Benzodiazepines Drive Alteration of Chromatin at the Integrated HIV-1 LTR

Antiretroviral therapy (ART) lowers human immunodeficiency virus type 1 (HIV-1) viral load to undetectable levels, but does not eliminate the latent reservoir. One of the factors controlling the latent reservoir is transcriptional silencing of the integrated HIV-1 long terminal repeat (LTR). The molecular mechanisms that control HIV-1 transcription are not completely understood. We have previously shown that RUNX1, a host transcription factor, may play a role in the establishment and maintenance of HIV-1 latency. Prior work has demonstrated that inhibition of RUNX1 by the benzodiazepine (BDZ) Ro5-3335 synergizes with suberanilohydroxamic acid (SAHA) to activate HIV-1 transcription. In this current work, we examine the effect of RUNX1 inhibition on the chromatin state of the integrated HIV-1 LTR. Using chromatin immunoprecipitation (ChIP), we found that Ro5-3335 significantly increased the occupancy of STAT5 at the HIV-1 LTR. We also screened other BDZs for their ability to regulate HIV-1 transcription and demonstrate their ability to increase transcription and alter chromatin at the LTR without negatively affecting Tat activity. These findings shed further light on the mechanism by which RUNX proteins control HIV-1 transcription and suggest that BDZ compounds might be useful in activating HIV-1 transcription through STAT5 recruitment to the HIV-1 LTR.

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Interaction between the Human Immunodeficiency Virus Type 1 Gag Matrix Domain and Phosphatidylinositol-(4,5)-Bisphosphate Is Essential for Efficient Gag Membrane Binding

Journal of Virology ◽

10.1128/jvi.01614-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2405-2417 ◽

Cited By ~ 168

Author(s):

Vineela Chukkapalli ◽

Ian B. Hogue ◽

Vitaly Boyko ◽

Wei-Shau Hu ◽

Akira Ono

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Molecular Mechanisms ◽

Membrane Binding ◽

Particle Assembly ◽

Immunodeficiency Virus ◽

The Impact ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) particle assembly mediated by the viral structural protein Gag occurs predominantly on the plasma membrane (PM). Although it is known that the matrix (MA) domain of Gag plays a major role in PM localization, molecular mechanisms that determine the location of assembly remain to be elucidated. We observed previously that overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV) that depletes PM phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] impairs virus particle production and redirects processed Gag to intracellular compartments. In this study, we examined the impact of PI(4,5)P2 depletion on the subcellular localization of the entire Gag population using Gag-fluorescent protein chimeras. Upon 5ptaseIV overexpression, in addition to perinuclear localization, Gag also showed a hazy cytosolic signal, suggesting that PI(4,5)P2 depletion impairs Gag membrane binding. Indeed, Gag was less membrane bound in PI(4,5)P2-depleted cells, as assessed by biochemical analysis. These observations are consistent with the hypothesis that Gag interacts with PI(4,5)P2. To examine a putative Gag interaction with PI(4,5)P2, we developed an in vitro binding assay using full-length myristoylated Gag and liposome-associated PI(4,5)P2. Using this assay, we observed that PI(4,5)P2 significantly enhances liposome binding of wild-type Gag. In contrast, a Gag derivative lacking MA did not require PI(4,5)P2 for efficient liposome binding. To analyze the involvement of MA in PI(4,5)P2 binding further, we examined MA basic amino acid substitution mutants. These mutants, previously shown to localize in perinuclear compartments, bound PI(4,5)P2-containing liposomes weakly. Altogether, these results indicate that HIV-1 Gag binds PI(4,5)P2 on the membrane and that the MA basic domain mediates this interaction.

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Characterization of Chemokine Receptor Utilization of Viruses in the Latent Reservoir for Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.74.17.7824-7833.2000 ◽

2000 ◽

Vol 74 (17) ◽

pp. 7824-7833 ◽

Cited By ~ 115

Author(s):

Theodore Pierson ◽

Trevor L. Hoffman ◽

Joel Blankson ◽

Diana Finzi ◽

Karen Chadwick ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Chemokine Receptor ◽

