regimen. In the guinea pigs that underwent the DNA-rTV-gp140 regimen, the highest V1V2-gp70 antibody was induced in the 12-week-interval group. However, the avidity of antibodies was improved in the 4-week-interval group. Using the rTV-gp140 immunization strategy, guinea pigs boosted at 8 or 12 weeks after rTV priming elicited stronger humoral responses than those boosted at 4 weeks after priming. In conclusion, this study shows that the immunization strategy of HIV-1 DNA vaccine priming, followed by rTV and protein vaccine boosting, could strengthen the humoral response against HIV-1. Longer intervals were better to induce V1V2-gp70-specific antibodies, while shorter intervals were more beneficial to enhance the avidity of antibodies.

  17. Nucleoporin NUP153 phenylalanine-glycine motifs engage a common binding pocket within the HIV-1 capsid protein to mediate lentiviral infectivity.

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    Kenneth A Matreyek

    Full Text Available Lentiviruses can infect non-dividing cells, and various cellular transport proteins provide crucial functions for lentiviral nuclear entry and integration. We previously showed that the viral capsid (CA protein mediated the dependency on cellular nucleoporin (NUP 153 during HIV-1 infection, and now demonstrate a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG-repeat enriched NUP153 C-terminal domain (NUP153(C. NUP153(C fused to the effector domains of the rhesus Trim5α restriction factor (Trim-NUP153(C potently restricted HIV-1, providing an intracellular readout for the NUP153(C-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV bound NUP153(C under these conditions, results that correlated with direct binding between purified proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 protein during virus infection. Mutagenesis experiments concordantly identified NUP153(C and CA residues important for binding and lentiviral infectivity. Different FG motifs within NUP153(C mediated binding to HIV-1 versus EIAV capsids. HIV-1 CA binding mapped to residues that line the common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74 inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6 protein, with Asn57 (Asp58 in EIAV playing a particularly important role. PF74 and CPSF6 accordingly each competed with NUP153(C for binding to the HIV-1 CA pocket, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153(C expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicates that the NUP153(C-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. Our results highlight similar mechanisms of binding for disparate host factors

  18. Nedd4-mediated increase in HIV-1 Gag and Env proteins and immunity following DNA-vaccination of BALB/c mice.

    Science.gov (United States)

    Lewis, Brad; Whitney, Stephen; Hudacik, Lauren; Galmin, Lindsey; Huaman, Maria Cecilia; Cristillo, Anthony D

    2014-01-01

    The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation.

  19. Nedd4-mediated increase in HIV-1 Gag and Env proteins and immunity following DNA-vaccination of BALB/c mice.

    Directory of Open Access Journals (Sweden)

    Brad Lewis

    Full Text Available The late assembly domain of many viruses is critical for budding. Within these domains, encoded in viral structural proteins, are the conserved motifs PTAP, PPxY and YPxL. These sequences are the key determinants for association of viral proteins with intracellular molecules such as Tsg101, Nedd4 and AIP1/ALIX. While roles for Tsg101 and AIP1/ALIX in HIV-1 budding have been well established, less is known about the role of Nedd4. Recent studies, however, have identified a function for Nedd4-like protein in HIV-1 release. In this study, we investigated post-transcriptional changes of Nedd4 following SHIVSF162P3 infection of rhesus macaques, its role on HIV-1 p24 and gp120 levels in vitro and its potential as an immune modulator in HIV vaccination of BALB/c mice. Increased Nedd4 protein levels were noted in both CD4+ and CD8+ T cells following SHIVSF162P3-infection of naïve macaques. Transient co-transfection studies in 293 cells with HXB2 and Nedd4 demonstrated a Nedd4-mediated increase in p24 and gp120 levels. This increase was found to be dependent on the Ca2+/calmodulin-regulated phospholipid binding C2 domain and not ubiquitin ligase activity or HIV LTR activity. Co-transfection of Nedd4 with plasmid DNA expressing Gag or Env was further shown to augment both intracellular and extracellular Gag or Env proteins. To assess the potential of Nedd4 as an immune modulator, BALB/c mice were immunized intramuscularly with plasmid DNA encoding HIV gag, env and Nedd4. Nedd4 co-administration was found to increase serum anti-p24 but not anti-gp120 antibodies. Nedd4 co-injection was found to have no affect on Gag- or Env-specific IFNγ but had a trend of increased Gag-specific IL-6, IL-17A and TNFα that was not seen following Env stimulation. Based on our initial findings, Nedd4-mediated changes in HIV protein levels and its potential use in HIV-1 vaccine development warrants further investigation.

  20. Flexible and rigid structures in HIV-1 p17 matrix protein monitored by relaxation and amide proton exchange with NMR.

    Science.gov (United States)

    Ohori, Yuka; Okazaki, Honoka; Watanabe, Satoru; Tochio, Naoya; Arai, Munehito; Kigawa, Takanori; Nishimura, Chiaki

    2014-03-01

    The HIV-1 p17 matrix protein is a multifunctional protein that interacts with other molecules including proteins and membranes. The dynamic structure between its folded and partially unfolded states can be critical for the recognition of interacting molecules. One of the most important roles of the p17 matrix protein is its localization to the plasma membrane with the Gag polyprotein. The myristyl group attached to the N-terminus on the p17 matrix protein functions as an anchor for binding to the plasma membrane. Biochemical studies revealed that two regions are important for its function: D14-L31 and V84-V88. Here, the dynamic structures of the p17 matrix protein were studied using NMR for relaxation and amide proton exchange experiments at the physiological pH of 7.0. The results revealed that the α12-loop, which includes the 14-31 region, was relatively flexible, and that helix 4, including the 84-88 region, was the most protected helix in this protein. However, the residues in the α34-loop near helix 4 had a low order parameter and high exchange rate of amide protons, indicating high flexibility. This region is probably flexible because this loop functions as a hinge for optimizing the interactions between helices 3 and 4. The C-terminal long region of K113-Y132 adopted a disordered structure. Furthermore, the C-terminal helix 5 appeared to be slightly destabilized due to the flexible C-terminal tail based on the order parameters. Thus, the dynamic structure of the p17 matrix protein may be related to its multiple functions.

  1. Use of silver nanoparticles increased inhibition of cell-associated HIV-1 infection by neutralizing antibodies developed against HIV-1 envelope proteins

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    Garza Treviño Elsa N

    2011-09-01

    Full Text Available Abstract Background HIV/AIDS pandemic is a worldwide public health issue. There is a need for new approaches to develop new antiviral compounds or other therapeutic strategies to limit viral transmission. The envelope glycoproteins gp120 and gp41 of HIV are the main targets for both silver nanoparticles (AgNPs and neutralizing antibodies. There is an urgency to optimize the efficiency of the neutralizing antibodies (NABs. In this study, we demonstrated that there is an additive effect between the four NABs and AgNPs when combined against cell-associated HIV-1 infection in vitro Results Four NABs (Monoclonal antibody to HIV-1 gp41 126-7, HIV-1 gp120 Antiserum PB1 Sub 2, HIV-1 gp120 Antiserum PB1, HIV-1 gp120 Monoclonal Antibody F425 B4e8 with or without AgNPs of 30-50 nm in size were tested against cell free and cell-associated HIVIIIB virus. All NABs inhibited HIV-1 cell free infection at a dose response manner, but with AgNPs an antiviral additive effect was not achieved Although there was no inhibition of infection with cell-associated virus by the NABs itself, AgNPs alone were able to inhibit cell associated virus infection and more importantly, when mixed together with NABs they inhibited the HIV-1 cell associated infection in an additive manner. Discussion The most attractive strategies to deal with the HIV problem are the development of a prophylactic vaccine and the development of effective topical vaginal microbicide. For two decades a potent vaccine that inhibits transmission of infection of HIV has been searched. There are vaccines that elicit NABs but none of them has the efficacy to stop transmission of HIV-1 infection. We propose that with the addition of AgNPs, NABs will have an additive effect and become more potent to inhibit cell-associated HIV-1 transmission/infection. Conclusions The addition of AgNPs to NABs has significantly increased the neutralizing potency of NABs in prevention of cell-associated HIV-1 transmission

  2. Using Cellular Proteins to Reveal Mechanisms of HIV Infection | Center for Cancer Research

    Science.gov (United States)

    A vital step in HIV infection is the insertion of viral DNA into the genome of the host cell. In order for the insertion to occur, viral nucleic acid must be transported through the membrane that separates the main cellular compartment (the cytoplasm) from the nucleus, where the host DNA is located. Scientists are actively studying the mechanism used to transport viral DNA into the nucleus in the hopes of targeting this step with future anti-HIV treatments. Up to this point, researchers have identified some of the viral components that play a role in nuclear transport, but they have not determined how viral interactions with other molecules in the cell contribute to the process.

  3. Plasma Concentration of the Neurofilament Light Protein (NFL) is a Biomarker of CNS Injury in HIV Infection: A Cross-Sectional Study.

    Science.gov (United States)

    Gisslén, Magnus; Price, Richard W; Andreasson, Ulf; Norgren, Niklas; Nilsson, Staffan; Hagberg, Lars; Fuchs, Dietmar; Spudich, Serena; Blennow, Kaj; Zetterberg, Henrik

    2016-01-01

    Cerebrospinal fluid (CSF) neurofilament light chain protein (NFL) is a sensitive marker of neuronal injury in a variety of neurodegenerative conditions, including the CNS dysfunction injury that is common in untreated HIV infection. However, an important limitation is the requirement for lumbar puncture. For this reason, a sensitive and reliable blood biomarker of CNS injury would represent a welcome advance in both clinical and research settings. To explore whether plasma concentrations of NFL might be used to detect CNS injury in HIV infection, an ultrasensitive Single molecule array (Simoa) immunoassay was developed. Using a cross-sectional design, we measured NFL in paired CSF and plasma samples from 121 HIV-infected subjects divided into groups according to stage of their systemic disease, presence of overt HIV-associated dementia (HAD), and after antiretroviral treatment (ART)-induced viral suppression. HIV-negative controls were also examined. Plasma and CSF NFL concentrations were very highly correlated (r = 0.89, P NFL was more than 50-fold lower plasma than CSF it was within the quantifiable range of the new plasma assay in all subjects, including the HIV negatives and the HIV positives with normal CSF NFL concentrations. The pattern of NFL changes were almost identical in plasma and CSF, both exhibiting similar age-related increases in concentrations along with highest values in HAD and substantial elevations in ART-naïve neuroasymptomatic subjects with low blood CD4(+) T cells. These results show that plasma NFL may prove a valuable tool to evaluate ongoing CNS injury in HIV infection that may be applied in the clinic and in research settings to assess the presence if active CNS injury. Because CSF NFL is also elevated in a variety of other CNS disorders, sensitive measures of plasma NFL may similarly prove useful in other settings.

  4. Interleukin 6 Is a Stronger Predictor of Clinical Events Than High-Sensitivity C-Reactive Protein or D-Dimer During HIV Infection

    DEFF Research Database (Denmark)

    Borges, Alvaro Humberto Diniz; O'Connor, Jemma L; Phillips, Andrew N

    2016-01-01

    BACKGROUND: Interleukin 6 (IL-6), high-sensitivity C-reactive protein (hsCRP), and D-dimer levels are linked to adverse outcomes in human immunodeficiency virus (HIV) infection, but the strength of their associations with different clinical end points warrants investigation. METHODS: Participants...

  5. Lower HIV provirus levels are associated with more APOBEC3G protein in blood resting memory CD4+ T lymphocytes of controllers in vivo.

    Directory of Open Access Journals (Sweden)

    Mariapia De Pasquale

    Full Text Available Immunodeficiency does not progress for prolonged periods in some HLA B57- and/or B27-positive subjects with human immunodeficiency virus type 1 (HIV infection, even in the absence of antiretroviral therapy (ART. These "controllers" have fewer HIV provirus-containing peripheral blood mononuclear cells than "non-controller" subjects, but lymphocytes that harbor latent proviruses were not specifically examined in studies to date. Provirus levels in resting memory cells that can serve as latent reservoirs of HIV in blood were compared here between controllers and ART-suppressed non-controllers. APOBEC3G (A3G, a cellular factor that blocks provirus formation at multiple steps if not antagonized by HIV virion infectivity factor (Vif, was also studied. HLA-linked HIV control was associated with less provirus and more A3G protein in resting CD4+ T central memory (Tcm and effector memory (Tem lymphocytes (provirus: p = 0.01 for Tcm and p = 0.02 for Tem; A3G: p = 0.02 for Tcm and p = 0.02 for Tem. Resting memory T cells with the highest A3G protein levels (>0.5 RLU per unit of actin had the lowest levels of provirus (<1,000 copies of DNA per million cells in vivo (p = 0.03, Fisher's exact test. Using two different experimental approaches, Vif-positive viruses with more A3G were found to have decreased virion infectivity ex vivo. These results raise the hypothesis that HIV control is associated with increased cellular A3G that may be packaged into Vif-positive virions to add that mode of inhibition of provirus formation to previously described adaptive immune mechanisms for HIV control.

  6. Developing HIV-1 Protease Inhibitors through Stereospecific Reactions in Protein Crystals

    Directory of Open Access Journals (Sweden)

    Folasade M. Olajuyigbe

    2016-10-01

    Full Text Available Protease inhibitors are key components in the chemotherapy of HIV infection. However, the appearance of viral mutants routinely compromises their clinical efficacy, creating a constant need for new and more potent inhibitors. Recently, a new class of epoxide-based inhibitors of HIV-1 protease was investigated and the configuration of the epoxide carbons was demonstrated to play a crucial role in determining the binding affinity. Here we report the comparison between three crystal structures at near-atomic resolution of HIV-1 protease in complex with the epoxide-based inhibitor, revealing an in-situ epoxide ring opening triggered by a pH change in the mother solution of the crystal. Increased pH in the crystal allows a stereospecific nucleophile attack of an ammonia molecule onto an epoxide carbon, with formation of a new inhibitor containing amino-alcohol functions. The described experiments open a pathway for the development of new stereospecific protease inhibitors from a reactive lead compound.

  7. The effect of tenidap on cytokines, acute-phase proteins, and virus load in human immunodeficiency virus (HIV)-infected patients: correlation between plasma HIV-1 RNA and proinflammatory cytokine levels.

    Science.gov (United States)

    Dezube, B J; Lederman, M M; Chapman, B; Georges, D L; Dogon, A L; Mudido, P; Reis-Lishing, J; Cheng, S L; Silberman, S L; Crumpacker, C S

    1997-09-01

    Proinflammatory cytokines may be important in the pathogenesis of human immunodeficiency virus type 1 (HIV-1) disease. Tenidap decreases interleukin (IL)-6, IL-1, and tumor necrosis factor (TNF) production by peripheral blood mononuclear cells and decreases IL-6 plasma levels in rheumatoid arthritis patients. In this randomized double-blind study, 43 HIV-1-infected patients received tenidap (120 mg) or placebo daily for 6 weeks and then crossed over to the alternative therapy for an additional 6 weeks. Mean entry CD4 cell count was 140/microL. Analyses were performed on cytokines, acute-phase proteins, virus load, and CD4 cell counts. With the exception of small differences in plasma TNF levels, tenidap had no significant effect on these indices. Significant correlations of plasma IL-6 and TNF levels with HIV-1 RNA were noted. Six patients discontinued tenidap due to rash. The effects of tenidap in HIV-1 infection contrast to results in arthritis patients, in whom tenidap decreased plasma levels of IL-6 and acute-phase proteins.

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

    Science.gov (United States)

    Taniguchi, Ichiro; Mabuchi, Naoto; Ohno, Mutsuhito

    2014-06-01

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

  9. Molecular mechanism: the human dopamine transporter histidine 547 regulates basal and HIV-1 Tat protein-inhibited dopamine transport.

    Science.gov (United States)

    Quizon, Pamela M; Sun, Wei-Lun; Yuan, Yaxia; Midde, Narasimha M; Zhan, Chang-Guo; Zhu, Jun

    2016-12-14

    Abnormal dopaminergic transmission has been implicated as a risk determinant of HIV-1-associated neurocognitive disorders. HIV-1 Tat protein increases synaptic dopamine (DA) levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Through integrated computational modeling prediction and experimental validation, we identified that histidine547 on human DAT (hDAT) is critical for regulation of basal DA uptake and Tat-induced inhibition of DA transport. Compared to wild type hDAT (WT hDAT), mutation of histidine547 (H547A) displayed a 196% increase in DA uptake. Other substitutions of histidine547 showed that DA uptake was not altered in H547R but decreased by 99% in H547P and 60% in H547D, respectively. These mutants did not alter DAT surface expression or surface DAT binding sites. H547 mutants attenuated Tat-induced inhibition of DA transport observed in WT hDAT. H547A displays a differential sensitivity to PMA- or BIM-induced activation or inhibition of DAT function relative to WT hDAT, indicating a change in basal PKC activity in H547A. These findings demonstrate that histidine547 on hDAT plays a crucial role in stabilizing basal DA transport and Tat-DAT interaction. This study provides mechanistic insights into identifying targets on DAT for Tat binding and improving DAT-mediated dysfunction of DA transmission.

  10. Hydrogen Exchange Mass Spectrometry of Related Proteins with Divergent Sequences: A Comparative Study of HIV-1 Nef Allelic Variants

    Science.gov (United States)

    Wales, Thomas E.; Poe, Jerrod A.; Emert-Sedlak, Lori; Morgan, Christopher R.; Smithgall, Thomas E.; Engen, John R.

    2016-06-01

    Hydrogen exchange mass spectrometry can be used to compare the conformation and dynamics of proteins that are similar in tertiary structure. If relative deuterium levels are measured, differences in sequence, deuterium forward- and back-exchange, peptide retention time, and protease digestion patterns all complicate the data analysis. We illustrate what can be learned from such data sets by analyzing five variants (Consensus G2E, SF2, NL4-3, ELI, and LTNP4) of the HIV-1 Nef protein, both alone and when bound to the human Hck SH3 domain. Regions with similar sequence could be compared between variants. Although much of the hydrogen exchange features were preserved across the five proteins, the kinetics of Nef binding to Hck SH3 were not the same. These observations may be related to biological function, particularly for ELI Nef where we also observed an impaired ability to downregulate CD4 surface presentation. The data illustrate some of the caveats that must be considered for comparison experiments and provide a framework for investigations of other protein relatives, families, and superfamilies with HX MS.

  11. Quantitative Characterization of Configurational Space Sampled by HIV-1 Nucleocapsid Using Solution NMR, X-ray Scattering and Protein Engineering.

    Science.gov (United States)

    Deshmukh, Lalit; Schwieters, Charles D; Grishaev, Alexander; Clore, G Marius

    2016-06-03

    Nucleic-acid-related events in the HIV-1 replication cycle are mediated by nucleocapsid, a small protein comprising two zinc knuckles connected by a short flexible linker and flanked by disordered termini. Combining experimental NMR residual dipolar couplings, solution X-ray scattering and protein engineering with ensemble simulated annealing, we obtain a quantitative description of the configurational space sampled by the two zinc knuckles, the linker and disordered termini in the absence of nucleic acids. We first compute the conformational ensemble (with an optimal size of three members) of an engineered nucleocapsid construct lacking the N- and C-termini that satisfies the experimental restraints, and then validate this ensemble, as well as characterize the disordered termini, using the experimental data from the full-length nucleocapsid construct. The experimental and computational strategy is generally applicable to multidomain proteins. Differential flexibility within the linker results in asymmetric motion of the zinc knuckles which may explain their functionally distinct roles despite high sequence identity. One of the configurations (populated at a level of ≈40 %) closely resembles that observed in various ligand-bound forms, providing evidence for conformational selection and a mechanistic link between protein dynamics and function.

  12. Packageable antiviral therapeutics against human immunodeficiency virus type 1: virion-targeted virus inactivation by incorporation of a single-chain antibody against viral integrase into progeny virions.

    Science.gov (United States)

    Okui, N; Sakuma, R; Kobayashi, N; Yoshikura, H; Kitamura, T; Chiba, J; Kitamura, Y

    2000-03-01

    To determine their activities as an antiviral agent packageable within virions and suitable for continued expression in cells, we tested a single-chain antibody (scAb) against human immunodeficiency virus type 1 (HIV-1) integrase and its three fusion proteins: fused to viral protein R (scab-Vpr), a double-cassette of the WXXF motif binding to Vpr (scAb-WXXF), and viral major capsid protein (scAb-CA), respectively. Cotransfection of human 293T cells with expression plasmid for scAb-Vpr or -WXXF along with HIV-1 clone pLAI resulted in the production of a normal amount of progeny virions with infectivity decreased by more than 10(3)-fold. Immunoblot analyses showed that scAb-Vpr or -WXXF was associated with virions, whereas scAb or scAb-CA was not, suggesting that scAb-Vpr or -WXXF was incorporated into virions. The incorporation of scAb-WXXF appeared to be Vpr dependent, because the fusion protein was associated with the wild-type but not with Vpr-truncated HIV-1 virions. Since G418-selected HeLa clones carrying expression plasmid for scAb-WXXF were obtained much more frequently than those for scAb-Vpr, scAb-WXXF was inferred to be less toxic to cells than scAb-Vpr. These results suggest that scAb-WXXF may serve as a novel class of antiviral therapeutic that inactivates progeny HIV virions from within.

  13. The HIV-1 associated protein, Tat1–86, impairs dopamine transporters and interacts with cocaine to reduce nerve terminal function: a no-net-flux microdialysis study

    Science.gov (United States)

    Ferris, Mark J.; Frederick-Duus, Danielle; Fadel, Jim; Mactutus, Charles F.; Booze, Rosemarie M.

    2009-01-01

    Injection drug use accounts for approximately one-third of HIV-infections in the United States. HIV associated proteins have been shown to interact with various drugs of abuse to incite concerted neurotoxicity. One common area for their interaction is the nerve terminal, including dopamine transporter (DAT) systems. However, results regarding DAT function and regulation in HIV-infection, regardless of drug use, are mixed. Thus, the present experiments were designed to explicitly control Tat and cocaine administration in an in vivo model in order to reconcile differences that exist in the literature to date. We examined Tat plus cocaine-induced alterations using no-net-flux microdialysis, which is sensitive to alterations in DAT function, in order to test the potential for DAT as an early mediator of HIV-induced oxidative stress and neurodegeneration in vivo. Within 5 hours of intra-accumbal administration of the HIV-associated protein, Tat, we noted a significant reduction in local DAT efficiency with little change in DA overflow/release dynamics. Further, at 48 hrs post-Tat administration, we demonstrated a concerted effect of the HIV-protein Tat with cocaine on both uptake and release function. Finally, we discuss the extent to which DAT dysfunction may be considered a predecessor to generalized nerve terminal dysfunction. Characterization of DAT dysfunction in vivo may provide an early pharamacotherapeutic target, which in turn may prevent or attenuate downstream mediators of neurotoxicity (i.e., reactive species) to DA systems occurring in NeuroAIDS. PMID:19344635

  14. Safety and immunogenicity of an adjuvanted protein therapeutic HIV-1 vaccine in subjects with HIV-1 infection: a randomised placebo-controlled study.

    Science.gov (United States)

    Harrer, Thomas; Plettenberg, Andreas; Arastéh, Keikawus; Van Lunzen, Jan; Fätkenheuer, Gerd; Jaeger, Hans; Janssens, Michel; Burny, Wivine; Collard, Alix; Roman, François; Loeliger, Alfred; Koutsoukos, Marguerite; Bourguignon, Patricia; Lavreys, Ludo; Voss, Gerald

    2014-05-07

    The human immunodeficiency virus type-1 (HIV-1) vaccine candidate F4/AS01 has previously been shown to induce potent and persistent polyfunctional CD4(+) T-cell responses in HIV-1-seronegative volunteers. This placebo-controlled study evaluated two doses of F4/AS01 1-month apart in antiretroviral treatment (ART)-experienced and ART-naïve HIV-1-infected subjects (1:1 randomisation in each cohort). Safety, HIV-1-specific CD4(+) and CD8(+) T-cell responses, absolute CD4(+) T-cell counts and HIV-1 viral load were monitored for 12 months post-vaccination. Reactogenicity was clinically acceptable and no vaccine-related serious adverse events were reported. The frequency of HIV-1-specific CD4(+) T-cells 2 weeks post-dose 2 was significantly higher in the vaccine group than in the placebo group in both cohorts (pVaccine-induced HIV-1-specific CD4(+) T-cells exhibited a polyfunctional phenotype, expressing at least CD40L and IL-2. No increase in HIV-1-specific CD8(+) T-cells or change in CD8(+) T-cell activation marker expression profile was detected. Absolute CD4(+) T-cell counts were variable over time in both cohorts. Viral load remained suppressed in ART-experienced subjects. In ART-naïve subjects, a transient reduction in viral load from baseline was observed 2 weeks after the second F4/AS01 dose, which was concurrent with a higher frequency of HIV-1-specific CD4(+) T-cells expressing at least IL-2 in this cohort. In conclusion, F4/AS01 showed a clinically acceptable reactogenicity and safety profile, and induced polyfunctional HIV-1-specific CD4(+) T-cell responses in ART-experienced and ART-naïve subjects. These findings support further clinical investigation of F4/AS01 as a potential HIV-1 vaccine for therapeutic use in individuals with HIV-1 infection. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. HIV-1 Env DNA vaccine plus protein boost delivered by EP expands B- and T-cell responses and neutralizing phenotype in vivo.

    Directory of Open Access Journals (Sweden)

    Kar Muthumani

    Full Text Available An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs, and the elicitation of antibody-dependent cellular cytotoxicity (ADCC. Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP. However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted.

  16. Cytotoxity of HIV-1 gp120 protein on human blood-retinal barrier ceils%HIV-1 gp120蛋白对人血-视网膜屏障细胞的毒性作用

    Institute of Scientific and Technical Information of China (English)

    林浩添; 张振平; 余秋蓉; 晏丕松; 汪琪璘; 柏凌

    2009-01-01

    Objective To study the mechanism of human blood-retina barrier (BRB) destroyed by HIV- 1 gp120 protein. Methods Human blood-retina barrier cells (HBRBCs) including human retina capillary endothelial cells (HRCECs), human retina capillary perieytes (HRCPCs), human retinal pigment epithelium (HRPE) were primarily cultured. Culture media were regarded as eontrol. MTT method was used to observe the inhibition effect of HIV-1 gp120 protein on eell viability at 7 different concentrations (0.01 to 0.15 mg/L) for 24 h, and at a fixed concentration(0.08 mg/L) for different times (4-72 h). After 0.08, 0.1, 0.12 and 0.15 mg/L HIV- 1 gp120 protein were applied in those cells for 24 h, cell apoptotie rates and membrane potential of mitochondria (△ψm) were measured by flow eytometry. Activation of Cleaved caspase-9 was detected by Western blot. Change of cell mierostructure with 0.08mg/L HIV-1 gp120 protein before and after 24 h was detected by transmission electron microscopy (TEM). Results Concentration of HIV-1 gp120 protein less than 0.08 mg/L did not influence cell viability at 24 h. But at the concentration of more than 0.08 mg/L, HIV-1 gp120 protein could obviously inhibit HBRBCs proliferation with a concentration-dependent manner(HRCECs: r=-0.763, P<0.01 ; HRCPCs: r=-0.804, P<0.01 ; HRPE: r=-0.698, P<0.01). HIV-1 gp120 protein(0.08 mg/L) significantly inhibited cells proliferation at 12 h, and this inhibition effect was more stronger at 24,48,72 h with a time-dependent increase (HRCECs: r=-0.833, P<0.01 ; HRCPCs: r=-0.784, P<0.01 ; HRPE: r=-0.701, P<0.01). The relative growth rates were HRCECs: 84%, 70%, 41%, 22% ; HRCPCs: 80%, 69%, 38%, 18% ; HRPE: 86%, 73%, 45%, 26% respectively. Compared with control group, the ratio of apoptotic cells of HBRBCs and expression level of Cleaved caspase-9 protein increased but △ψm decreased at different concentrations with HIV-1 gp120 protein treatment for 24 h. The changes of these indexes were all concentration-dependent manner

  17. HIV-1 Vif Protein Mediates the Degradation of APOBEC3G in Fission Yeast When Over-expressed Using Codon Optimization

    Institute of Scientific and Technical Information of China (English)

    Lin LI; Jing-yun LI; Hong-shuai SUI; Richard Y. Zhao; Yong-jian LIU; Zuo-yi BAO; Si-yang LIU; Dao-min ZHUANG

    2008-01-01

    Interaction between the HIV-1 Vif protein and the cellular host APOBEC3G protein is a promising target for inhibition of HIV-1 replication. Considering that human cells are a very complicated environment for the study of protein interactions, the goal of this study was to check whether fission yeast could be used as a model cell for studying the Vif-APOBEC3G interaction. Vif and APOBEC3G were expressed in fusion with GFP protein in the S. pombe SP223 strain. Subcellular localizations of Vif and APOBEC3G were observed with fluorescent microscopy. Codon optimization was used to over express the Vif protein in S. pombe cells. The degradation of APOBEC3G mediated by Vif was tested through expressing Vif and GFP-APOBEC3G proteins in the same cell. Western Blot analysis was used to measure the corresponding protein levels under different experimental conditions. The results showed that the Vif protein was predominantly localized in the nucleus of S.pombe cells, APOBEC3G was localized in the cytoplasm and concentrated at punctate bodies that were often in close proximity to the nucleus but were not necessarily restricted from other regions in the cytoplasm. Vif protein expression levels were increased significantly by using codon optimization and APOBEC3G was degraded when Vif was over-expressed in the same S. pombe cells. These results indicate that fission yeast is a good model for studying the interaction between the Vif and APOBEC3G proteins.

  18. Combined antiretroviral therapy attenuates hepatic extracellular matrix remodeling in HIV patients assessed by novel protein fingerprint markers

    DEFF Research Database (Denmark)

    Leeming, Diana J; Anadol, Evrim; Schierwagen, Robert

    2014-01-01

    OBJECTIVES: Combined antiretroviral therapy (cART) attenuates hepatic fibrosis in hepatitis C virus and HIV coinfected patients. However, the role of HIV or cART on hepatic fibrosis in HIV monoinfection is discussed controversially. During liver fibrosis, matrix metalloproteinases (MMPs) degrade ...

  19. The structure and function of HIV-1 accessory protein Vif%HIV-1辅助蛋白Vif的结构与功能

    Institute of Scientific and Technical Information of China (English)

    李震宇; 刘新泳

    2008-01-01

    HIV-1 Vif(viral infectivity factor)蛋白是由保守的vif基因编码的碱性蛋白质,是HIV-1病毒的辅助调节蛋白之一.研究表明Vif蛋白具有调节病毒侵入、组装、出芽和成熟等功能.此外,Vif蛋白能够特异性地与体内抗病毒因子APOBEC3G相互作用,增强病毒的感染性.因此,针对HIV-1Vif蛋白进行抑制剂设计已经成为抗HIV药物研究的热点之一.本文对HIV-1Vif蛋白的结构与功能研究的最新进展进行了综述.

  20. Exploring the binding of d(GGGT)4 to the HIV-1 integrase: An approach to investigate G-quadruplex aptamer/target protein interactions.

    Science.gov (United States)

    Esposito, Veronica; Pirone, Luciano; Mayol, Luciano; Pedone, Emilia; Virgilio, Antonella; Galeone, Aldo

    2016-08-01

    The aptamer d(GGGT)4 (T30923 or T30695) forms a 5'-5' dimer of two stacked parallel G-quadruplexes, each characterized by three G-tetrads and three single-thymidine reversed-chain loops. This aptamer has been reported to exhibit anti-HIV activity by targeting the HIV integrase, a viral enzyme responsible for the integration of viral DNA into the host-cell genome. However, information concerning the aptamer/target interaction is still rather limited. In this communication we report microscale thermophoresis investigations on the interaction between the HIV-1 integrase and d(GGGT)4 aptamer analogues containing abasic sites singly replacing thymidines in the original sequence. This approach has allowed the identification of which part of the aptamer G-quadruplex structure is mainly involved in the interaction with the protein.

  1. Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Weizao, E-mail: chenw3@mail.nih.gov [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Feng, Yang [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Wang, Yanping [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); The Basic Research Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Zhu, Zhongyu; Dimitrov, Dimiter S. [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. Black-Right-Pointing-Pointer We hypothesize that CD4i antibodies could induce conformational changes in gp120. Black-Right-Pointing-Pointer CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. Black-Right-Pointing-Pointer CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.

  2. Expression of the IL-7 receptor alpha-chain is down regulated on the surface of CD4 T-cells by the HIV-1 Tat protein.

    Directory of Open Access Journals (Sweden)

    Denny McLaughlin

    Full Text Available HIV infection elicits defects in CD4 T-cell homeostasis in both a quantitative and qualitative manner. Interleukin-7 (IL-7 is essential to T-cell homeostasis and several groups have shown reduced levels of the IL-7 receptor alpha-chain (CD127 on both CD4 and CD8 T-cells in viremic HIV+ patients. We have shown previously that soluble HIV Tat protein specifically down regulates cell surface expression of CD127 on human CD8 T-cells in a paracrine fashion. The effects of Tat on CD127 expression in CD4 T-cells has yet to be described. To explore this effect, CD4 T-cells were isolated from healthy individuals and expression levels of CD127 were examined on cells incubated in media alone or treated with Tat protein. We show here that, similar to CD8 T-cells, the HIV-1 Tat protein specifically down regulates CD127 on primary human CD4 T-cells and directs the receptor to the proteasome for degradation. Down regulation of CD127 in response to Tat was seen on both memory and naive CD4 T-cell subsets and was blocked using either heparin or anti-Tat antibodies. Tat did not induce apoptosis in cultured primary CD4 T-cells over 72 hours as determined by Annexin V and PI staining. Pre-incubation of CD4 T-cells with HIV-1 Tat protein did however reduce the ability of IL-7 to up regulate Bcl-2 expression. Similar to exogenous Tat, endogenously expressed HIV Tat protein also suppressed CD127 expression on primary CD4 T-cells. In view of the important role IL-7 plays in lymphocyte proliferation, homeostasis and survival, down regulation of CD127 by Tat likely plays a central role in immune dysregulation and CD4 T-cell decline. Understanding this effect could lead to new approaches to mitigate the CD4 T-cell loss evident in HIV infection.

  3. UV-inactivated vaccinia virus (VV) in a multi-envelope DNA-VV-protein (DVP) HIV-1 vaccine protects macaques from lethal challenge with heterologous SHIV

    Science.gov (United States)

    Jones, Bart G; Sealy, Robert E; Zhan, Xiaoyan; Freiden, Pamela J; Surman, Sherri L; Blanchard, James L.; Hurwitz, Julia L

    2012-01-01

    The pandemic of HIV-1 has continued for decades, yet there remains no licensed vaccine. Previous research has demonstrated the effectiveness of a multi-envelope, multi-vectored HIV-1 vaccine in a macaque-SHIV model, illustrating a potential means of combating HIV-1. Specifically, recombinant DNA, vaccinia virus (VV) and purified protein (DVP) delivery systems were used to vaccinate animals with dozens of antigenically-distinct HIV-1 envelopes for induction of immune breadth. The vaccinated animals controlled disease following challenge with a heterologous SHIV. This demonstration suggested that the antigenic cocktail vaccine strategy, which has succeeded in several other vaccine fields (e.g. pneumococcus), might also succeed against HIV-1. The strategy remains untested in an advanced clinical study, in part due to safety concerns associated with the use of replication-competent VV. To address this concern, we designed a macaque study in which psoralen/ultraviolet light-inactivated VV (UV VV) was substituted for replication-competent VV in the multi-envelope DVP protocol. Control animals received a vaccine encompassing no VV, or no vaccine. All VV vaccinated animals generated an immune response toward VV, and all vaccinated animals generated an immune response toward HIV-1 envelope. After challenge with heterologous SHIV 89.6P, animals that received replication-competent VV or UV VV experienced similar outcomes. They exhibited reduced peak viral loads, maintenance of CD4+ T cell counts and improved survival compared to control animals that received no VV or no vaccine; there were 0/15 deaths among all animals that received VV and 5/9 deaths among controls. Results define a practical means of improving VV safety, and encourage advancement of a promising multi-envelope DVP HIV-1 vaccine candidate. PMID:22425790

  4. Visualization of X4- and R5-Tropic HIV-1 Viruses Expressing Fluorescent Proteins in Human Endometrial Cells: Application to Tropism Study.

    Science.gov (United States)

    Terrasse, Rachel; Memmi, Meriam; Palle, Sabine; Heyndrickx, Leo; Vanham, Guido; Pozzetto, Bruno; Bourlet, Thomas

    2017-01-01

    Worldwide most HIV infections occur through heterosexual transmission, involving complex interactions of cell-free and cell-associated particles with cells of the female genital tract mucosa. The ability of HIV-1 to "infect" epithelial cells remains poorly understood. To address this question, replicative-competent chimeric constructs expressing fluorescent proteins and harboring the envelope of X4- or R5-tropic HIV-1 strains were used to "infect" endometrial HEC1-A cells. The virus-cell interactions were visualized using confocal microscopy (CM) at various times post infection. Combined with quantification of viral RNA and total HIV DNA in infected cells, the CM pictures suggest that epithelial cells do not support a complete viral replication cycle: X4-tropic viruses are imported into the nucleus in a non-productive way, whereas R5-tropic viruses transit through the cytoplasm without replication and are preferentially transmitted to susceptible activated peripheral blood mononuclear cells. Within the limit of experiments conducted in vitro on a continued cell line, these results indicate that the epithelial mucosa may participate to the selection of HIV-1 strains at the mucosal level.

  5. Induction of heat-shock protein 70 by prostaglandin A₁ inhibits HIV-1 Vif-mediated degradation of APOBEC3G.

    Science.gov (United States)

    Sugiyama, Ryuichi; Abe, Makoto; Nishitsuji, Hironori; Murakami, Yuko; Takeuchi, Hiroaki; Takaku, Hiroshi

    2013-09-01

    Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit human immunodeficiency virus type 1 (HIV-1) replication in various cell types. This antiviral activity has been associated with the induction of heat-shock protein 70 (HSP70) in infected cells. We investigated a new role of prostaglandin A₁ (PGA₁) in the replication of HIV-1 in non-permissive cells. Because overexpression of HSP70 blocks the viral infectivity factor (Vif)-mediated degradation of APOBEC3G (A3G) via the ubiquitin-proteasome pathway, we examined the effects of PGA₁ on A3G and HIV-1 replication. The induction of HSP70 synthesis by PGA₁ blocked Vif-mediated A3G degradation and enhanced the incorporation of A3G into both wild-type and Vif-deficient viruses. Furthermore, we determined the viral titer of HIV-1 particles produced from PGA₁-treated 293T cells. The induction of HSP70 synthesis by PGA₁ significantly reduced the viral titer in the presence of A3G. Additionally, the p24 Gag antigen levels were dramatically reduced in non-permissive cells treated once or repeatedly with PGA₁. Thus, we showed that PGA₁ inhibits HIV-1 replication, at least in part, by blocking Vif-mediated A3G degradation.