Latent Reservoir ◽

Immunodeficiency Virus ◽

Latently Infected ◽

Hiv 1

ABSTRACT Latently infected resting CD4+ T cells provide a long-term reservoir for human immunodeficiency virus type 1 (HIV-1) and are likely to represent the major barrier to virus eradication in patients on combination antiretroviral therapy. The mechanisms by which viruses enter the latent reservoir and the nature of the chemokine receptors involved have not been determined. To evaluate the phenotype of the virus in this compartment with respect to chemokine receptor utilization, full-length HIV-1 env genes were cloned from latently infected cells and assayed functionally. We demonstrate that the majority of the viruses in the latent reservoir utilize CCR5 during entry, although utilization of several other receptors, including CXCR4, was observed. No alternative coreceptors were shown to be involved in a systematic fashion. Although R5 viruses are present in the latent reservoir, CCR5 was not expressed at high levels on resting CD4+ T cells. To understand the mechanism by which R5 viruses enter latent reservoir, the ability of an R5 virus, HIV-1 Ba-L, to infect highly purified resting CD4+ T lymphocytes from uninfected donors was evaluated. Entry of Ba-L could be observed when virus was applied at a multiplicity approaching 1. However, infection was limited to a subset of cells expressing low levels of CCR5 and markers of immunologic memory. Naive cells could not be infected by an R5 virus even when challenged with a large inoculum. Direct cell fractionation studies showed that latent virus is present predominantly in resting memory cells but also at lower levels in resting naive cells. Taken together, these findings provide support for the hypothesis that the direct infection of naive T cells is not the major mechanism by which the latent infection of resting T cells is established.

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Persistence of Wild-Type Virus and Lack of Temporal Structure in the Latent Reservoir for Human Immunodeficiency Virus Type 1 in Pediatric Patients with Extensive Antiretroviral Exposure

Journal of Virology ◽

10.1128/jvi.76.18.9481-9492.2002 ◽

2002 ◽

Vol 76 (18) ◽

pp. 9481-9492 ◽

Cited By ~ 89

Author(s):

Christian T. Ruff ◽

Stuart C. Ray ◽

Patricia Kwon ◽

Rebekah Zinn ◽

Amanda Pendleton ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Temporal Structure ◽

Latent Reservoir ◽

Drug Resistant ◽

Wild Type ◽

Time Points ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Although highly active antiretroviral therapy (HAART) for human immunodeficiency virus type 1 (HIV-1) infection can reduce levels of HIV-1 RNA in plasma to below the limit of detection, replication-competent forms of the virus persist in all infected individuals. One form of persistence involves a stable reservoir of latent but potentially infectious virus that resides in resting memory CD4+ T cells. The mechanisms involved in maintaining this latent reservoir are incompletely understood. In the present study, we examined the dynamic characteristics of this reservoir in a cohort of children who developed drug-resistant HIV-1 as a result of extensive exposure to inadequately suppressive one- or two-drug regimens prior to the advent of HAART. We have previously shown that drug-resistant viruses selected by nonsuppressive pre-HAART regimens can enter and persist in this reservoir. We have extended these findings here by demonstrating that archival wild-type HIV-1 persists in this reservoir despite the fact that in these patients drug-resistant mutants have been favored by the selective conditions for many years. Phylogenetic analysis of replication-competent viruses persisting in resting CD4+ T cells revealed a striking lack of temporal structure in the sense that isolates obtained at later time points did not show greater sequence divergence than isolates from earlier time points. The persistence of drug-sensitive virus and the lack of temporal structure in the latent reservoir provide genetic evidence for the idea that HIV-1 can persist in a latent form free of selective pressure from antiretroviral drugs in long-lived resting memory CD4+ T cells. Although there may be other mechanisms for viral persistence, this stable pool of latently infected cells is of significant concern because of its potential to serve as a lasting source of replication-competent viruses, including the infecting wild-type form and all drug-resistant variants that have arisen subsequently.

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c-Myc and Sp1 Contribute to Proviral Latency by Recruiting Histone Deacetylase 1 to the Human Immunodeficiency Virus Type 1 Promoter

Journal of Virology ◽

10.1128/jvi.01208-07 ◽

2007 ◽

Vol 81 (20) ◽

pp. 10914-10923 ◽

Cited By ~ 151

Author(s):

Guochun Jiang ◽

Amy Espeseth ◽

Daria J. Hazuda ◽

David M. Margolis

Keyword(s):

Human Immunodeficiency Virus ◽

Rna Polymerase Ii ◽

Histone Deacetylase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Molecular Mechanisms ◽