  6. Experimental Estimation of the Effects of All Amino-Acid Mutations to HIV's Envelope Protein on Viral Replication in Cell Culture.

    Science.gov (United States)

    Haddox, Hugh K; Dingens, Adam S; Bloom, Jesse D

    2016-12-01

    HIV is notorious for its capacity to evade immunity and anti-viral drugs through rapid sequence evolution. Knowledge of the functional effects of mutations to HIV is critical for understanding this evolution. HIV's most rapidly evolving protein is its envelope (Env). Here we use deep mutational scanning to experimentally estimate the effects of all amino-acid mutations to Env on viral replication in cell culture. Most mutations are under purifying selection in our experiments, although a few sites experience strong selection for mutations that enhance HIV's replication in cell culture. We compare our experimental measurements of each site's preference for each amino acid to the actual frequencies of these amino acids in naturally occurring HIV sequences. Our measured amino-acid preferences correlate with amino-acid frequencies in natural sequences for most sites. However, our measured preferences are less concordant with natural amino-acid frequencies at surface-exposed sites that are subject to pressures absent from our experiments such as antibody selection. Our data enable us to quantify the inherent mutational tolerance of each site in Env. We show that the epitopes of broadly neutralizing antibodies have a significantly reduced inherent capacity to tolerate mutations, rigorously validating a pervasive idea in the field. Overall, our results help disentangle the role of inherent functional constraints and external selection pressures in shaping Env's evolution.

  7. HIV Transmission

    Science.gov (United States)

    ... Abroad Treatment Basic Statistics Get Tested Find an HIV testing site near you. Enter ZIP code or city Follow HIV/AIDS CDC HIV CDC HIV/AIDS See RSS | ... on HIV Syndicated Content Website Feedback HIV/AIDS HIV Transmission Language: English (US) Español (Spanish) Recommend ...

  8. Immunogenicities of Env glycoproteins from circulating HIV-1 isolates in China focusing on the strategy of "DNA prime plus protein boost"

    Institute of Scientific and Technical Information of China (English)

    WANG Zheng; WANG Shi-xia; LIU Si-yang; BAO Zuo-yi; ZHUANG Dao-min; LI Lin; ZHANG Chun-hua; ZHANG Lu; LI Jing-yun; LU Shan

    2009-01-01

    Background The adenovirus-based HIV-1 vaccine developed by Merck Company suffered from an unexpected failure in September 2007. This generated a big shift in the strategy of HIV vaccine development with renewed focus on the induction of neutralizing antibodies. A major challenge in developing an HIV-1 vaccine is to identify immunogens and adopt delivery methods that can elicit broadly neutralizing antibodies against primary isolates of different genetic subtypes.Methods Most circulating HIV-1 isolates in China are composed of clades Thai-B, CRF_BC and CRF01_AE. In order to construct DNA vaccines against these 3 HIV-1 subtypes, DNA vaccines carrying the gp120 regions from HIV-1 isolates of GX48(AE), GX79(AE), NX22(BC), GS22(BC), HN24(Thai-B) were constructed. Expression of gp120 from these DNA vaccines was detected by Western blotting in transiently transfected 293T cells. Pilot immunizations of New Zealand white rabbits were performed using the strategy of "DNA prime plus protein boost" and the neutralizing antibody response was detected in a Tzm-bl cell based assay against different HIV-1 strains.Results Response of gp120-specific antibody was relatively low after DNA primes (mean titer=10~(4.72)); however, the titer of gp120-specific antibody went up with 2 protein boosts (mean titer=10~(6.81)). Above all, neutralizing antibody (Nab) titers induced by this combined approach were much better than those elicited by DNA or protein used alone (P <0.01). Neutralizing activities of immunized rabbit sera against several pseudoviruses and laboratorial strains were evaluated, most rabbit sera primed with monovalent vaccine were capable of neutralizing only 1 of 5 viruses, however, sera primed with the polyvalent DNA vaccines were able to neutralize at least 2 of 5 viruses.Conclusion Polyvalent DNA prime plus protein boost is an effective immunization strategy to broaden the neutralization breadth and further research should be performed on the basis of this pilot study.

  9. Production and purification of immunologically active core protein p24 from HIV-1 fused to ricin toxin B subunit in E. coli

    Directory of Open Access Journals (Sweden)

    Gómez-Lim Miguel A

    2009-02-01

    Full Text Available Abstract Background Gag protein from HIV-1 is a polyprotein of 55 kDa, which, during viral maturation, is cleaved to release matrix p17, core p24 and nucleocapsid proteins. The p24 antigen contains epitopes that prime helper CD4 T-cells, which have been demonstrated to be protective and it can elicit lymphocyte proliferation. Thus, p24 is likely to be an integral part of any multicomponent HIV vaccine. The availability of an optimal adjuvant and carrier to enhance antiviral responses may accelerate the development of a vaccine candidate against HIV. The aim of this study was to investigate the adjuvant-carrier properties of the B ricin subunit (RTB when fused to p24. Results A fusion between ricin toxin B subunit and p24 HIV (RTB/p24 was expressed in E. coli. Affinity chromatography was used for purification of p24 alone and RTB/p24 from cytosolic fractions. Biological activity of RTB/p24 was determined by ELISA and affinity chromatography using the artificial receptor glycoprotein asialofetuin. Both assays have demonstrated that RTB/p24 is able to interact with complex sugars, suggesting that the chimeric protein retains lectin activity. Also, RTB/p24 was demonstrated to be immunologically active in mice. Two weeks after intraperitoneal inoculation with RTB/p24 without an adjuvant, a strong anti-p24 immune response was detected. The levels of the antibodies were comparable to those found in mice immunized with p24 alone in the presence of Freund adjuvant. RTB/p24 inoculated intranasally in mice, also elicited significant immune responses to p24, although the response was not as strong as that obtained in mice immunized with p24 in the presence of the mucosal adjuvant cholera toxin. Conclusion In this work, we report the expression in E. coli of HIV-1 p24 fused to the subunit B of ricin toxin. The high levels of antibodies obtained after intranasal and intraperitoneal immunization of mice demonstrate the adjuvant-carrier properties of RTB when

  10. Specific amplification of gene encoding N-terminal region of catalase-peroxidase protein (KatG-N) for diagnosis of disseminated MAC disease in HIV patients.

    Science.gov (United States)

    Latawa, Romica; Singh, Krishna Kumar; Wanchu, Ajay; Sethi, Sunil; Sharma, Kusum; Sharma, Aman; Laal, Suman; Verma, Indu

    2014-10-01

    Disseminated Mycobacterium avium-intracellulare complex (MAC) infection is considered as severe complication of advanced HIV/AIDS disease. Currently available various laboratory investigations have not only limited ability to discriminate between MAC infection and tuberculosis but are also laborious and time consuming. The aim of this study was, therefore, to design a molecular-based strategy for specific detection of MAC and its differentiation from Mycobacterium tuberculosis (M. tb) isolated from the blood specimens of HIV patients. A simple PCR was developed based on the amplification of 120-bp katG-N gene corresponding to the first 40 amino acids of N-terminal catalase-peroxidase (KatG) protein of Mycobacterium avium that shows only ~13% sequence homology by clustal W alignment to N-terminal region of M. tb KatG protein. This assay allowed the accurate and rapid detection of MAC bacteremia, distinguishing it from M. tb in a single PCR reaction without any need for sequencing or hybridization protocol to be performed thereafter. This study produced enough evidence that a significant proportion of Indian HIV patients have disseminated MAC bacteremia, suggesting the utility of M. avium katG-N gene PCR for early detection of MAC disease in HIV patients. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Association of VH4-59 Antibody Variable Gene Usage with Recognition of an Immunodominant Epitope on the HIV-1 Gag Protein.

    Directory of Open Access Journals (Sweden)

    Valentine U Chukwuma

    Full Text Available The human antibody response against HIV-1 infection recognizes diverse antigenic subunits of the virion, and includes a high level of antibodies to the Gag protein. We report here the isolation and characterization of a subset of Gag-specific human monoclonal antibodies (mAbs that were prevalent in the antibody repertoire of an HIV-infected individual. Several lineages of Gag-specifc mAbs were encoded by a single antibody heavy chain variable region, VH4-59, and a representative antibody from this group designated mAb 3E4 recognized a linear epitope on the globular head of the p17 subunit of Gag. We found no evidence that mAb 3E4 exhibited any function in laboratory studies aimed at elucidating the immunologic activity, including assays for neutralization, Ab-dependent cell-mediated virus inhibition, or enhanced T cell reactivity caused by Gag-3E4 complexes. The findings suggest this immunodominant epitope in Gag protein, which is associated with VH4-59 germline gene usage, may induce a high level of B cells that encode binding but non-functional antibodies that occupy significant repertoire space following HIV infection. The studies define an additional specific molecular mechanism in the immune distraction activity of the HIV virion.

  12. Immunogenicity of three Haemophilus influenzae type b protein conjugate vaccines in HIV seropositive adults and analysis of predictors of vaccine response.

    Science.gov (United States)

    Dockrell, D H; Poland, G A; Steckelberg, J M; Wollan, P C; Strickland, S R; Pomeroy, C

    1999-07-16

    HIV-seropositive adults may be at increased risk of infection due to Haemophilus influenzae type b (Hib) as compared with HIV-seronegative adults. Protein conjugate vaccines have been demonstrated to induce protective levels of antibodies against Hib in immunocompetent infants and also in HIV-seropositive infants. In this study we determined the immunogenicity of three protein conjugate Hib vaccines (PRP-D, HbOC, HbNOMP) in 135 HIV-seropositive adults who received one dose of Hib vaccine. Anti-polyribosylribitol phosphate (PRP) antibodies were measured at 0, 1, 3 and 12 months postimmunization by the Farr method. We demonstrate that all three vaccines are highly immunogenic and result in protective (> 1.0 microg/ml) levels of antibody. Overall the anti-PRP antibody level was > 1.0 microg/ml in 26% of patients preimmunization, 91% at both 1 and 3 months, and 79% at 12 months postvaccination. Comparison of responses to the three vaccines over time demonstrated differences in the mean geometric anti-PRP antibody level at 1 month (p=0.03) and the 12 month time points (p=0.03) with lower geometric mean levels in the HbNOMP group, though baseline differences in groups limit the interpretation of these findings. In a univariate analysis of baseline characteristics which predicted poor vaccine response, low total IgG2 levels preimmunization predicted a poor antibody response at 1 month (p < 0.01) and at 12 months (p=0.05), while low CD4 T-cell count predicted poor response at 12 months (p < 0.01). We conclude that all three US licensed protein conjugate Hib vaccines are immunogenic in HIV-seropositive adults, and that baseline CD4 T-cell count and IgG2 levels predict the likelihood of antibody response to vaccine.

  13. The prevalence and predictive value of dipstick urine protein in HIV-positive persons in Europe

    DEFF Research Database (Denmark)

    Mocroft, Amanda; Ryom, Lene; Lapadula, Giuseppe

    2014-01-01

    INTRODUCTION: Proteinuria (PTU) is an important marker for the development and progression of renal disease, cardiovascular disease and death, but there is limited information about the prevalence and factors associated with confirmed PTU in predominantly white European HIV+ persons, especially...... consecutive DUP >1+ (>30 mg/dL) >3 months apart; persons with eGFR persons were included, participants were mainly white (n=1,517, 92.5%), male (n=1296, 79.0%) and men having...... sex with men (n=809; 49.3%). Median age at baseline was 45 (IQR 37-52 years), and CD4 was 570 (IQR 406-760/mm(3)). The median baseline date was 2/12 (IQR 11/11-6/12), and median eGFR was 99 (IQR 88-109 mL/min/1.73 m(2)). Sixty-nine persons had PTU (4.2%, 95% CI 3.2-4.7%). Persons with diabetes had...

  14. Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat

    Directory of Open Access Journals (Sweden)

    Muller Sylviane

    2008-07-01

    Full Text Available Abstract Background During HIV-1 infection, the Tat protein plays a key role by transactivating the transcription of the HIV-1 proviral DNA. In addition, Tat induces apoptosis of non-infected T lymphocytes, leading to a massive loss of immune competence. This apoptosis is notably mediated by the interaction of Tat with microtubules, which are dynamic components essential for cell structure and division. Tat binds two Zn2+ ions through its conserved cysteine-rich region in vitro, but the role of zinc in the structure and properties of Tat is still controversial. Results To investigate the role of zinc, we first characterized Tat apo- and holo-forms by fluorescence correlation spectroscopy and time-resolved fluorescence spectroscopy. Both of the Tat forms are monomeric and poorly folded but differ by local conformational changes in the vicinity of the cysteine-rich region. The interaction of the two Tat forms with tubulin dimers and microtubules was monitored by analytical ultracentrifugation, turbidity measurements and electron microscopy. At 20°C, both of the Tat forms bind tubulin dimers, but only the holo-Tat was found to form discrete complexes. At 37°C, both forms promoted the nucleation and increased the elongation rates of tubulin assembly. However, only the holo-Tat increased the amount of microtubules, decreased the tubulin critical concentration, and stabilized the microtubules. In contrast, apo-Tat induced a large amount of tubulin aggregates. Conclusion Our data suggest that holo-Tat corresponds to the active form, responsible for the Tat-mediated apoptosis.

  15. Binding of recombinant HIV coat protein gp120 to human monocytes

    Energy Technology Data Exchange (ETDEWEB)

    Finbloom, D.S.; Hoover, D.L.; Meltzer, M.S. (Food and Drug Administration, Bethesda, MD (USA))

    1991-02-15

    Inasmuch as the exact level of CD4 Ag expression on macrophages is controversial and because HIV may interact with macrophages in a manner different from that on T cells, we analyzed the binding of gp120 to freshly isolated and cultured monocytes. rgp120 was iodinated using the lactoperoxidase method to a sp. act. of 600 Ci/mmol. Highly purified monocytes (greater than 90%) were isolated from the leukapheresed blood of normal volunteers by Ficoll-Hypaque sedimentation followed by countercurrent centrifugal elutriation and cultured 7 days in DMEM supplemented with 1000 U/ml macrophage CSF in 10% human serum. Whereas MOLT/4 cells consistently bound freshly prepared 125I-rgp120 at 80% specificity with 5100 +/- 700 mol/cell, MCSF cultured monocytes bound rgp120 at only 0 to 20% specificity and 420 +/- 200 mol/cell. Most of the radioactivity bound by these cells could not be blocked by the addition of unlabeled rgp120. In contrast, the U937 myeloid cell line bound rgp120 with 50% specificity and about 2500 mol/cell. Whereas the antibody OKT4a (anti-CD4) blocked 80% of the binding on MOLT/4 cells and 50% on U937 cells, binding was only inhibited on the average of 6% on cultured monocytes. When soluble rCD4 was used as an inhibitor, binding to MOLT/4 cells was blocked by 80%. In contrast, binding to cultured monocytes was inhibited by 28%. HIV infectivity was blocked by similar concentrations of OKT4a. These observations suggest that although most binding of gp120 to cultured monocytes is not to the CD4 determinant, several hundred molecules do bind to a CD4-like molecule which promotes virus entry and replication.

  16. Bioorthogonal mimetics of palmitoyl-CoA and myristoyl-CoA and their subsequent isolation by click chemistry and characterization by mass spectrometry reveal novel acylated host-proteins modified by HIV-1 infection.

    Science.gov (United States)

    Colquhoun, David R; Lyashkov, Alexey E; Ubaida Mohien, Ceereena; Aquino, Veronica N; Bullock, Brandon T; Dinglasan, Rhoel R; Agnew, Brian J; Graham, David R M

    2015-06-01

    Protein acylation plays a critical role in protein localization and function. Acylation is essential for human immunodeficiency virus 1 (HIV-1) assembly and budding of HIV-1 from the plasma membrane in lipid raft microdomains and is mediated by myristoylation of the Gag polyprotein and the copackaging of the envelope protein is facilitated by colocalization mediated by palmitoylation. Since the viral accessory protein NEF has been shown to alter the substrate specificity of myristoyl transferases, and alter cargo trafficking lipid rafts, we hypothesized that HIV-1 infection may alter protein acylation globally. To test this hypothesis, we labeled HIV-1 infected cells with biomimetics of acyl azides, which are incorporated in a manner analogous to natural acyl-Co-A. A terminal azide group allowed us to use a copper catalyzed click chemistry to conjugate the incorporated modifications to a number of substrates to carry out SDS-PAGE, fluorescence microscopy, and enrichment for LC-MS/MS. Using LC-MS/MS, we identified 103 and 174 proteins from the myristic and palmitic azide enrichments, with 27 and 45 proteins respectively that differentiated HIV-1 infected from uninfected cells. This approach has provided us with important insights into HIV-1 biology and is widely applicable to many virological systems.

  17. Kinetics of interaction of HIV fusion protein (gp41) with lipid membranes studied by real-time AFM imaging

    Energy Technology Data Exchange (ETDEWEB)

    Bitler, Arkady, E-mail: arkady.bitler@weizmann.ac.il [Department of Chemical Research Support (Israel); Lev, Naama; Fridmann-Sirkis, Yael; Blank, Lior [Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100 (Israel); Cohen, Sidney R. [Department of Chemical Research Support (Israel); Shai, Yechiel [Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100 (Israel)

    2010-05-15

    One of the most important steps in the process of viral infection is a fusion between cell membrane and virus, which is mediated by the viral envelope glycoprotein. The study of activity of the glycoprotein in the post-fusion state is important for understanding the progression of infection. Here we present a first real-time kinetic study of the activity of gp41 (the viral envelope glycoprotein of human immunodeficiency virus-HIV) and its two mutants in the post-fusion state with nanometer resolution by atomic force microscopy (AFM). Tracking the changes in the phosphatidylcholine (PC) and phosphatidylcholine-phosphatidylserine (PC:PS) membrane integrity over one hour by a set of AFM images revealed differences in the interaction of the three types of protein with zwitterionic and negatively charged membranes. A quantitative analysis of the slow kinetics of hole formation in the negatively charged lipid bilayer is presented. Specifically, analysis of the rate of roughness change for the three types of proteins suggests that they exhibit different types of kinetic behavior.