Histone Deacetylase 1 ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Histone deacetylase (HDAC) inhibitors such as valproic acid (VPA) induce the expression of quiescent proviral human immunodeficiency virus type 1 (HIV-1) and may deplete proviral infection in vivo. To uncover novel molecular mechanisms that maintain HIV latency, we sought cellular mRNAs whose expression was diminished in resting CD4+ T cells of HIV-1-infected patients exposed to VPA. c-Myc was prominent among genes markedly downregulated upon exposure to VPA. c-Myc expression repressed HIV-1 expression in chronically infected cell lines. Chromatin immunoprecipitation (ChIP) assays revealed that c-Myc and HDAC1 are coordinately resident at the HIV-1 long terminal repeat (LTR) promoter and absent from the promoter after VPA treatment in concert with histone acetylation, RNA polymerase II recruitment, and LTR expression. Sequential ChIP assays demonstrated that c-Myc, Sp1, and HDAC1 coexist in the same DNA-protein complex at the HIV promoter. Short hairpin RNA inhibition of c-Myc reduces both c-Myc and HDAC1 occupancy, blocks c-Myc repression of Tat activation, and increases LTR expression. These results expand the understanding of mechanisms that recruit HDAC and maintain the latency of HIV-1, suggesting novel therapeutic approaches against latent proviral HIV infection.

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A Novel Assay Allows Genotyping of the Latent Reservoir for Human Immunodeficiency Virus Type 1 in the Resting CD4+ T Cells of Viremic Patients

Journal of Virology ◽

10.1128/jvi.79.8.5185-5202.2005 ◽

2005 ◽

Vol 79 (8) ◽

pp. 5185-5202 ◽

Cited By ~ 25

Author(s):

Daphne Monie ◽

Rachel P. Simmons ◽

Richard E. Nettles ◽

Tara L. Kieffer ◽

Yan Zhou ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Latent Reservoir ◽

Drug Resistant ◽

Plasma Virus ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT A latent reservoir for human immunodeficiency virus type 1 (HIV-1) consisting of integrated provirus in resting memory CD4+ T cells prevents viral eradication in patients on highly active antiretroviral therapy (HAART). It is difficult to analyze the nature and dynamics of this reservoir in untreated patients and in patients failing therapy, because it is obscured by an excess of unintegrated viral DNA that constitutes the majority of viral species in resting CD4+ T cells from viremic patients. Therefore, we developed a novel culture assay that stimulates virus production from latent, integrated HIV-1 in resting CD4+ T cells in the presence of antiretroviral drugs that prevent the replication of unintegrated virus. Following activation, resting CD4+ T cells with integrated HIV-1 DNA produced virus particles for several days, with peak production at day 5. Using this assay, HIV-1 pol sequences from the resting CD4+ T cells of viremic patients were found to be genetically distinct from contemporaneous plasma virus. Despite the predominance of a relatively homogeneous population of drug-resistant viruses in the plasma of patients failing HAART, resting CD4+ T cells harbored a diverse array of wild-type and archival drug-resistant viruses that were less fit than plasma virus in the context of current therapy. These results provide the first direct evidence that resting CD4+ T cells serve as a stable reservoir for HIV-1 even in the setting of high levels of viremia. The ability to analyze archival species in viremic patients may have clinical utility in detecting drug-resistant variants not present in the plasma.

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TRIM28 promotes HIV-1 latency by SUMOylating CDK9 and inhibiting P-TEFb

eLife ◽

10.7554/elife.42426 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 16

Author(s):

Xiancai Ma ◽

Tao Yang ◽

Yuewen Luo ◽

Liyang Wu ◽

Yawen Jiang ◽

...

Keyword(s):

Molecular Mechanisms ◽

Kinase Activity ◽

Latent Reservoir ◽

Functional Cure ◽

Site Specific ◽

Cyclin T1 ◽

Immunodeficiency Virus ◽

Cdk9 Kinase Activity ◽

Hiv 1

Comprehensively elucidating the molecular mechanisms of human immunodeficiency virus type 1 (HIV-1) latency is a priority to achieve a functional cure. As current 'shock' agents failed to efficiently reactivate the latent reservoir, it is important to discover new targets for developing more efficient latency-reversing agents (LRAs). Here, we found that TRIM28 potently suppresses HIV-1 expression by utilizing both SUMO E3 ligase activity and epigenetic adaptor function. Through global site-specific SUMO-MS study and serial SUMOylation assays, we identified that P-TEFb catalytic subunit CDK9 is significantly SUMOylated by TRIM28 with SUMO4. The Lys44, Lys56 and Lys68 residues on CDK9 are SUMOylated by TRIM28, which inhibits CDK9 kinase activity or prevents P-TEFb assembly by directly blocking the interaction between CDK9 and Cyclin T1, subsequently inhibits viral transcription and contributes to HIV-1 latency. The manipulation of TRIM28 and its consequent SUMOylation pathway could be the target for developing LRAs.