  18. Science challenging HIV infection.

    Science.gov (United States)

    Rao, R R; Lakshi, V

    1993-04-01

    The first accepted report of a novel human, slow virus disease belonging to "lentivirus" known as acquired immunodeficiency syndrome can be traced to reports of June 1981. HIV-1 and HIV-2 were later found over the period 1984-86 to be unequivocally associated with AIDS. They are two serologically distinct viruses belonging to the same family with the unique properties of integration and latency in the host cell genome and the presence of reverse transcriptase. Typical of all retroviruses, the HIV genome comprises three genes governing the synthesis of all core proteins, replication protein encoding, and envelope proteins. HIV uses the CD4 antigen on T-helper cells, and about 40% of blood monocytes and tissue macrophages as a cell surface receptor. HIV may, however, also infect cells which contain no CD4. Macrophages serve as the main reservoir of HIV and may carry the virus to different organs. Very recently a rare type of white blood cell called the dendritic cell has been found to allow for direct infection by HIV during sexual intercourse. These cells are prominently present in the anal and vaginal mucosa. The authors discuss facts and figures on the HIV epidemic, the Indian scenario, classification of the clinical spectrum, the enzyme immunoassay HIV testing format, Western blot, immunofluorescence antibody, HIV culture, flow cytometry, radio immuno precipitation assay, and the detection of HIV DNA. Significant advances have been made over the last ten years in understanding the pathogenesis of HIV infection and accurately diagnosing infected individuals, with recombinant technology, polymerase chain reaction, and the construction of synthetic hybrid virus rapidly becoming part of routine diagnostics. More sensitive, specific, and rapid techniques are, however, needed for the early diagnosis and management of AIDS cases. The need for more ideal antibody incorporating both regulatory and structural proteins of the virion, preferably manufactured using

  19. Design and synthesis of discontinuous protein binding site mimics of HIV gp120

    NARCIS (Netherlands)

    Mulder, G.E.

    2013-01-01

    Essentially all cellular processes are mediated by protein-protein interactions and detailed knowledge about these interactions can aid in determining biological functions or provide opportunities for the treatment of human disease. Extensive research has been performed on the development of

  20. Structure of the Bro1 domain protein BROX and functional analyses of the ALIX Bro1 domain in HIV-1 budding.

    Directory of Open Access Journals (Sweden)

    Qianting Zhai

    Full Text Available BACKGROUND: Bro1 domains are elongated, banana-shaped domains that were first identified in the yeast ESCRT pathway protein, Bro1p. Humans express three Bro1 domain-containing proteins: ALIX, BROX, and HD-PTP, which function in association with the ESCRT pathway to help mediate intraluminal vesicle formation at multivesicular bodies, the abscission stage of cytokinesis, and/or enveloped virus budding. Human Bro1 domains share the ability to bind the CHMP4 subset of ESCRT-III proteins, associate with the HIV-1 NC(Gag protein, and stimulate the budding of viral Gag proteins. The curved Bro1 domain structure has also been proposed to mediate membrane bending. To date, crystal structures have only been available for the related Bro1 domains from the Bro1p and ALIX proteins, and structures of additional family members should therefore aid in the identification of key structural and functional elements. METHODOLOGY/PRINCIPAL FINDINGS: We report the crystal structure of the human BROX protein, which comprises a single Bro1 domain. The Bro1 domains from BROX, Bro1p and ALIX adopt similar overall structures and share two common exposed hydrophobic surfaces. Surface 1 is located on the concave face and forms the CHMP4 binding site, whereas Surface 2 is located at the narrow end of the domain. The structures differ in that only ALIX has an extended loop that projects away from the convex face to expose the hydrophobic Phe105 side chain at its tip. Functional studies demonstrated that mutations in Surface 1, Surface 2, or Phe105 all impair the ability of ALIX to stimulate HIV-1 budding. CONCLUSIONS/SIGNIFICANCE: Our studies reveal similarities in the overall folds and hydrophobic protein interaction sites of different Bro1 domains, and show that a unique extended loop contributes to the ability of ALIX to function in HIV-1 budding.

  1. HIV-1gp41蛋白的表达及其免疫反应性%Expression and purification of recombinant HIV-1 gp41 protein and determination of its immunoreactivity

    Institute of Scientific and Technical Information of China (English)

    梁权; 罗春贞; 毛培菊; 王缦

    2011-01-01

    Objective Expressions of HIV-1 gp41 antigen for in-vitro immunoassay.Methods HIV-1 gp41 gene was amplified by PCR by using the plasmid X1 as a template, and then inserted into the plasmid E1 to construct recombinant plasmids E1-1. The expression of the recombinant plasmid in competent E.coli cell Bl21DE3 was induced with IPTG and analyzed with SDS-PAGE. After being purified by Ni-NTA affinity chromatography, the recombinant protein was further evaluated with ELISA for their functional characteristics.Results The result from SDS-PAGE showed expression of E1-1 in the competent E.coli cell. The purity of the purified recombinant protein was over 95%, and the protein has excellent immunoreactivity and specificity with ELISA.Conclusions Recombinant protein E1-1 can be expressed with a high immunoactivity, which can be qualified for HIV ELISA production.%目的 高效表达HIV-1 gp41基因片段,满足生产HIV体外检测试剂的需要.方法 以含HIV-1 gp41基因的质粒X1为模板,采用PCR方法扩增出gp41基因,克隆入E1载体构建重组表达质粒E1-1,转化E.coli BL21DE3感受态细胞,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导后,使用镍螯合琼脂糖亲和层析柱(Ni-NTA Agaros)纯化重组蛋白,通过ELISA法检测重组蛋白的免疫反应性和特异性.结果 SDS -PAGE鉴定结果表明,重组E1-1蛋白获得了正确的表达;纯化的重组E1-1蛋白纯度>95%; ELISA检测结果表明,重组E1-1蛋白具有较优的免疫反应性和特异性.结论 成功表达出具有较高免疫反应性的重组E1-1蛋白,可应用于HIV体外检测试剂.

  2. Reciprocal functional pseudotyping of HIV-1 and HTLV-1 viral genomes by the heterologous counterpart envelope proteins.

    Science.gov (United States)

    Klase, Zachary; Jeang, Kuan-Teh

    2013-08-15

    HIV-1 and HTLV-1 can infect CD4+ T cells and can co-infect the same individual. In principle, it is possible that both viruses can infect the same CD4+ T cells in dually infected persons. Currently, how efficiently HTLV-1 and HIV-1 co-infects the same cell and the full extent of their biological interactions are not well-understood. Here, we report evidence confirming that both viruses can infect the same cells and that HTLV-1 envelope (Env) can pseudotype HIV-1 viral particles and HIV-1 envelope (Env) can pseudotype HTLV-1 virions to mediate subsequent infections of substrate cells. We also show that the construction of a chimeric HTLV-1 molecular clone carrying the HIV-1 Env in place of its HTLV-1 counterpart results in a replication competent moiety. These findings raise new implications of viral complementation and assortment between HIV-1 and HTLV-1 in dually infected persons.

  3. Pertussis toxin B-oligomer suppresses IL-6 induced HIV-1 and chemokine expression in chronically infected U1 cells via inhibition of activator protein 1.

    Science.gov (United States)

    Rizzi, Chiara; Crippa, Massimo P; Jeeninga, Rienk E; Berkhout, Ben; Blasi, Francesco; Poli, Guido; Alfano, Massimo

    2006-01-15

    Pertussis toxin B-oligomer (PTX-B) inhibits HIV replication in T lymphocytes and monocyte-derived macrophages by interfering with multiple steps of the HIV life cycle. PTX-B prevents CCR5-dependent (R5) virus entry in a noncompetitive manner, and it also exerts suppressive effects on both R5- and CXCR4-dependent HIV expression at a less-characterized postentry level. We demonstrate in this study that PTX-B profoundly inhibits HIV expression in chronically infected promonocytic U1 cells stimulated with several cytokines and, particularly, the IL-6-mediated effect, a cytokine that triggers viral production in these cells independently of NF-kappaB activation. From U1 cells we have subcloned a cell line, named U1-CR1, with increased responsiveness to IL-6. In these cells, PTX-B neither down-regulated the IL-6R nor prevented IL-6 induced signaling in terms of STAT3 phosphorylation and DNA binding. In contrast, PTX-B inhibited AP-1 binding to target DNA and modified its composition with a proportional increases in FosB, Fra2, and ATF2. PTX-B inhibited IL-6-induced HIV-1 long-terminal repeat-driven transcription from A, C, E, and F viral subtypes, which contain functional AP-1 binding sites, but failed to inhibit transcription from subtypes B and D LTR devoid of these sites. In addition, PTX-B inhibited the secretion of IL-6-induced, AP-1-dependent genes, including urokinase-type plasminogen activator, CXCL8/IL-8, and CCL2/monocyte chemotactic protein-1. Thus, PTX-B suppression of IL-6 induced expression of HIV and cellular genes in chronically infected promonocytic cells is strongly correlated to inhibition of AP-1.

  4. Specific VpU Codon Changes were Significantly associated with gp120 V3 Tropic Signatures in HIV-1 B-subtype

    Institute of Scientific and Technical Information of China (English)

    Salvatore Dimonte; Muhammed Babakir-Mina; Stefano Aquaro; Carlo-Federico Perno

    2012-01-01

    After infection and integration steps,HIV-1 transcriptions increase sharply and singly-spliced mRNAs are produced.These encode Env (gpl20 and gp41) and auxiliary proteins Vif,Vpr and VpU.The same localization within the unique structure of the mRNAs suggests that the VpU sequence prior to the Env could affect the Env polyprotein expression.The HIV-1 infection process begins when the gp120 subunit of the envelope glycoprotein complex interacts with its receptor(s) on the target cell.The V3 domain of gp120 is the major determinant of cellular co-receptor binding.According to phenotypic information of HIV-1 isolates,sequences from the VpU to V3 regions (119 in R5-and 120 X4-tropic viruses; oneper patient) were analysed.The binomial correlation phi coefficient was used to assess covariation among VpU and gp120v3 signatures.Subsequently,average linkage hierarchical agglomerative clustering was performed.Beyond the classical V3 signatures (R5-viruses:S11,E25D; X4-viruses:S11KR,E25KRQ),other specific V3 and novel VpU signatures were found to be statistically associated with co-receptor usage.Several statistically significant associations between V3 and VpU mutations were also observed.The dendrogram showed two distinct large clusters:one associated with R5-tropic sequences (bootstrap=0.94),involving:(a) H13NPV3,E25DV3,S11V3,T22AV3 and Q61HVpU,(b) E25AV3 and L12FVpU:,(c) D44EVpU,R18QV3 and D80NVpU; and another associated with X4-tropic sequences (bootstrap=0.97),involving:(i) E25Iv3 and V10AvpU,(ii)0-1insVVpU,H13Rv3,146LVpU,I30Mv3 and 60-62delVpU,(iii) S11KRV3 and E25KRQV3.Some of these pairs of mutations were encoded always by one specific codon.These data indicate the possible VpU mutational patterns contributing to regulation of HIV-1 tropism.

  5. Core Binding Factor β Protects HIV, Type 1 Accessory Protein Viral Infectivity Factor from MDM2-mediated Degradation.

    Science.gov (United States)

    Matsui, Yusuke; Shindo, Keisuke; Nagata, Kayoko; Yoshinaga, Noriyoshi; Shirakawa, Kotaro; Kobayashi, Masayuki; Takaori-Kondo, Akifumi

    2016-11-25

    HIV, type 1 overcomes host restriction factor apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins by organizing an E3 ubiquitin ligase complex together with viral infectivity factor (Vif) and a host transcription cofactor core binding factor β (CBFβ). CBFβ is essential for Vif to counteract APOBEC3 by enabling the recruitment of cullin 5 to the complex and increasing the steady-state level of Vif protein; however, the mechanisms by which CBFβ up-regulates Vif protein remains unclear. Because we have reported previously that mouse double minute 2 homolog (MDM2) is an E3 ligase for Vif, we hypothesized that CBFβ might protect Vif from MDM2-mediated degradation. Co-immunoprecipitation analyses showed that Vif mutants that do not bind to CBFβ preferentially interact with MDM2 and that overexpression of CBFβ disrupts the interaction between MDM2 and Vif. Knockdown of CBFβ reduced the steady-state level of Vif in MDM2-proficient cells but not in MDM2-null cells. Cycloheximide chase analyses revealed that Vif E88A/W89A, which does not interact with CBFβ, degraded faster than wild-type Vif in MDM2-proficient cells but not in MDM2-null cells, suggesting that Vif stabilization by CBFβ is mainly caused by impairing MDM2-mediated degradation. We identified Vif R93E as a Vif variant that does not bind to MDM2, and the virus with this substitution mutation was more resistant to APOBEC3G than the parental virus. Combinatory substitution of Vif residues required for CBFβ binding and MDM2 binding showed full recovery of Vif steady-state levels, supporting our hypothesis. Our data provide new insights into the mechanism of Vif augmentation by CBFβ.

  6. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: novel insights into the regulation of Rev nuclear import.

    LENUS (Irish Health Repository)

    Gu, Lili

    2011-01-01

    The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s) predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised.

  7. Fc Receptor-Mediated Activities of Env-Specific Human Monoclonal Antibodies Generated from Volunteers Receiving the DNA Prime-Protein Boost HIV Vaccine DP6-001.

    Science.gov (United States)

    Costa, Matthew R; Pollara, Justin; Edwards, Regina Whitney; Seaman, Michael S; Gorny, Miroslaw K; Montefiori, David C; Liao, Hua-Xin; Ferrari, Guido; Lu, Shan; Wang, Shixia

    2016-11-15

    HIV-1 is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years of infection, and therefore, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that moderate protection is possible and that this protection may correlate with antibody-dependent cellular cytotoxicity (ADCC) activity. Our previous studies demonstrated that in an HIV vaccine phase I trial, the DP6-001 trial, a polyvalent Env DNA prime-protein boost formulation could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities. Here we report on the production and analysis of HIV-1 Env-specific human monoclonal antibodies (hMAbs) isolated from vaccinees in the DP6-001 trial. For this initial report, 13 hMAbs from four vaccinees in the DP6-001 trial showed broad binding to gp120 proteins of diverse subtypes both autologous and heterologous to vaccine immunogens. Equally cross-reactive Fc receptor-mediated functional activities, including ADCC and antibody-dependent cellular phagocytosis (ADCP) activities, were present with both immune sera and isolated MAbs, confirming the induction of nonneutralizing functional hMAbs by the DNA prime-protein boost vaccination. Elicitation of broadly reactive hMAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV vaccine design. The roles of Fc receptor-mediated protective antibody responses are gaining more attention due to their potential contribution to the low-level protection against HIV-1 infection that they provided in the RV144 trial. At the same time, information about hMabs from other human HIV vaccine studies is very limited. In the current study, both immune sera and monoclonal antibodies from vaccinated humans showed not only high-level ADCC and ADCP activities but also cross-subtype ADCC and ADCP activities when a polyvalent DNA prime-protein boost

  8. 高效表达重组HIV-1 gp41及在尿液检测中的应用%Overexpression, Purification of recombinant HIV-1 gp41 protein and detection of HIV antibody in urine

    Institute of Scientific and Technical Information of China (English)

    张晓光; 戚其平; 马晶; 张晓梅; 王自春; 李红霞; 曾毅

    2008-01-01

    目的 应用优化的HIV-1 gp41基因,通过原核表达得到高纯度的重组HIV-1 gp41抗原,制备基于重组gp41抗原的尿液HIV-1抗体检测试剂盒,并评价此试剂盒在尿液抗体检测中的灵敏度和特异性.方法 将编码HIV-1B亚型gp41主要抗原表位的基因插入到原核表达载体pET22b中,构建表达质粒pET22b-mgp41.将表达质粒转化B121(DE3)后经IPTG诱导其表达重组抗原rgp41.重组抗原经镍离子亲和层析和分子筛层析得到纯化.将纯化后重组抗原包被ELISA板,对4796份正常人群尿液、641份HIV-1抗体阳件感染者尿液进行HIV-1抗体检测.结果重组抗原经纯化后纯度95%.用本实验中制备的HIV-1抗体尿液检测试剂盒的检测结果显示,此试剂盒灵敏度为100%,特异性为98.52%.结论 本研究中制备的HIV-1尿液诊断试剂盒可以满足HIV感染者初筛实验的需要.%Objective To establish a specific and sensitive Enzyme-linked immunosorbent Assay(ELISA)kit for detection of HIV-1 antibody in urine using Escherichia coli expression products as coating antigen. Methods The truncated HIV-1 gp41 gene fragment of major antigenic epitopes was inserted into the plasmid pET22b to obtain expression plasmid pET22b-mgp41. The recombinant antigen was expressed in BL21 (DE3) strains of Escherichia coli and was purified by immobilized metal chelation and gel filtration chromatography. Using this antigen as coating antigen, a HIV-1 urine antibody ELISA kit was developed. In order to examine the clinical utility of the kit, 5437 urine samples were assayed, which consisted of 641 urine samples from HIV infected patients and 4796 samples from normal subjects. Results The purity of purified antigen is up to 95%. Anti-HIV antibodies were detected in all the urine samples from HIV infected patients, and the diagnostic sensitivity for HIV-1 infection was 100%. In healthy control group, 71 cases showed false positive, the specificity was 98.52%.Conclusion The HIV-1

  9. Direct interaction of the human I-mfa domain-containing protein, HIC, with HIV-1 Tat results in cytoplasmic sequestration and control of Tat activity.

    Science.gov (United States)

    Gautier, Virginie W; Sheehy, Noreen; Duffy, Margaret; Hashimoto, Kenichi; Hall, William W

    2005-11-08

    The primary function of the HIV-1 regulatory protein Tat, activation of transcription from the viral LTR, is highly regulated by complex interactions between Tat and a number of host cell proteins. Tat nuclear import, a process mediated by importin beta, is a prerequisite for its activity. Here, we report and characterize the interaction of the human inhibitor of MyoD family domain-containing protein (I-mfa), HIC, with Tat at a biochemical and a functional level. This interaction was shown to occur in vivo and in vitro and to involve the nuclear localization signal and the transactivation responsive element-binding domains of Tat and the I-mfa domain of HIC. Coexpression of HIC and Tat resulted in the down-regulation of transactivation of the HIV-1 LTR, and colocalization studies revealed the cytoplasmic sequestration of Tat by HIC. Functionally this sequestration appears to be the underlying mechanism of LTR transcriptional repression by HIC and represents a unique mechanism for the control of Tat activity and regulation of HIV-1 replication.

  10. High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

    Directory of Open Access Journals (Sweden)

    Bianco Linda

    2009-11-01

    Full Text Available Abstract Background In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein. In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. Results The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19 gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. Conclusion We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor

  11. HIV-1 Tat Protein Activates both the MyD88 and TRIF Pathways To Induce Tumor Necrosis Factor Alpha and Interleukin-10 in Human Monocytes.

    Science.gov (United States)

    Planès, Rémi; Ben Haij, Nawal; Leghmari, Kaoutar; Serrero, Manutea; BenMohamed, Lbachir; Bahraoui, Elmostafa

    2016-07-01

    In this study, we show that the HIV-1 Tat protein interacts with rapid kinetics to engage the Toll-like receptor 4 (TLR4) pathway, leading to the production of proinflammatory and anti-inflammatory cytokines. The pretreatment of human monocytes with Tat protein for 10 to 30 min suffices to irreversibly engage the activation of the TLR4 pathway, leading to the production of tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10), two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection. Therefore, this study analyzed whether the HIV-1 Tat protein is able to activate these two pathways separately or simultaneously. Using three complementary approaches, including mice deficient in the MyD88, TIRAP/MAL, or TRIF adaptor, biochemical analysis, and the use of specific small interfering RNAs (siRNAs), we demonstrated (i) that Tat was able to activate both the MyD88 and TRIF pathways, (ii) the capacity of Tat to induce TIRAP/MAL degradation, (iii) the crucial role of the MyD88 pathway in the production of Tat-induced TNF-α and IL-10, (iv) a reduction but not abrogation of IL-10 and TNF-α by Tat-stimulated macrophages from mice deficient in TIRAP/MAL, and (v) the crucial role of the TRIF pathway in Tat-induced IL-10 production. Further, we showed that downstream of the MyD88 and TRIF pathways, the Tat protein activated the protein kinase C (PKC) βII isoform, the mitogen-activated protein (MAP) kinases p38 and extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB in a TLR4-dependent manner. Collectively, our data show that by recruiting the TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both the MyD88 and TRIF pathways and to the activation of PKC, MAP kinase, and NF-κB signaling to induce the production of TNF-α and IL-10. In this study, we demonstrate that by recruiting the TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement

  12. UV and X-ray structural studies of a 101-residue long Tat protein from a HIV-1 primary isolate and of its mutated, detoxified, vaccine candidate.

    Science.gov (United States)

    Foucault, Marine; Mayol, Katia; Receveur-Bréchot, Véronique; Bussat, Marie-Claire; Klinguer-Hamour, Christine; Verrier, Bernard; Beck, Alain; Haser, Richard; Gouet, Patrice; Guillon, Christophe

    2010-05-01

    The 101-residue long Tat protein of primary isolate 133 of the human immunodeficiency virus type 1 (HIV-1), wt-Tat(133) displays a high transactivation activity in vitro, whereas the mutant thereof, STLA-Tat(133), a vaccine candidate for HIV-1, has none. These two proteins were chemically synthesized and their biological activity was validated. Their structural properties were characterized using circular dichroism (CD), fluorescence emission, gel filtration, dynamic light scattering, and small angle X-ray scattering (SAXS) techniques. SAXS studies revealed that both proteins were extended and belong to the family of intrinsically unstructured proteins. CD measurements showed that wt-Tat(133) or STLA-Tat(133) underwent limited structural rearrangements when complexed with specific fragments of antibodies. Crystallization trials have been performed on the two forms, assuming that the Tat(133) proteins might have a better propensity to fold in supersaturated conditions, and small crystals have been obtained. These results suggest that biologically active Tat protein is natively unfolded and requires only a limited gain of structure for its function.

  13. A Response Surface Methodology Approach to Investigate the Effect of Sulfur Dioxide, pH, and Ethanol on DbCD and DbVPR Gene Expression and on the Volatile Phenol Production in Dekkera/Brettanomyces bruxellensis CBS2499.

    Science.gov (United States)

    Valdetara, Federica; Fracassetti, Daniela; Campanello, Alessia; Costa, Carlo; Foschino, Roberto; Compagno, Concetta; Vigentini, Ileana

    2017-01-01

    Dekkera/Brettanomyces bruxellensis, the main spoilage yeast in barrel-aged wine, metabolize hydroxycinnamic acids into off-flavors, namely ethylphenols. Recently, both the enzymes involved in this transformation, the cinnamate decarboxylase (DbCD) and the vinylphenol reductase (DbVPR), have been identified. To counteract microbial proliferation in wine, sulfur dioxide (SO2) is used commonly to stabilize the final product, but limiting its use is advised to preserve human health and boost sustainability in winemaking. In the present study, the influence of SO2 was investigated in relation with pH and ethanol factors on the expression of DbCD and DbVPR genes and volatile phenol production in D. bruxellensis CBS2499 strain under different model wines throughout a response surface methodology (RSM). In order to ensure an exact quantification of DbCD and DbVPR expression, an appropriate housekeeping gene was sought among DbPDC, DbALD, DbEF, DbACT, and DbTUB genes by GeNorm and Normfinder algorithms. The latter gene showed the highest expression stability and it was chosen as the reference housekeeping gene in qPCR assays. Even though SO2 could not be commented as main factor because of its statistical irrelevance on the response of DbCD gene, linear interactions with pH and ethanol concurred to define a significant effect (p bruxellensis spoilage.

  14. HIV-infected patients with lipodystrophy have higher rates of carotid atherosclerosis: The role of monocyte chemoattractant protein-1

    NARCIS (Netherlands)

    B. Coll; S. Parra; C. Alonso-Villaverde; E. de Groot; G. Aragones; M. Montero-Sieburth; M. Tous; J. Camps; J. Joven; L. Masana

    2006-01-01

    Individuals with HIV-1 infection are at increased risk for cardiovascular events, and lipodystrophy is generally associated with proatheroizenic metabolic disturbances. We conducted a case-control study to assess the presence of sub-clinical atherosclerosis in HIV-1-infected patients with or without

  15. PknE, a serine/threonine protein kinase of Mycobacterium tuberculosis initiates survival crosstalk that also impacts HIV coinfection.

    Directory of Open Access Journals (Sweden)

    Dinesh Kumar Parandhaman

    Full Text Available Serine threonine protein kinases (STPK play a major role in the pathogenesis of Mycobacterium tuberculosis. Here, we examined the role of STPK pknE, using a deletion mutant ΔpknE in the modulation of intracellular signaling events that favor M. tuberculosis survival. Phosphorylation kinetics of MAPK (p38MAPK, Erk½ and SAPK/JNK was defective in ΔpknE compared to wild-type infected macrophages. This defective signaling dramatically delayed and reduced the phosphorylation kinetics of transcription factors ATF-2 and c-JUN in ΔpknE infected macrophages. MAPK inhibitors instead of reducing the phosphorylation in ΔpknE infected macrophages, revealed crosstalks with Erk½ signaling influenced by SAPK/JNK and p38 pathways independently. Modulations in intra cellular signaling altered the expression of coreceptors CCR5 and CXCR4 in ΔpknE infected macrophages. In conclusion, pknE plays a role in MAPK crosstalks that enables intracellular survival of M. tuberculosis. This survival strategy also impacts HIV/TB coinfection.

  16. ReFlexIn: a flexible receptor protein-ligand docking scheme evaluated on HIV-1 protease.

    Directory of Open Access Journals (Sweden)

    Simon Leis

    Full Text Available For many targets of pharmaceutical importance conformational changes of the receptor protein are relevant during the ligand binding process. A new docking approach, ReFlexIn (Receptor Flexibility by Interpolation, that combines receptor flexibility with the computationally efficient potential grid representation of receptor molecules has been evaluated on the retroviral HIV-1 (Human Immunodeficiency Virus 1 protease system. An approximate inclusion of receptor flexibility is achieved by using interpolation between grid representations of individual receptor conformations. For the retroviral protease the method was tested on an ensemble of protease structures crystallized in the presence of different ligands and on a set of structures obtained from morphing between the unbound and a ligand-bound protease structure. Docking was performed on ligands known to bind to the protease and several non-binders. For the binders the ReFlexIn method yielded in almost all cases ligand placements in similar or closer agreement with experiment than docking to any of the ensemble members without degrading the discrimination with respect to non-binders. The improved docking performance compared to docking to rigid receptors allows for systematic virtual screening applications at very small additional computational cost.

  17. LINE-1 retrotransposable element DNA accumulates in HIV-1-infected cells.

    Science.gov (United States)

    Jones, R Brad; Song, Haihan; Xu, Yang; Garrison, Keith E; Buzdin, Anton A; Anwar, Naveed; Hunter, Diana V; Mujib, Shariq; Mihajlovic, Vesna; Martin, Eric; Lee, Erika; Kuciak, Monika; Raposo, Rui André Saraiva; Bozorgzad, Ardalan; Meiklejohn, Duncan A; Ndhlovu, Lishomwa C; Nixon, Douglas F; Ostrowski, Mario A

    2013-12-01

    Type 1 long-interspersed nuclear elements (L1s) are autonomous retrotransposable elements that retain the potential for activity in the human genome but are suppressed by host factors. Retrotransposition of L1s into chromosomal DNA can lead to genomic instability, whereas reverse transcription of L1 in the cytosol has the potential to activate innate immune sensors. We hypothesized that HIV-1 infection would compromise cellular control of L1 elements, resulting in the induction of retrotransposition events. Here, we show that HIV-1 infection enhances L1 retrotransposition in Jurkat cells in a Vif- and Vpr-dependent manner. In primary CD4(+) cells, HIV-1 infection results in the accumulation of L1 DNA, at least the majority of which is extrachromosomal. These data expose an unrecognized interaction between HIV-1 and endogenous retrotransposable elements, which may have implications for the innate immune response to HIV-1 infection, as well as for HIV-1-induced genomic instability and cytopathicity.

  18. Water molecules inside protein structure affect binding of monosaccharides with HIV-1 antibody 2G12.

    Science.gov (United States)

    Ueno-Noto, Kaori; Takano, Keiko

    2016-10-05

    Water molecules inside biomolecules constitute integral parts of their structure and participate in the functions of the proteins. Some of the X-ray crystallographic data are insufficient for analyzing a series of ligand-protein complexes in the same condition. We theoretically investigated antibody binding abilities of saccharide ligands and the effects of the inner water molecules of ligand-antibody complexes. Classical molecular dynamics and quantum chemical simulations using a model with possible water molecules inside the protein were performed with saccharide ligands and Human Immunodeficiency Virus 1 neutralizing antibody 2G12 complexes to estimate how inner water molecules of the protein affect the dynamics of the complexes as well as the ligand-antibody interaction. Our results indicate the fact that d-fructose's strong affinity to the antibody was partly due to the good retentiveness of solvent water molecules of the ligand and its stability of the ligand's conformation and relative position in the active site. © 2016 Wiley Periodicals, Inc.

  19. Use of Mutual Information Arrays to Predict Coevolving Sites in the Full Length HIV gp120 Protein for Subtypes B and C

    Institute of Scientific and Technical Information of China (English)

    Bo Wei; Na Han; Hai-zhou Liu; Anthony Rayner; Simon Rayner

    2011-01-01

    It is well established that different sites within a protein evolve at different rates according to their role within the protein; identification of these correlated mutations can aid in tasks such as ab initio protein structure,structure function analysis or sequence alignment.Mutual Information is a standard measure for coevolution between two sites but its application is limited by signal to noise ratio.In this work we report a preliminary study to investigate whether larger sequence sets could circumvent this problem by calculating mutual information arrays for two sets of drug naive sequences from the HIV gp120 protein for the B and C subtypes.Our results suggest that while the larger sequences sets can improve the signal to noise ratio,the gain is offset by the high mutation rate of the HIV virus which makes it more difficult to achieve consistent alignments.Nevertheless,we were able to predict a number of coevolving sites that were supported by previous experimental studies as well as a region close to the C terminal of the protein that was highly variable in the C subtype but highly conserved in the B subtype.

  20. HIV-1 Envelope Proteins and V1/V2 Domain Scaffolds with Mannose-5 to Improve the Magnitude and Quality of Protective Antibody Responses to HIV-1*

    Science.gov (United States)

    Morales, Javier F.; Morin, Trevor J.; Yu, Bin; Tatsuno, Gwen P.; O'Rourke, Sara M.; Theolis, Richard; Mesa, Kathryn A.; Berman, Phillip W.

    2014-01-01

    Two lines of investigation have highlighted the importance of antibodies to the V1/V2 domain of gp120 in providing protection from HIV-1 infection. First, the recent RV144 HIV-1 vaccine trial documented a correlation between non-neutralizing antibodies to the V2 domain and protection. Second, multiple broadly neutralizing monoclonal antibodies to the V1/V2 domain (e.g. PG9) have been isolated from rare infected individuals, termed elite neutralizers. Interestingly, the binding of both types of antibodies appears to depend on the same cluster of amino acids (positions 167–171) adjacent to the junction of the B and C strands of the four-stranded V1/V2 domain β-sheet structure. However, the broadly neutralizing mAb, PG9, additionally depends on mannose-5 glycans at positions 156 and 160 for binding. Because the gp120 vaccine immunogens used in previous HIV-1 vaccine trials were enriched for complex sialic acid-containing glycans, and lacked the high mannose structures required for the binding of PG9-like mAbs, we wondered if these immunogens could be improved by limiting glycosylation to mannose-5 glycans. Here, we describe the PG9 binding activity of monomeric gp120s from multiple strains of HIV-1 produced with mannose-5 glycans. We also describe the properties of glycopeptide scaffolds from the V1/V2 domain also expressed with mannose-5 glycans. The V1/V2 scaffold from the A244 isolate was able to bind the PG9, CH01, and CH03 mAbs with high affinity provided that the proper glycans were present. We further show that immunization with A244 V1/V2 fragments alone, or in a prime/boost regimen with gp120, enhanced the antibody response to sequences in the V1/V2 domain associated with protection in the RV144 trial. PMID:24872420

  1. Sugar-binding proteins potently inhibit dendritic cell human immunodeficiency virus type 1 (HIV-1) infection and dendritic-cell-directed HIV-1 transfer.

    Science.gov (United States)

    Turville, Stuart G; Vermeire, Kurt; Balzarini, Jan; Schols, Dominique

    2005-11-01

    Both endocytic uptake and viral fusion can lead to human immunodeficiency virus type 1 (HIV-1) transfer to CD4+ lymphocytes, either through directional regurgitation (infectious transfer in trans [I-IT]) or through de novo viral production in dendritic cells (DCs) resulting in a second-phase transfer to CD4+ lymphocytes (infectious second-phase transfer [I-SPT]). We have evaluated in immature monocyte-derived DCs both pathways of transfer with regard to their susceptibilities to being blocked by potential microbicidal compounds, including cyanovirin (CNV); the plant lectins Hippeastrum hybrid agglutinin, Galanthus nivalis agglutinin, Urtica dioica agglutinin, and Cymbidium hybrid agglutinin; and the glycan mannan. I-IT was a relatively inefficient means of viral transfer compared to I-SPT at both high and low levels of the viral inoculum. CNV was able to completely block I-IT at 15 microg/ml. All other compounds except mannan could inhibit I-IT by at least 90% when used at doses of 15 microg/ml. In contrast, efficient inhibition of I-SPT was remarkably harder to achieve, as 50% effective concentration levels for plant lectins and CNV to suppress this mode of HIV-1 transfer increased significantly. Thus, our findings indicate that I-SPT may be more elusive to targeting by antiviral drugs and stress the need for drugs affecting the pronounced inhibition of the infection of DCs by HIV-1.

  2. Serum Zinc Concentration and C-Reactive Protein in Individuals with Human Immunodeficiency Virus Infection: the Positive Living with HIV (POLH) Study.

    Science.gov (United States)

    Poudel, Krishna C; Bertone-Johnson, Elizabeth R; Poudel-Tandukar, Kalpana

    2016-05-01

    Low zinc levels and chronic inflammation are common in individuals infected with human immunodeficiency virus (HIV). Zinc deficiency may promote systemic inflammation, but research on the role of zinc in inflammation among HIV-positive individuals taking account of anti-retroviral therapy is lacking. We assessed the association between serum zinc and C-reactive protein (CRP) concentration in a cohort of HIV-positive individuals. A cross-sectional survey was conducted among 311 HIV-positive individuals (177 men and 134 women) aged 18-60 years residing in Kathmandu, Nepal. High-sensitive or regular serum CRP concentrations were measured by the latex agglutination nephelometry or turbidimetric method, and zinc concentrations were measured by the atomic absorption method. Relationships were assessed using multiple linear regression analysis. The geometric means of zinc in men and women were 73.83 and 71.93 ug/dL, respectively, and of CRP were 1.64 and 0.96 mg/L, respectively. Mean serum CRP concentration was significantly decreased with increasing serum zinc concentration across zinc tertiles (P for trend = 0.010), with mean serum CRP concentration in the highest tertile of serum zinc concentration was 44.2 % lower than that in the lowest tertile. The mean serum CRP concentrations in men and women in the highest tertile of serum zinc concentrations were 30 and 35.9 % lower, respectively, than that in the lowest tertile (P for trend = 0.263 and 0.162, respectively). We found a significant inverse relation between log zinc and log CRP concentrations (beta for 1 unit change in log zinc; β = -1.79, p = 0.0003). Serum zinc concentration may be inversely associated with serum CRP concentration in HIV-positive individuals.

  3. Unbiased proteomic analysis of proteins interacting with the HIV-1 5′LTR sequence: role of the transcription factor Meis

    Science.gov (United States)

    Tacheny, A.; Michel, S.; Dieu, M.; Payen, L.; Arnould, T.; Renard, P.

    2012-01-01

    To depict the largest picture of a core promoter interactome, we developed a one-step DNA-affinity capture method coupled with an improved mass spectrometry analysis process focused on the identification of low abundance proteins. As a proof of concept, this method was developed through the analysis of 230 bp contained in the 5′long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1). Beside many expected interactions, many new transcriptional regulators were identified, either transcription factors (TFs) or co-regulators, which interact directly or indirectly with the HIV-1 5′LTR. Among them, the homeodomain-containing TF myeloid ectopic viral integration site was confirmed to functionally interact with a specific binding site in the HIV-1 5′LTR and to act as a transcriptional repressor, probably through recruitment of the repressive Sin3A complex. This powerful and validated DNA-affinity approach could also be used as an efficient screening tool to identify a large set of proteins that physically interact, directly or indirectly, with a DNA sequence of interest. Combined with an in silico analysis of the DNA sequence of interest, this approach provides a powerful approach to select the interacting candidates to validate functionally by classical approaches. PMID:22904091

  4. A triclinic crystal structure of the carboxy-terminal domain of HIV-1 capsid protein with four molecules in the asymmetric unit reveals a novel packing interface

    Science.gov (United States)

    Lampel, Ayala; Yaniv, Oren; Berger, Or; Bacharach, Eran; Gazit, Ehud; Frolow, Felix

    2013-01-01

    The Gag precursor is the major structural protein of the virion of human immunodeficiency virus-1 (HIV-1). Capsid protein (CA), a cleavage product of Gag, plays an essential role in virus assembly both in Gag-precursor multimerization and in capsid core formation. The carboxy-terminal domain (CTD) of CA contains 20 residues that are highly conserved across retroviruses and constitute the major homology region (MHR). Genetic evidence implies a role for the MHR in interactions between Gag precursors during the assembly of the virus, but the structural basis for this role remains elusive. This paper describes a novel triclinic structure of the HIV-1 CA CTD at 1.6 Å resolution with two canonical dimers of CA CTD in the asymmetric unit. The canonical dimers form a newly identified packing interface where interactions of four conserved MHR residues take place. This is the first structural indication that these MHR residues participate in the putative CTD–CTD interactions. These findings suggest that the molecules forming this novel interface resemble an intermediate structure that participates in the early steps of HIV-1 assembly. This interface may therefore provide a novel target for antiviral drugs. PMID:23722834

  5. HIV Molecular Immunology 2014

    Energy Technology Data Exchange (ETDEWEB)

    Yusim, Karina [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Korber, Bette Tina Marie [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Barouch, Dan [Beth Israel Deaconess Medical Center, Boston, MA (United States); Koup, Richard [Vaccine Research Center National Institutes of Health (United States); de Boer, Rob [Utrecht Univ. (Netherlands). Dept. of Biology; Moore, John P. [Cornell Univ., Ithaca, NY (United States). Weill Medical College; Brander, Christian [Institucioi Catalana de Recerca i Estudis Avancats (ICREA), Barcelona (Spain); Haynes, Barton F. [Duke Univ., Durham, NC (United States). Duke Human Vaccine Institute and Departments of Medicine, Surgery and Immunology; Walker, Bruce D. [Ragon Institute of Massachusetts General Hospital, Cambridge, MA (United States); Harvard Univ., Cambridge, MA (United States); Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States)

    2015-02-03

    HIV Molecular Immunology is a companion volume to HIV Sequence Compendium. This publication, the 2014 edition, is the PDF version of the web-based HIV Immunology Database (http://www.hiv.lanl.gov/content/immunology/). The web interface for this relational database has many search options, as well as interactive tools to help immunologists design reagents and interpret their results. In the HIV Immunology Database, HIV-specific B-cell and T-cell responses are summarized and annotated. Immunological responses are divided into three parts, CTL, T helper, and antibody. Within these parts, defined epitopes are organized by protein and binding sites within each protein, moving from left to right through the coding regions spanning the HIV genome. We include human responses to natural HIV infections, as well as vaccine studies in a range of animal models and human trials. Responses that are not specifically defined, such as responses to whole proteins or monoclonal antibody responses to discontinuous epitopes, are summarized at the end of each protein section. Studies describing general HIV responses to the virus, but not to any specific protein, are included at the end of each part. The annotation includes information such as crossreactivity, escape mutations, antibody sequence, TCR usage, functional domains that overlap with an epitope, immune response associations with rates of progression and therapy, and how specific epitopes were experimentally defined. Basic information such as HLA specificities for T-cell epitopes, isotypes of monoclonal antibodies, and epitope sequences are included whenever possible. All studies that we can find that incorporate the use of a specific monoclonal antibody are included in the entry for that antibody. A single T-cell epitope can have multiple entries, generally one entry per study. Finally, maps of all defined linear epitopes relative to the HXB2 reference proteins are provided.