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The role of viral coreceptors and enhanced macrophage tropism in human immunodeficiency virus type 1 disease progression

Sexual Health ◽

10.1071/sh03006 ◽

2004 ◽

Vol 1 (1) ◽

pp. 23 ◽

Cited By ~ 14

Author(s):

Paul R. Gorry ◽

Jasminka Sterjovski ◽

Melissa Churchill ◽

Kristie Witlox ◽

Lachlan Gray ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Disease Progression ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Molecular Mechanisms ◽

Coreceptor Usage ◽

Immunodeficiency Virus ◽

The Impact ◽

Hiv 1

Despite numerous studies on the impact of viral diversity, human immunodeficiency virus type 1 (HIV-1)-specific immune responses and host factors on disease progression, we still do not have a firm understanding of the long-term pathogenesis of HIV-1 infection. Rapid depletion of CD4+ T-lymphocytes has been associated with a switch in viral coreceptor usage from CCR5 to CXCR4 in ~40 to 50% of infected individuals. However, the majority of infected individuals who progress to AIDS harbour only CCR5-dependent (R5) viral strains. The progression HIV-1 disease is associated with an enhanced tropism of R5 viral strains for monocyte/macrophage lineage cells (enhanced M-tropism). However, the underlying molecular mechanisms contributing to enhanced M-tropism by R5 HIV-1 strains, and how HIV-1 variants with enhanced M-tropism cause CD4+ T-cell depletion in vivo are unknown. This review examines the relationship between viral coreceptor usage, M-tropism, and pathogenicity of HIV-1. We highlight evidence supporting the hypothesis that enhanced M-tropism of R5 HIV-1 results from adaptive viral evolution, resulting in HIV-1 variants that have increased ability to utilise relatively low levels of CCR5 expressed on macrophages, by way of increased CCR5 affinity. The evidence also suggests that these late-emerging, R5 viral strains have reduced sensitivity to entry inhibitors, and increased ability to cause CD4+ T-lymphocyte loss. These variants are likely to impact HIV-1 disease progression, especially in patients who persistently harbour only R5 viral strains.

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The Pathogenicity of Human Immunodeficiency Virus (HIV) Type 1 Nef in CD4C/HIV Transgenic Mice Is Abolished by Mutation of Its SH3-Binding Domain, and Disease Development Is Delayed in the Absence of Hck

Journal of Virology ◽

10.1128/jvi.75.19.9378-9392.2001 ◽

2001 ◽

Vol 75 (19) ◽

pp. 9378-9392 ◽

Cited By ~ 88

Author(s):

Zaher Hanna ◽

Xiaoduan Weng ◽

Denis G. Kay ◽

Johanne Poudrier ◽

Clifford Lowell ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Molecular Mechanisms ◽

Surface Expression ◽

Binding Domain ◽

Cell Surface Expression ◽

Disease Development ◽

Pathogenic Potential ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Nef protein is an important determinant of AIDS pathogenesis. We have previously reported that HIV-1 Nef is responsible for the induction of a severe AIDS-like disease in CD4C/HIV transgenic (Tg) mice. To understand the molecular mechanisms of this Nef-induced disease, we generated Tg mice expressing a mutated Nef protein in which the SH3 ligand-binding domain (P72XXP75XXP78) was mutated to A72XXA75XXQ78. This mutation completely abolished the pathogenic potential of Nef, although a partial downregulation of the CD4 cell surface expression was still observed in these Tg mice. We also studied whether Hck, one of the effectors previously found to bind to this PXXP motif of Nef, was involved in disease development. Breeding of Tg mice expressing wild-type Nef on an hck −/− (knockout) background did not abolish any of the pathological phenotypes. However, the latency of disease development was prolonged. These data indicate that an intact PXXP domain is essential for inducing an AIDS-like disease in CD4C/HIV Tg mice and suggest that interaction of a cellular effector(s) with this domain is required for the induction of this multiorgan disease. Our findings indicate that Hck is an important, but not an essential, effector of Nef and suggest that another factor(s), yet to be identified, may be more critical for disease development.

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