  6. Inhibition of catechol-O-methyl transferase (COMT) by tolcapone restores reductions in microtubule-associated protein 2 (MAP2) and synaptophysin (SYP) following exposure of neuronal cells to neurotropic HIV.

    Science.gov (United States)

    Lee, Ting Ting; Chana, Gursharan; Gorry, Paul R; Ellett, Anne; Bousman, Chad A; Churchill, Melissa J; Gray, Lachlan R; Everall, Ian P

    2015-10-01

    This investigation aimed to assess whether inhibition of cathecol-O-methyl transferase (COMT) by tolcapone could provide neuroprotection against HIV-associated neurodegenerative effects. This study was conducted based on a previous work, which showed that a single nucleotide polymorphism (SNP) at position 158 (val158met) in COMT, resulted in 40 % lower COMT activity. Importantly, this reduction confers a protective effect against HIV-associated neurocognitive disorders (HAND), which have been linked to HIV-associated brain changes. SH-SY5Y-differentiated neurons were exposed to macrophage-propagated HIV (neurotropic MACS2-Br strain) in the presence or absence of tolcapone for 6 days. RNA was extracted, and qPCR was performed using Qiagen RT2 custom array consisting of genes for neuronal and synaptic integrity, COMT and pro-inflammatory markers. Immunofluorescence was conducted to validate the gene expression changes at the protein level. Our findings demonstrated that HIV significantly increased the messenger RNA (mRNA) expression of COMT while reducing the expression of microtubule-associated protein 2 (MAP2) (p = 0.0015) and synaptophysin (SYP) (p = 0.012) compared to control. A concomitant exposure of tolcapone ameliorated the perturbed expression of MAP2 (p = 0.009) and COMT (p = 0.024) associated with HIV. Immunofluorescence revealed a trend reduction of SYP and MAP2 with exposure to HIV and that concomitant exposure of tolcapone increased SYP (p = 0.016) compared to HIV alone. Our findings demonstrated in vitro that inhibition of COMT can ameliorate HIV-associated neurodegenerative changes that resulted in the decreased expression of the structural and synaptic components MAP2 and SYP. As HIV-associated dendritic and synaptic damage are contributors to HAND, inhibition of COMT may represent a potential strategy for attenuating or preventing some of the symptoms of HAND.

  7. Inhibition of Catechol-O-methyl Transferase (COMT) by Tolcapone Restores Reductions in Microtubule-associated Protein 2 (MAP2) and Synaptophysin (SYP) Following Exposure of Neuronal Cells to Neurotropic HIV

    Science.gov (United States)

    Lee, Ting Ting; Chana, Gursharan; Gorry, Paul R.; Ellett, Anne; Bousman, Chad A.; Churchill, Melissa J.; Gray, Lachlan R.; Everall, Ian P.

    2015-01-01

    This investigation aimed to assess whether inhibition of cathecol-O-methyl transferase (COMT) by Tolcapone could provide neuroprotection against HIV-associated neurodegenerative effects. This study was conducted based on previous work, which showed that a single nucleotide polymorphism (SNP) at position 158 (val158met) in COMT, resulted in 40% lower COMT activity. Importantly, this reduction confers a protective effect against HIV-associated neurocognitive disorders (HAND), which have been linked to HIV-associated brain changes. SH-SY5Y differentiated neurons were exposed to macrophage-propagated HIV (neurotropic MACS2-Br strain) in the presence or absence of Tolcapone for 6 days. RNA was extracted and qPCR was performed using Qiagen RT2-custom-array consisting of genes for neuronal and synaptic integrity, COMT and pro-inflammatory markers. Immunofluorescence was conducted to validate the gene expression changes at the protein level. Our findings demonstrated that HIV significantly increased the mRNA expression of COMT while reduced the expression of microtubule-associated protein 2 (MAP2) (p=0.0015) and synaptophysin (SYP) (p=0.012) compared to control. A concomitant exposure of Tolcapone ameliorated the perturbed expression of MAP2 (p=0.009) and COMT (p=0.024) associated with HIV. Immunofluorescence revealed a trend reduction of SYP and MAP2 with exposure to HIV, and that concomitant exposure of Tolcapone increased SYP (p=0.016) compared to HIV alone. Our findings demonstrated in vitro that inhibition of COMT can ameliorate HIV-associated neurodegenerative changes that resulted in the decreased expression of the structural and synaptic components MAP2 and SYP. As HIV-associated dendritic and synaptic damage are contributors to HAND, inhibition of COMT may represent a potential strategy for attenuating or preventing some of the symptoms of HAND. PMID:26037113

  8. GPG-NH2 acts via the metabolite αHGA to target HIV-1 Env to the ER-associated protein degradation pathway

    Directory of Open Access Journals (Sweden)

    Vahlne Anders

    2010-03-01

    Full Text Available Abstract Background The synthetic peptide glycyl-prolyl-glycine amide (GPG-NH2 was previously shown to abolish the ability of HIV-1 particles to fuse with the target cells, by reducing the content of the viral envelope glycoprotein (Env in progeny HIV-1 particles. The loss of Env was found to result from GPG-NH2 targeting the Env precursor protein gp160 to the ER-associated protein degradation (ERAD pathway during its maturation. However, the anti-viral effect of GPG-NH2 has been shown to be mediated by its metabolite α-hydroxy-glycineamide (αHGA, which is produced in the presence of fetal bovine serum, but not human serum. In accordance, we wanted to investigate whether the targeting of gp160 to the ERAD pathway by GPG-NH2 was attributed to its metabolite αHGA. Results In the presence of fetal bovine serum, GPG-NH2, its intermediary metabolite glycine amide (G-NH2, and final metabolite αHGA all induced the degradation of gp160 through the ERAD pathway. However, when fetal bovine serum was replaced with human serum only αHGA showed an effect on gp160, and this activity was further shown to be completely independent of serum. This indicated that GPG-NH2 acts as a pro-drug, which was supported by the observation that it had to be added earlier to the cell cultures than αHGA to induce the degradation of gp160. Furthermore, the substantial reduction of Env incorporation into HIV-1 particles that occurs during GPG-NH2 treatment was also achieved by treating HIV-1 infected cells with αHGA. Conclusions The previously observed specificity of GPG-NH2 towards gp160 in HIV-1 infected cells, resulting in the production of Env (gp120/gp41 deficient fusion incompetent HIV-1 particles, was most probably due to the action of the GPG-NH2 metabolite αHGA.

  9. Antiretrovirals, Methamphetamine, and HIV-1 Envelope Protein gp120 Compromise Neuronal Energy Homeostasis in Association with Various Degrees of Synaptic and Neuritic Damage.

    Science.gov (United States)

    Sanchez, Ana B; Varano, Giuseppe P; de Rozieres, Cyrus M; Maung, Ricky; Catalan, Irene C; Dowling, Cari C; Sejbuk, Natalia E; Hoefer, Melanie M; Kaul, Marcus

    2015-10-19

    HIV-1 infection frequently causes HIV-associated neurocognitive disorders (HAND) despite combination antiretroviral therapy (cART). Evidence is accumulating that components of cART can themselves be neurotoxic upon long-term exposure. In addition, abuse of psychostimulants, such as methamphetamine, seems to aggravate HAND and compromise antiretroviral therapy. However, the combined effect of virus and recreational and therapeutic drugs on the brain is poorly understood. Therefore, we exposed mixed neuronal-glial cerebrocortical cells to antiretrovirals (ARVs) (zidovudine [AZT], nevirapine [NVP], saquinavir [SQV], and 118-D-24) of four different pharmacological categories and to methamphetamine and, in some experiments, the HIV-1 gp120 protein for 24 h and 7 days. Subsequently, we assessed neuronal injury by fluorescence microscopy, using specific markers for neuronal dendrites and presynaptic terminals. We also analyzed the disturbance of neuronal ATP levels and assessed the involvement of autophagy by using immunofluorescence and Western blotting. ARVs caused alterations of neurites and presynaptic terminals primarily during the 7-day incubation and depending on the specific compounds and their combinations with and without methamphetamine. Similarly, the loss of neuronal ATP was context specific for each of the drugs or combinations thereof, with and without methamphetamine or viral gp120. Loss of ATP was associated with activation of AMP-activated protein kinase (AMPK) and autophagy, which, however, failed to restore normal levels of neuronal ATP. In contrast, boosting autophagy with rapamycin prevented the long-term drop of ATP during exposure to cART in combination with methamphetamine or gp120. Our findings indicate that the overall positive effect of cART on HIV infection is accompanied by detectable neurotoxicity, which in turn may be aggravated by methamphetamine.

  10. Plasma proteomic profiling in HIV-1 infected methamphetamine abusers.

    Directory of Open Access Journals (Sweden)

    Gwenael Pottiez

    Full Text Available We wanted to determine whether methamphetamine use affects a subset of plasma proteins in HIV-infected persons. Plasma samples from two visits were identified for subjects from four groups: HIV+, ongoing, persistent METH use; HIV+, short-term METH abstinent; HIV+, long term METH abstinence; HIV negative, no history of METH use. Among 390 proteins identified, 28 showed significant changes in expression in the HIV+/persistent METH+ group over the two visits, which were not attributable to HIV itself. These proteins were involved in complement, coagulation pathways and oxidative stress. Continuous METH use is an unstable condition, altering levels of a number of plasma proteins.

  11. Particle-based transcutaneous administration of HIV-1 p24 protein to human skin explants and targeting of epidermal antigen presenting cells.

    Science.gov (United States)

    Rancan, Fiorenza; Amselgruber, Sarah; Hadam, Sabrina; Munier, Sevérine; Pavot, Vincent; Verrier, Bernard; Hackbarth, Steffen; Combadiere, Behazine; Blume-Peytavi, Ulrike; Vogt, Annika

    2014-02-28

    Transcutaneous immunization is a promising vaccination strategy for the treatment of infectious diseases and cancer. In this study, we investigate the combination of cyanoacrylate skin surface stripping (CSSS) and particle-based antigen delivery to target the HIV-1 p24 protein to skin antigen presenting cells (APC). The CSSS treatment pre-activates skin APC and opens hair follicles, where protein-loaded particles accumulate and allow for sustained delivery of the loaded antigen to perifollicular APC. We found that poly-lactic acid (PLA) and polystyrene (PS) particles targeted the adsorbed HIV-1 p24 protein to the hair follicles. Small amounts of PS and PLA particles were found to translocate to the epidermis and be internalized by skin cells, whereas most of the particles aggregated in the hair follicle canal, where they released the loaded antigen. The p24 protein diffused to the epidermis and dermis and was detected in skin cells, especially in Langerhans cells and dermal dendritic cells. Furthermore, the combination of CSSS and particle-based delivery resulted in activation and maturation of Langerhans cells (HLA-DR, CD80 and CD83). We conclude that particle-based antigen delivery across partially disrupted skin barrier is a feasible and effective approach to needle-free transcutaneous vaccination.

  12. Contribution of TLR4 and MyD88 for adjuvant monophosphoryl lipid A (MPLA) activity in a DNA prime-protein boost HIV-1 vaccine.

    Science.gov (United States)

    Pouliot, Kimberly; Buglione-Corbett, Rachel; Marty-Roix, Robyn; Montminy-Paquette, Sara; West, Kim; Wang, Shixia; Lu, Shan; Lien, Egil

    2014-09-03

    Recombinant protein vaccines are commonly formulated with an immune-stimulatory compound, or adjuvant, to boost immune responses to a particular antigen. Recent studies have shown that, through recognition of molecular motifs, receptors of the innate immune system are involved in the functions of adjuvants to generate and direct adaptive immune responses. However, it is not clear to which degree those receptors are also important when the adjuvant is used as part of a novel heterologous prime-boost immunization process in which the priming and boosting components are not the same type of vaccines. In the current study, we compared the immune responses elicited by a pentavalent HIV-1 DNA prime-protein boost vaccine in mice deficient in either Toll-like receptor 4 (TLR4) or myeloid differentiation primary response gene 88 (MyD88) to wildtype mice. HIV gp120 protein administered in the boost phase was formulated with either monophosphoryl lipid A (MPLA), QS-21, or Al(OH)3. Endpoint antibody titer, serum cytokine response and T-cell memory response were assessed. Neither TLR4 nor MyD88 deficiency had a significant effect on the immune response of mice given vaccine formulated with QS-21 or Al(OH)3. However, TLR4- and MyD88-deficiency decreased both the antibody and T-cell responses in mice administered HIV gp120 formulated with MPLA. These results further our understanding of the activation of TLR4 and MyD88 by MPLA in the context of a DNA prime/protein boost immunization strategy. Copyright © 2014. Published by Elsevier Ltd.

  13. Biophysical analysis of the MHR motif in folding and domain swapping of the HIV capsid protein C-terminal domain

    National Research Council Canada - National Science Library

    Bocanegra, Rebeca; Fuertes, Miguel Ángel; Rodríguez-Huete, Alicia; Neira, José Luis; Mateu, Mauricio G

    2015-01-01

    Infection by human immunodeficiency virus (HIV) depends on the function, in virion morphogenesis and other stages of the viral cycle, of a highly conserved structural element, the major homology region (MHR...

  14. HIV-1 induces DCIR expression in CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Alexandra A Lambert

    Full Text Available The C-type lectin receptor DCIR, which has been shown very recently to act as an attachment factor for HIV-1 in dendritic cells, is expressed predominantly on antigen-presenting cells. However, this concept was recently challenged by the discovery that DCIR can also be detected in CD4(+ T cells found in the synovial tissue from rheumatoid arthritis (RA patients. Given that RA and HIV-1 infections share common features such as a chronic inflammatory condition and polyclonal immune hyperactivation status, we hypothesized that HIV-1 could promote DCIR expression in CD4(+ T cells. We report here that HIV-1 drives DCIR expression in human primary CD4(+ T cells isolated from patients (from both aviremic/treated and viremic/treatment naive persons and cells acutely infected in vitro (seen in both virus-infected and uninfected cells. Soluble factors produced by virus-infected cells are responsible for the noticed DCIR up-regulation on uninfected cells. Infection studies with Vpr- or Nef-deleted viruses revealed that these two viral genes are not contributing to the mechanism of DCIR induction that is seen following acute infection of CD4(+ T cells with HIV-1. Moreover, we report that DCIR is linked to caspase-dependent (induced by a mitochondria-mediated generation of free radicals and -independent intrinsic apoptotic pathways (involving the death effector AIF. Finally, we demonstrate that the higher surface expression of DCIR in CD4(+ T cells is accompanied by an enhancement of virus attachment/entry, replication and transfer. This study shows for the first time that HIV-1 induces DCIR membrane expression in CD4(+ T cells, a process that might promote virus dissemination throughout the infected organism.

  15. HIV-1 Vif蛋白的体内表达及其生物学功能%In Vivo Expression and Biological Function of HIV-1 Vif Protein

    Institute of Scientific and Technical Information of China (English)

    张文艳; 孔维; 于湘晖

    2008-01-01

    目的 分别构建带有6His和mbp tag的HIV-1 Vif基因真核表达载体,在体内表达融合蛋白,并检测其生物学功能.方法 PCR扩增带有6His和mbp tag的HIV-1 Vif基因,克隆至真核表达载体VRl012上.将抗病毒因子APOBEC3G(A3G)分别与空载体VR1012、HIV-1野生型Vif/VR1012、Vif-mbp/VR1012和Vif-6His/VR1012共同转染293T细胞,检测Vif-mbp和Vif-6His的表达,并进行A3G体内降解试验.将A3G和/或可产生病毒的质粒HXB2Neo △ Aif分别与HIV-1野生型Vif/VR1012、Vif-mbp/VR1012和Vif-6His/VR1012共同转染293T细胞,通过A3G包装进病毒和Magi细胞感染试验,进一步检测HIV-1 Vif-mbp和Vif-6His融合蛋白的生物学功能.结果 重组表达质粒Vif-mbp/VR1012和Vif-6His/VR1012经双酶切鉴定证明构建正确.转染293T细胞48 h后,Vif-mbp和Vif-6His融合蛋白均有表达,但表达的Vff-mbp有不同程度的降解.Vif-6His可降解A3G,使其表达量下降至10%左右,而Vif-mbp几乎不能降解A3G.Vif-6His可阻止A3G包装进病毒颗粒中,而Vif-mbp无此功能.Vif-mbp可使HXB2Neo △ Vif病毒感染能力恢复至15%,而Vif-6His可使其恢复至86%.结论 已成功构建了带有6His和mbp tag的HIV-1 Vif基因真核表达载体,表达的Vif-mbp融合蛋白影响了Vif的生物学功能,但Vif-6His融合蛋白不影响Vif的生物学功能.

  16. Transmitted/founder and chronic HIV-1 envelope proteins are distinguished by differential utilization of CCR5.

    Science.gov (United States)

    Parker, Zahra F; Iyer, Shilpa S; Wilen, Craig B; Parrish, Nicholas F; Chikere, Kelechi C; Lee, Fang-Hua; Didigu, Chuka A; Berro, Reem; Klasse, Per Johan; Lee, Benhur; Moore, John P; Shaw, George M; Hahn, Beatrice H; Doms, Robert W

    2013-03-01

    Infection by HIV-1 most often results from the successful transmission and propagation of a single virus variant, termed the transmitted/founder (T/F) virus. Here, we compared the attachment and entry properties of envelope (Env) glycoproteins from T/F and chronic control (CC) viruses. Using a panel of 40 T/F and 47 CC Envs, all derived by single genome amplification, we found that 52% of clade C and B CC Envs exhibited partial resistance to the CCR5 antagonist maraviroc (MVC) on cells expressing high levels of CCR5, while only 15% of T/F Envs exhibited this same property. Moreover, subtle differences in the magnitude with which MVC inhibited infection on cells expressing low levels of CCR5, including primary CD4(+) T cells, were highly predictive of MVC resistance when CCR5 expression levels were high. These results are consistent with previous observations showing a greater sensitivity of T/F Envs to MVC inhibition on cells expressing very high levels of CCR5 and indicate that CC Envs are often capable of recognizing MVC-bound CCR5, albeit inefficiently on cells expressing physiologic levels of CCR5. When CCR5 expression levels are high, this phenotype becomes readily detectable. The utilization of drug-bound CCR5 conformations by many CC Envs was seen with other CCR5 antagonists, with replication-competent viruses, and did not obviously correlate with other phenotypic traits. The striking ability of clade C and B CC Envs to use MVC-bound CCR5 relative to T/F Envs argues that the more promiscuous use of CCR5 by these Env proteins is selected against at the level of virus transmission and is selected for during chronic infection.

  17. HIV Testing

    Science.gov (United States)

    ... Abroad Treatment Basic Statistics Get Tested Find an HIV testing site near you. Enter ZIP code or city Follow HIV/AIDS CDC HIV CDC HIV/AIDS See RSS | ... All Collapse All Should I get tested for HIV? CDC recommends that everyone between the ages of ...

  18. Specific protein profile in cerebrospinal fluid from HIV-1-positive cART-treated patients affected by neurological disorders.

    Science.gov (United States)

    Zanin, Valentina; Delbue, Serena; Marcuzzi, Annalisa; Tavazzi, Eleonora; Del Savio, Rossella; Crovella, Sergio; Marchioni, Enrico; Ferrante, Pasquale; Comar, Manola

    2012-10-01

    Cytokines/chemokines are involved in the immune response of infections, including HIV-1. We defined the profile of 48 cytokines/chemokines in cerebrospinal fluid from 18 cART patients with chronic HIV-1 infection by Luminex technology. Nine patients were affected with leukoencephalopathies: five with John Cunningham virus (JCV) + progressive multifocal leukoencephalopathy (PML) and four with JCV-not determined leukoencephalopathy (NDLE). In addition, nine HIV-1-positive patients with no neurological signs (NND) and five HIV-1-negative patients affected with acute disseminated encephalomyelitis (ADEM) were enrolled. Ten cytokines (IL-15, IL-3, IL-16, IL-18, CTACK, GRO1, SCF, MCP-1, MIF, SDF) were highly expressed in HIV-1-positive patients while IL-1Ra and IL-17 were present at a lower level. In addition, the levels of IL-17, IL-9, FGF-basic, MIP-1β, and MCP-1 were significantly higher (p < 0.05) in patients with neurological diseases (PML, NDLE, ADEM) with respect to NND. Focusing the attention to the cytokine profile in JCV + PML patients with respect to JCV-NDLE patients, only TNF-β was significantly downregulated (p < 0.05) in JCV + PML patients. This pilot study emphasized the role of immunoregulation in HIV-1-related neurological disorders during cART treatment.

  19. sCD4-17b bifunctional protein: Extremely broad and potent neutralization of HIV-1 Env pseudotyped viruses from genetically diverse primary isolates

    Directory of Open Access Journals (Sweden)

    Dey Barna

    2010-02-01

    Full Text Available Abstract Background We previously described a potent recombinant HIV-1 neutralizing protein, sCD4-17b, composed of soluble CD4 attached via a flexible polypeptide linker to an SCFv of the 17b human monoclonal antibody directed against the highly conserved CD4-induced bridging sheet of gp120 involved in coreceptor binding. The sCD4 moiety of the bifunctional protein binds to gp120 on free virions, thereby enabling the 17b SCFv moiety to bind and block the gp120/coreceptor interaction required for entry. The previous studies using the MAGI-CCR5 assay system indicated that sCD4-17b (in concentrated cell culture medium, or partially purified potently neutralized several genetically diverse HIIV-1 primary isolates; however, at the concentrations tested it was ineffective against several other strains despite the conservation of binding sites for both CD4 and 17b. To address this puzzle, we designed variants of sCD4-17b with different linker lengths, and tested the neutralizing activities of the immunoaffinity purified proteins over a broader concentration range against a large number of genetically diverse HIV-1 primary isolates, using the TZM-bl Env pseudotype assay system. We also examined the sCD4-17b sensitivities of isogenic viruses generated from different producer cell types. Results We observed that immunoaffinity purified sCD4-17b effectively neutralized HIV-1 pseudotypes, including those from HIV-1 isolates previously found to be relatively insensitive in the MAGI-CCR5 assay. The potencies were equivalent for the original construct and a variant with a longer linker, as observed with both pseudotype particles and infectious virions; by contrast, a construct with a linker too short to enable simultaneous binding of the sCD4 and 17b SCFv moieties was much less effective. sCD4-17b displayed potent neutralizing activity against 100% of nearly 4 dozen HIV-1 primary isolates from diverse genetic subtypes (clades A, B, C, D, F, and circulating

  20. Truncation and expression of HIV-1 trans-membrane protein gp41%HIV-1跨膜蛋白gp41的截短及表达

    Institute of Scientific and Technical Information of China (English)

    端义坤; 张涛; 余模松

    2003-01-01

    HIV-1跨膜蛋白gp41进行截短,在大肠杆菌中进行表达并纯化.PCR扩增gp41的部分编码基因,回收的PCR产物纯化后克隆到连接载体pGEM-T上,然后用EcoR I和Sa1 I切下目的基因,并构建到表达载体pGEX-4T3上,导入宿主细胞BL21(DE3),用IPTG诱导表达,表达产物用亲和层析进行纯化并作相应鉴定.截短的HIV-1跨膜蛋白gp41能直接在大肠杆菌内进行表达,利用亲和层析能方便地将目的蛋白进行纯化,为跨膜蛋白的进一步应用打下基础.

  1. Long-term passage of Vif-null HIV-1 in CD4(+) T cells expressing sub-lethal levels of APOBEC proteins fails to develop APOBEC resistance.

    Science.gov (United States)

    Miyagi, Eri; Kao, Sandra; Fumitaka, Miyoshi; Buckler-White, Alicia; Plishka, Ron; Strebel, Klaus

    2017-04-01

    APOBEC3G (A3G) is a cytidine deaminase with potent antiviral activity that is antagonized by Vif. A3G is expressed in a cell type-specific manner and some semi-permissive cells, including A3.01, express A3G but fail to block replication of Vif-null HIV-1. Here we explored the semi-permissive nature of A3.01 cells and found it to be defined exclusively by the levels of A3G. Indeed, minor changes in A3G levels rendered A3.01 cells either fully permissive or non-permissive for Vif-null HIV-1. Our data indicate that A3.01 cells express sub-lethal levels of catalytically active A3G that affects Vif-null HIV-1 at the proviral level but does not completely block virus replication due to purifying selection. Attempts to use the selective pressure exerted by such sub-lethal levels of A3G to select for APOBEC-resistant Vif-null virus capable of replicating in H9 cells failed despite passaging virus for five months, demonstrating that Vif is a critical viral accessory protein.

  2. Design and synthesis of á,á-trehalose derivatives bearing guanidino groups as inhibitors for the Tat Protein-TAR RNA interaction of HIV-1

    Institute of Scientific and Technical Information of China (English)

    Wang Min; Xu Zhidong; Tu Pengfei; Xiao Sulong; Yu Xiaolin; Yang Ming

    2004-01-01

    Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the transactivation responsive region (TAR) RNA. Disruption of Tat-TAR RNA interaction could inhibit HIV-1 replication. Here four target compounds were designed and synthesized to bind to TAR RNA for blocking the interaction of Tat-TAR RNA. The core molecule 6,6′-diamino-6,6′ -dideoxy-á,á-trehalose was obtained from selective bromination of a,a-trehalose at C-6,6′, followed by acetylation, azide displacement, deacetylation and reduction.Coupling of the core molecule with the protected amino acid, then deprotection and guanidinylation generated 6,6′-bis(L-arginylamino)-6,6′-dideoxy-á,á-trehalose,6,6′-bisguanidino-6,6′-dideoxy-á ,á-trehalose, 6,6′-bis [((a)-guanidinobutyryl)amino]-6,6′-dideoxy-á,á-trehalose and 6,6′-bis(guanidinoacetylamino)-6,6′-dideoxy-á,á-trehalose, respectively.Their abilities to inhibit Tat-TAR RNA interaction were determined by a Tat-dependent HIV-1LTR-driven CAT assays.

  3. HIV Prevention

    Science.gov (United States)

    ... on HIV Syndicated Content Website Feedback HIV/AIDS Prevention Language: English (US) Español (Spanish) Recommend on ... All Is abstinence the only 100% effective HIV prevention option? Yes. Abstinence means not having oral, vaginal, ...

  4. Enhanced production of functional extracellular single chain variable fragment against HIV-1 matrix protein from Escherichia coli by sequential simplex optimization.

    Science.gov (United States)

    Intachai, Kannaporn; Singboottra, Panthong; Leksawasdi, Noppol; Kasinrerk, Watchara; Tayapiwatana, Chatchai; Butr-Indr, Bordin

    2015-01-01

    The optimal culture condition for extracellular recombinant single chain variable fragment anti HIV-1 p17 protein (scFv anti-p17) production in Escherichia coli HB2151 was investigated by the sequential simplex optimization (SS) method. Five variable parameters were submitted in the fermentation process. The most favorable condition obtained from 19 independent experiments was as followed: 58 µM of IPTG induction to 1.7 OD600 nm at 25.5°C for 16 h with 202 rpm agitation rate. The amount of secreted scFv anti-p17 at the optimal condition was 38% higher than under the control condition. The binding activity of soluble extracellular scFv anti-p17 protein increased 95.5% and 73.2% in comparison with the control condition and non-optimized condition respectively. The soluble scFv anti-p17 from crude HB2151 lysated was subsequently purified by immobilized metal ion affinity chromatography (IMAC) with His-tag. The purified scFv anti-p17 was intact and retained its antigen-binding affinity against HIV-1 p17. We demonstrated that the sequential simplex optimization method was a key for exertion of high yield with fewer experimental requirements for acquiring of large scale secretory protein production.

  5. Platelet-derived growth factor (PDGF-BB-mediated induction of monocyte chemoattractant protein 1 in human astrocytes: implications for HIV-associated neuroinflammation

    Directory of Open Access Journals (Sweden)

    Bethel-Brown Crystal

    2012-12-01

    Full Text Available Abstract Chemokine (C-C motif ligand 2, also known as monocyte chemoattractant protein 1 (MCP-1 is an important factor for the pathogenesis of HIV-associated neurocognitive disorders (HAND. The mechanisms of MCP-1-mediated neuropathogenesis, in part, revolve around its neuroinflammatory role and the recruitment of monocytes into the central nervous system (CNS via the disrupted blood-brain barrier (BBB. We have previously demonstrated that HIV-1/HIV-1 Tat upregulate platelet-derived growth factor (PDGF-BB, a known cerebrovascular permeant; subsequently, the present study was aimed at exploring the regulation of MCP-1 by PDGF-BB in astrocytes with implications in HAND. Specifically, the data herein demonstrate that exposure of human astrocytes to HIV-1 LAI elevated PDGF-B and MCP-1 levels. Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels. MCP-1 induction was regulated by activation of extracellular-signal-regulated kinase (ERK1/2, c-Jun N-terminal kinase (JNK and p38 mitogen-activated protein (MAP kinases and phosphatidylinositol 3-kinase (PI3K/Akt pathways and the downstream transcription factor, nuclear factor κB (NFκB. Chromatin immunoprecipitation (ChIP assays demonstrated increased binding of NFκB to the human MCP-1 promoter following PDGF-BB exposure. Conditioned media from PDGF-BB-treated astrocytes increased monocyte transmigration through human brain microvascular endothelial cells (HBMECs, an effect that was blocked by STI-571, a tyrosine kinase inhibitor (PDGF receptor (PDGF-R blocker. PDGF-BB-mediated release of MCP-1 was critical for increased permeability in an in vitro BBB model as evidenced by blocking antibody assays. Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND. These results suggest that astrocyte

  6. Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies.

    Science.gov (United States)

    McGuire, Andrew T; Hoot, Sam; Dreyer, Anita M; Lippy, Adriana; Stuart, Andrew; Cohen, Kristen W; Jardine, Joseph; Menis, Sergey; Scheid, Johannes F; West, Anthony P; Schief, William R; Stamatatos, Leonidas

    2013-04-08

    Broadly neutralizing antibodies (bnAbs) against HIV are believed to be a critical component of the protective responses elicited by an effective HIV vaccine. Neutralizing antibodies against the evolutionarily conserved CD4-binding site (CD4-BS) on the HIV envelope glycoprotein (Env) are capable of inhibiting infection of diverse HIV strains, and have been isolated from HIV-infected individuals. Despite the presence of anti-CD4-BS broadly neutralizing antibody (bnAb) epitopes on recombinant Env, Env immunization has so far failed to elicit such antibodies. Here, we show that Env immunogens fail to engage the germline-reverted forms of known bnAbs that target the CD4-BS. However, we found that the elimination of a conserved glycosylation site located in Loop D and two glycosylation sites located in variable region 5 of Env allows Env-binding to, and activation of, B cells expressing the germline-reverted BCRs of two potent broadly neutralizing antibodies, VRC01 and NIH45-46. Our results offer a possible explanation as to why Env immunogens have been ineffective in stimulating the production of such bNAbs. Importantly, they provide key information as to how such immunogens can be engineered to initiate the process of antibody-affinity maturation against one of the most conserved Env regions.

  7. Determining the frequency and mechanisms of HIV-1 and HIV-2 RNA copackaging by single-virion analysis

    DEFF Research Database (Denmark)

    Dilley, Kari A; Ni, Na; Nikolaitchik, Olga A;

    2011-01-01

    HIV-1 and HIV-2 are derived from two distinct primate viruses and share only limited sequence identity. Despite this, HIV-1 and HIV-2 Gag polyproteins can coassemble into the same particle and their genomes can undergo recombination, albeit at an extremely low frequency, implying that HIV-1 and HIV......-2 RNA can be copackaged into the same particle. To determine the frequency of HIV-1 and HIV-2 RNA copackaging and to dissect the mechanisms that allow the heterologous RNA copackaging, we directly visualized the RNA content of each particle by using RNA-binding proteins tagged with fluorescent...... proteins to label the viral genomes. We found that when HIV-1 and HIV-2 RNA are present in viral particles at similar ratios, ∼10% of the viral particles encapsidate both HIV-1 and HIV-2 RNAs. Furthermore, heterologous RNA copackaging can be promoted by mutating the 6-nucleotide (6-nt) dimer initiation...

  8. HIV Molecular Immunology 2015

    Energy Technology Data Exchange (ETDEWEB)

    Yusim, Karina [Los Alamos National Lab. (LANL), Los Alamos, NM (United States). Theoretical Division; Korber, Bette Tina [Los Alamos National Lab. (LANL), Los Alamos, NM (United States). Theoretical Division; Brander, Christian [Institucio Catalana de Recerca i Estudis Avancats (ICREA), Barcelona (Spain); Barouch, Dan [Beth Israel Deaconess Medical Center, Boston, MA (United States). Division of Vaccine Research; de Boer, Rob [Utrecht University, Utrecht (Netherlands). Faculty of Biology; Haynes, Barton F. [Duke Univ., Durham, NC (United States). Duke Human Vaccine Institute and Departments of Medicine, Surgery and Immunology; Koup, Richard [National Inst. of Health (NIH), Bethesda, MD (United States). Vaccine Research Center; Moore, John P. [Cornell Univ., Ithaca, NY (United States). Weill Medical College; Walker, Bruce D. [Ragon Institute, Cambridge, MA (United States); Watkins, David [Wisconsin Regional Primate Research Center, Madison, WI (United States)

    2016-04-05

    The scope and purpose of the HIV molecular immunology database: HIV Molecular Immunology is a companion volume to HIV Sequence Compendium. This publication, the 2015 edition, is the PDF version of the web-based HIV Immunology Database (http://www.hiv.lanl.gov/ content/immunology/). The web interface for this relational database has many search options, as well as interactive tools to help immunologists design reagents and interpret their results. In the HIV Immunology Database, HIV-specific B-cell and T-cell responses are summarized and annotated. Immunological responses are divided into three parts, CTL, T helper, and antibody. Within these parts, defined epitopes are organized by protein and binding sites within each protein, moving from left to right through the coding regions spanning the HIV genome. We include human responses to natural HIV infections, as well as vaccine studies in a range of animal models and human trials. Responses that are not specifically defined, such as responses to whole proteins or monoclonal antibody responses to discontinuous epitopes, are summarized at the end of each protein section. Studies describing general HIV responses to the virus, but not to any specific protein, are included at the end of each part. The annotation includes information such as cross-reactivity, escape mutations, antibody sequence, TCR usage, functional domains that overlap with an epitope, immune response associations with rates of progression and therapy, and how specific epitopes were experimentally defined. Basic information such as HLA specificities for T-cell epitopes, isotypes of monoclonal antibodies, and epitope sequences are included whenever possible. All studies that we can find that incorporate the use of a specific monoclonal antibody are included in the entry for that antibody. A single T-cell epitope can have multiple entries, generally one entry per study. Finally, maps of all defined linear epitopes relative to the HXB2 reference proteins

  9. Identification of a novel HIV-1 inhibitor targeting Vif-dependent degradation of human APOBEC3G protein.

    Science.gov (United States)

    Pery, Erez; Sheehy, Ann; Nebane, N Miranda; Brazier, Andrew Jay; Misra, Vikas; Rajendran, Kottampatty S; Buhrlage, Sara J; Mankowski, Marie K; Rasmussen, Lynn; White, E Lucile; Ptak, Roger G; Gabuzda, Dana

    2015-04-17

    APOBEC3G (A3G) is a cellular cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA and by deamination-independent mechanisms. HIV-1 Vif binds to A3G, resulting in its degradation via the 26 S proteasome. Therefore, this interaction represents a potential therapeutic target. To identify compounds that inhibit interaction between A3G and HIV-1 Vif in a high throughput format, we developed a homogeneous time-resolved fluorescence resonance energy transfer assay. A 307,520 compound library from the NIH Molecular Libraries Small Molecule Repository was screened. Secondary screens to evaluate dose-response performance and off-target effects, cell-based assays to identify compounds that attenuate Vif-dependent degradation of A3G, and assays testing antiviral activity in peripheral blood mononuclear cells and T cells were employed. One compound, N.41, showed potent antiviral activity in A3G(+) but not in A3G(-) T cells and had an IC50 as low as 8.4 μM and a TC50 of >100 μM when tested against HIV-1Ba-L replication in peripheral blood mononuclear cells. N.41 inhibited the Vif-A3G interaction and increased cellular A3G levels and incorporation of A3G into virions, thereby attenuating virus infectivity in a Vif-dependent manner. N.41 activity was also species- and Vif-dependent. Preliminary structure-activity relationship studies suggest that a hydroxyl moiety located at a phenylamino group is critical for N.41 anti-HIV activity and identified N.41 analogs with better potency (IC50 as low as 4.2 μM). These findings identify a new lead compound that attenuates HIV replication by liberating A3G from Vif regulation and increasing its innate antiviral activity.

  10. Cloning of an SNF2/SWI2-related protein that binds specifically to the SPH motifs of the SV40 enhancer and to the HIV-1 promoter.

    Science.gov (United States)

    Sheridan, P L; Schorpp, M; Voz, M L; Jones, K A

    1995-03-03

    We have isolated a human cDNA clone encoding HIP116, a protein that binds to the SPH repeats of the SV40 enhancer and to the TATA/inhibitor region of the human immunodeficiency virus (HIV)-1 promoter. The predicted HIP116 protein is related to the yeast SNF2/SWI2 transcription factor and to other members of this extended family and contains seven domains similar to those found in the vaccinia NTP1 ATPase. Interestingly, HIP116 also contains a C3HC4 zinc-binding motif (RING finger) interspersed between the ATPase motifs in an arrangement similar to that found in the yeast RAD5 and RAD16 proteins. The HIP116 amino terminus is unique among the members of this family, and houses a specific DNA-binding domain. Antiserum raised against HIP116 recognizes a 116-kDa nuclear protein in Western blots and specifically supershifts SV40 and HIV-1 protein-DNA complexes in gel shift experiments. The binding site for HIP116 on the SV40 enhancer directly overlaps the site for TEF-1, and like TEF-1, binding of HIP116 to the SV40 enhancer is destroyed by mutations that inhibit SPH enhancer activity in vivo. Purified fractions of HIP116 display strong ATPase activity that is preferentially stimulated by SPH DNA and can be inhibited specifically by antibodies to HIP116. These findings suggest that HIP116 might affect transcription, directly or indirectly, by acting as a DNA binding site-specific ATPase.

  11. A Response Surface Methodology Approach to Investigate the Effect of Sulfur Dioxide, pH, and Ethanol on DbCD and DbVPR Gene Expression and on the Volatile Phenol Production in Dekkera/Brettanomyces bruxellensis CBS2499

    Directory of Open Access Journals (Sweden)

    Federica Valdetara

    2017-09-01

    Full Text Available Dekkera/Brettanomyces bruxellensis, the main spoilage yeast in barrel-aged wine, metabolize hydroxycinnamic acids into off-flavors, namely ethylphenols. Recently, both the enzymes involved in this transformation, the cinnamate decarboxylase (DbCD and the vinylphenol reductase (DbVPR, have been identified. To counteract microbial proliferation in wine, sulfur dioxide (SO2 is used commonly to stabilize the final product, but limiting its use is advised to preserve human health and boost sustainability in winemaking. In the present study, the influence of SO2 was investigated in relation with pH and ethanol factors on the expression of DbCD and DbVPR genes and volatile phenol production in D. bruxellensis CBS2499 strain under different model wines throughout a response surface methodology (RSM. In order to ensure an exact quantification of DbCD and DbVPR expression, an appropriate housekeeping gene was sought among DbPDC, DbALD, DbEF, DbACT, and DbTUB genes by GeNorm and Normfinder algorithms. The latter gene showed the highest expression stability and it was chosen as the reference housekeeping gene in qPCR assays. Even though SO2 could not be commented as main factor because of its statistical irrelevance on the response of DbCD gene, linear interactions with pH and ethanol concurred to define a significant effect (p < 0.05 on its expression. The DbCD gene was generally downregulated respect to a permissive growth condition (0 mg/L mol. SO2, pH 4.5 and 5% v/v ethanol; the combination of the factor levels that maximizes its expression (0.83-fold change was calculated at 0.25 mg/L mol. SO2, pH 4.5 and 12.5% (v/v ethanol. On the contrary, DbVPR expression was not influenced by main factors or by their interactions; however, its expression is maximized (1.80-fold change at the same conditions calculated for DbCD gene. While no linear interaction between factors influenced the off-flavor synthesis, ethanol and pH produced a significant effect as

  12. Zirconium phosphatidylcholine-based nanocapsules as an in vivo degradable drug delivery system of MAP30, a momordica anti-HIV protein.

    Science.gov (United States)

    Caizhen, Guo; Yan, Gao; Ronron, Chang; Lirong, Yang; Panpan, Chu; Xuemei, Hu; Yuanbiao, Qiao; Qingshan, Li

    2015-04-10

    An essential in vivo drug delivery system of a momordica anti-HIV protein, MAP30, was developed through encapsulating in chemically synthesized matrices of zirconium egg- and soy-phosphatidylcholines, abbreviated to Zr/EPC and Zr/SPC, respectively. Matrices were characterized by transmission electron microscopy and powder X-ray diffractometry studies. Zr/EPC granule at an approximate diameter of 69.43±7.78 nm was a less efficient encapsulator than the granule of Zr/SPC. Interlayer spacing of the matrices encapsulating MAP30 increased from 8.8 and 9.7 Å to 7.4 and 7.9 nm, respectively. In vivo kinetics on degradation and protein release was performed by analyzing the serum sampling of intravenously injected SPF chickens. The first order and biphasic variations were obtained for in vivo kinetics using equilibrium dialysis. Antimicrobial and anti-HIV assays yielded greatly decreased MIC50 and EC50 values of nanoformulated MAP30. An acute toxicity of MAP30 encapsulated in Zr/EPC occurred at a single intravenous dose above 14.24 mg/kg bw in NIH/KM/ICR mice. The folding of MAP30 from Zr/EPC sustained in vivo chickens for more than 8 days in high performance liquid chromatography assays. These matrices could protect MAP30 efficiently with strong structure retention, lowered toxicity and prolonged in vivo life.

  13. Mutations of Conserved Residues in the Major Homology Region Arrest Assembling HIV-1 Gag as a Membrane-Targeted Intermediate Containing Genomic RNA and Cellular Proteins.

    Science.gov (United States)

    Tanaka, Motoko; Robinson, Bridget A; Chutiraka, Kasana; Geary, Clair D; Reed, Jonathan C; Lingappa, Jaisri R

    2015-12-09

    The major homology region (MHR) is a highly conserved motif that is found within the Gag protein of all orthoretroviruses and some retrotransposons. While it is widely accepted that the MHR is critical for assembly of HIV-1 and other retroviruses, how the MHR functions and why it is so highly conserved are not understood. Moreover, consensus is lacking on when HIV-1 MHR residues function during assembly. Here, we first addressed previous conflicting reports by confirming that MHR deletion, like conserved MHR residue substitution, leads to a dramatic reduction in particle production in human and nonhuman primate cells expressing HIV-1 proviruses. Next, we used biochemical analyses and immunoelectron microscopy to demonstrate that conserved residues in the MHR are required after assembling Gag has associated with genomic RNA, recruited critical host factors involved in assembly, and targeted to the plasma membrane. The exact point of inhibition at the plasma membrane differed depending on the specific mutation, with one MHR mutant arrested as a membrane-associated intermediate that is stable upon high-salt treatment and other MHR mutants arrested as labile, membrane-associated intermediates. Finally, we observed the same assembly-defective phenotypes when the MHR deletion or conserved MHR residue substitutions were engineered into Gag from a subtype B, lab-adapted provirus or Gag from a subtype C primary isolate that was codon optimized. Together, our data support a model in which MHR residues act just after membrane targeting, with some MHR residues promoting stability and another promoting multimerization of the membrane-targeted assembling Gag oligomer. The retroviral Gag protein exhibits extensive amino acid sequence variation overall; however, one region of Gag, termed the major homology region, is conserved among all retroviruses and even some yeast retrotransposons, although the reason for this conservation remains poorly understood. Highly conserved residues

  14. Transactivation and signaling functions of Tat are not correlated: biological and immunological characterization of HIV-1 subtype-C Tat protein

    Science.gov (United States)

    Siddappa, Nagadenahalli Byrareddy; Venkatramanan, Mohanram; Venkatesh, Prasanna; Janki, Mohanbabu Vijayamma; Jayasuryan, Narayana; Desai, Anita; Ravi, Vasanthapuram; Ranga, Udaykumar

    2006-01-01

    Background Of the diverse subtypes of Human Immunodeficiency Virus Type-1 (HIV-1), subtype-C strains cause a large majority of infections worldwide. The reasons for the global dominance of HIV-1 subtype-C infections are not completely understood. Tat, being critical for viral infectivity and pathogenesis, may differentially modulate pathogenic properties of the viral subtypes. Biochemical studies on Tat are hampered by the limitations of the current purification protocols. Tat purified using standard protocols often is competent for transactivation activity but defective for a variety of other biological functions. Keeping this limitation in view, we developed an efficient protein purification strategy for Tat. Results Tat proteins obtained using the novel strategy described here were free of contaminants and retained biological functions as evaluated in a range of assays including the induction of cytokines, upregulation of chemokine coreceptor, transactivation of the viral promoter and rescue of a Tat-defective virus. Given the highly unstable nature of Tat, we evaluated the effect of the storage conditions on the biological function of Tat following purification. Tat stored in a lyophilized form retained complete biological activity regardless of the storage temperature. To understand if variations in the primary structure of Tat could influence the secondary structure of the protein and consequently its biological functions, we determined the CD spectra of subtype-C and -B Tat proteins. We demonstrate that subtype-C Tat may have a relatively higher ordered structure and be less flexible than subtype-B Tat. We show that subtype-C Tat as a protein, but not as a DNA expression vector, was consistently inferior to subtype-B Tat in a variety of biological assays. Furthermore, using ELISA, we evaluated the anti-Tat antibody titers in a large number of primary clinical samples (n = 200) collected from all four southern Indian states. Our analysis of the Indian

  15. HIV-1 p17 matrix protein interacts with heparan sulfate side chain of CD44v3, syndecan-2, and syndecan-4 proteoglycans expressed on human activated CD4+ T cells affecting tumor necrosis factor alpha and interleukin 2 production.

    Science.gov (United States)

    De Francesco, Maria A; Baronio, Manuela; Poiesi, Claudio

    2011-06-01

    HIV-1 p17 contains C- and N-terminal sequences with positively charged residues and a consensus cluster for heparin binding. We have previously demonstrated by affinity chromatography that HIV-1 p17 binds strongly to heparin-agarose at physiological pH and to human activated CD4(+) T cells. In this study we demonstrated that the viral protein binds to heparan sulfate side chains of syndecan-2, syndecan-4, and CD44v3 purified from HeLa cells and that these heparan sulfate proteoglycans (HSPGs) co-localize with HIV-1 p17 on activated human CD4(+) T cells by confocal fluorescence analysis. Moreover, we observed a stimulatory or inhibitory activity when CD4(+) T cells were activated with mitogens together with nanomolar or micromolar concentrations of the matrix protein.

  16. Teaching Foundational Topics and Scientific Skills in Biochemistry within the Conceptual Framework of HIV Protease

    Science.gov (United States)

    Johnson, R. Jeremy

    2014-01-01

    HIV protease has served as a model protein for understanding protein structure, enzyme kinetics, structure-based drug design, and protein evolution. Inhibitors of HIV protease are also an essential part of effective HIV/AIDS treatment and have provided great societal benefits. The broad applications for HIV protease and its inhibitors make it a…

  17. Structure of the HIV-1 Full-Length Capsid Protein in a Conformationally Trapped Unassembled State Induced by Small-Molecule Binding

    Energy Technology Data Exchange (ETDEWEB)

    Du, Shoucheng; Betts, Laurie; Yang, Ruifeng; Shi, Haibin; Concel, Jason; Ahn, Jinwoo; Aiken, Christopher; Zhang, Peijun; Yeh, Joanne I. (Pitt); (Vanderbilt); (UNC)

    2012-11-26

    The capsid (CA) protein plays crucial roles in HIV infection and replication, essential to viral maturation. The absence of high-resolution structural data on unassembled CA hinders the development of antivirals effective in inhibiting assembly. Unlike enzymes that have targetable, functional substrate-binding sites, the CA does not have a known site that affects catalytic or other innate activity, which can be more readily targeted in drug development efforts. We report the crystal structure of the HIV-1 CA, revealing the domain organization in the context of the wild-type full-length (FL) unassembled CA. The FL CA adopts an antiparallel dimer configuration, exhibiting a domain organization sterically incompatible with capsid assembly. A small compound, generated in situ during crystallization, is bound tightly at a hinge site ('H site'), indicating that binding at this interdomain region stabilizes the ADP conformation. Electron microscopy studies on nascent crystals reveal both dimeric and hexameric lattices coexisting within a single condition, in agreement with the interconvertibility of oligomeric forms and supporting the feasibility of promoting assembly-incompetent dimeric states. Solution characterization in the presence of the H-site ligand shows predominantly unassembled dimeric CA, even under conditions that promote assembly. Our structure elucidation of the HIV-1 FL CA and characterization of a potential allosteric binding site provides three-dimensional views of an assembly-defective conformation, a state targeted in, and thus directly relevant to, inhibitor development. Based on our findings, we propose an unprecedented means of preventing CA assembly, by 'conformationally trapping' CA in assembly-incompetent conformational states induced by H-site binding.

  18. Women and HIV

    Science.gov (United States)

    ... Consumer Information by Audience For Women Women and HIV Share Tweet Linkedin Pin it More sharing options ... HIV? What should pregnant women know about HIV? HIV Quick Facts What is HIV? HIV is the ...

  19. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: Novel insights into the regulation of Rev nuclear import.

    LENUS (Irish Health Repository)

    Gu, Lili

    2011-03-14

    Abstract Background The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s) predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised. Results In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein) as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion. Conclusions Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.

  20. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: Novel insights into the regulation of Rev nuclear import

    Directory of Open Access Journals (Sweden)

    Sheehy Noreen

    2011-03-01

    Full Text Available Abstract Background The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised. Results In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion. Conclusions Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.

  1. Intestinal Damage and Inflammatory Biomarkers in Human Immunodeficiency Virus (HIV)-Exposed and HIV-Infected Zimbabwean Infants.

    Science.gov (United States)

    Prendergast, Andrew J; Chasekwa, Bernard; Rukobo, Sandra; Govha, Margaret; Mutasa, Kuda; Ntozini, Robert; Humphrey, Jean H

    2017-09-15

    Disease progression is rapid in human immunodeficiency virus (HIV)-infected infants. Whether intestinal damage and inflammation underlie mortality is unknown. We measured plasma intestinal fatty acid binding protein (I-FABP), soluble CD14 (sCD14), interleukin 6 (IL-6), and C-reactive protein (CRP) at 6 weeks and 6 months of age in 272 HIV-infected infants who either died (cases) or survived (controls), and in 194 HIV-exposed uninfected (HEU) and 197 HIV-unexposed infants. We estimated multivariable odds ratios for mortality and postnatal HIV transmission for each biomarker using logistic regression. At 6 weeks, HIV-infected infants had higher sCD14 and IL-6 but lower I-FABP than HIV-exposed and HIV-unexposed infants (P HIV-exposed than HIV-unexposed infants (P = .02). At 6 months, HIV-infected infants had highest sCD14, IL-6, and CRP concentrations (P HIV-exposed vs HIV-unexposed infants (P = .04). No biomarker was associated with mortality in HIV-infected infants, or with odds of breast-milk HIV transmission in HIV-exposed infants. HIV-infected infants have elevated inflammatory markers by 6 weeks of age, which increase over time. In contrast to adults and older children, inflammatory biomarkers were not associated with mortality. HEU infants have higher inflammation than HIV-unexposed infants until at least 6 months, which may contribute to poor health outcomes.

  2. Protein kinase C-delta regulates HIV-1 replication at an early post-entry step in macrophages

    Directory of Open Access Journals (Sweden)

    Contreras Xavier

    2012-05-01

    Full Text Available Abstract Background Macrophages, which are CD4 and CCR5 positive, can sustain HIV-1 replication for long periods of time. Thus, these cells play critical roles in the transmission, dissemination and persistence of viral infection. Of note, current antiviral therapies do not target macrophages efficiently. Previously, it was demonstrated that interactions between CCR5 and gp120 stimulate PKC. However, the PKC isozymes involved were not identified. Results In this study, we identified PKC-delta as a major cellular cofactor for HIV-1 replication in macrophages. Indeed, PKC-delta was stimulated following the interaction between the virus and its target cell. Moreover, inhibition of PKC-delta blocked the replication of R5-tropic viruses in primary human macrophages. However, this inhibition did not have significant effects on receptor and co-receptor expression or fusion. Additionally, it did not affect the formation of the early reverse transcription product containing R/U5 sequences, but did inhibit the synthesis of subsequent cDNAs. Importantly, the inhibition of PKC-delta altered the redistribution of actin, a cellular cofactor whose requirement for the completion of reverse transcription was previously established. It also prevented the association of the reverse transcription complex with the cytoskeleton. Conclusion This work highlights the importance of PKC-delta during early steps of the replicative cycle of HIV-1 in human macrophages.

  3. Evidence of differential HLA class I-mediated viral evolution in functional and accessory/regulatory genes of HIV-1.

    Directory of Open Access Journals (Sweden)

    Zabrina L Brumme

    2007-07-01

    Full Text Available Despite the formidable mutational capacity and sequence diversity of HIV-1, evidence suggests that viral evolution in response to specific selective pressures follows generally predictable mutational pathways. Population-based analyses of clinically derived HIV sequences may be used to identify immune escape mutations in viral genes; however, prior attempts to identify such mutations have been complicated by the inability to discriminate active immune selection from virus founder effects. Furthermore, the association between mutations arising under in vivo immune selection and disease progression for highly variable pathogens such as HIV-1 remains incompletely understood. We applied a viral lineage-corrected analytical method to investigate HLA class I-associated sequence imprinting in HIV protease, reverse transcriptase (RT, Vpr, and Nef in a large cohort of chronically infected, antiretrovirally naïve individuals. A total of 478 unique HLA-associated polymorphisms were observed and organized into a series of "escape maps," which identify known and putative cytotoxic T lymphocyte (CTL epitopes under selection pressure in vivo. Our data indicate that pathways to immune escape are predictable based on host HLA class I profile, and that epitope anchor residues are not the preferred sites of CTL escape. Results reveal differential contributions of immune imprinting to viral gene diversity, with Nef exhibiting far greater evidence for HLA class I-mediated selection compared to other genes. Moreover, these data reveal a significant, dose-dependent inverse correlation between HLA-associated polymorphisms and HIV disease stage as estimated by CD4(+ T cell count. Identification of specific sites and patterns of HLA-associated polymorphisms across HIV protease, RT, Vpr, and Nef illuminates regions of the genes encoding these products under active immune selection pressure in vivo. The high density of HLA-associated polymorphisms in Nef compared to other

  4. Comparison of Interferon gamma inducible protein-10 and Interferon gamma based QuantiFERON TB Gold assays with tuberculin skin test in HIV infected subjects

    Science.gov (United States)

    Basirudeen, S; Rajasekaran, S; Alamelu, R

    2011-01-01

    We aimed to compare the positivity of the QuantiFERON TB gold in-tube (QFT-IT antigens) specific Interferon gamma (IFN-γ/QFT-IT) and IFN-γ nducible protein-10 (IP-10/QFT-IT) assays with tuberculin skin test (TST) among human immunodeficiency virus (HIV) infected individuals in a TB endemic setting. A total of 180 HIV infected subjects, with no evidence of active TB were recruited. IFN-γ nd IP-10 levels specific to QFT-IT antigens were measured in plasma from QFT-IT tubes. The overall positivity of TST at 5mm cut-off point (19%) was significantly lower when compared to IFN-γ/QFT-IT (38%) and IP-10/QFT-IT (45%) assays. The positivity of IP-10/QFT-IT was significantly higher than IFN-γ/QFT-IT (p=0.038). Indeterminate results for IFN-γ/QFT-IT and IP-10/QFT-IT were more frequent in subjects with CD4 count 1