Maintenance Of HIV-1 Latency Research Articles

New transcription regulatory mechanisms of latent HIV LTR

Despite the effectiveness of antiretroviral medication, the HIV virus persists in resting memory T cells of infected patients in a latent state, providing the main impediment to eradication of the virus. We are interested in identifying the molecular mechanism responsible for the establishment and maintenance of HIV latency and its re-activation. We recently used a cell system reflecting HIV latency in my lab to determine the high resolution nucleosomal landscape of the latent HIV LTR and examine its dynamic changes upon re-activation (Rafati et al., Nov 2011 PLoS Biology). We combined mathematical predictions of nucleosome positioning with a combinatorial biochemical approach based on formaldehyde crosslinking of latent and activated HIV infected cells (using FAIRE, ChIP and high resolution MNase nucleosomal mapping) to define LTR nucleosome positioning and regulation during active and latent HIV infections. We found that BAF, an ATP-dependent chromatin remodelling complex generates a chromatin structure at the LTR that is energetically unfavorable to its intrinsic histone-DNA sequence preferences. Specifically, we find that BAF positions a repressive nucleosome immediately downstream of the HIV transcription start site, abrogating transcription, and in this way contributes to the establishment and maintenance of HIV latency. Our data describe a novel molecular mechanism for the establishment and maintenance of HIV latency, and we identify the catalytic subunit of BAF, the enzyme BRG1, as a putative molecular target to deplete the latent reservoir in infected patients. We will also present preliminary data addressing the role of a novel signalling pathway in de-repression of latent HIV, and the effect of small molecules and ligands, which activate this pathway to study reactivation of latent HIV. We anticipate these experiments will further our understanding of HIV transcription regulation and identify both novel cofactors for targeting and molecules with potential to purge HIV latency.

Influence of Host Gene Transcription Level and Orientation on HIV-1 Latency in a Primary-Cell Model

Human immunodeficiency virus type 1 (HIV-1) establishes a latent reservoir in resting memory CD4(+) T cells. This latent reservoir is a major barrier to the eradication of HIV-1 in infected individuals and is not affected by highly active antiretroviral therapy (HAART). Reactivation of latent HIV-1 is a possible strategy for elimination of this reservoir. The mechanisms with which latency is maintained are unclear. In the analysis of the regulation of HIV-1 gene expression, it is important to consider the nature of HIV-1 integration sites. In this study, we analyzed the integration and transcription of latent HIV-1 in a primary CD4(+) T cell model of latency. The majority of integration sites in latently infected cells were in introns of transcription units. Serial analysis of gene expression (SAGE) demonstrated that more than 90% of those host genes harboring a latent integrated provirus were transcriptionally active, mostly at high levels. For latently infected cells, we observed a modest preference for integration in the same transcriptional orientation as the host gene (63.8% versus 36.2%). In contrast, this orientation preference was not observed in acutely infected or persistently infected cells. These results suggest that transcriptional interference may be one of the important factors in the establishment and maintenance of HIV-1 latency. Our findings suggest that disrupting the negative control of HIV-1 transcription by upstream host promoters could facilitate the reactivation of latent HIV-1 in some resting CD4(+) T cells.

Role of histone modification on transcriptional regulation and HIV-1 gene expression: possible mechanisms of periodontal diseases in AIDS progression

Although approximately 200 distinct cell types - including fibroblasts, neurons, and hematopoietic cells - possess the same DNA sequence, they have diverse functions in humans and exhibit considerably different gene expression patterns. It has become increasingly clear that epigenetic regulation plays an important role in gene expression. There are two major forms of epigenetic regulation: posttranslational modification of DNA-associated histone proteins in chromatin and methylation of DNA. These forms are regulated by distinct but coupled pathways. Notably, histone Lys acetylation by histone acetyltransferase and deacetylation by histone deacetylases play a crucial role in on-off regulation of gene expression. It is now understood that epigenetics plays an important role not only in the regulation of gene expression but also in the pathogenesis of a broad range of diseases such as cancer and microbial infections. We have determined that epigenetic regulation is involved in the establishment and maintenance of HIV-1 latency and in the reactivation of HIV-1 by periodontopathic bacteria. In this review, we focus on the effect of histone modification on transcriptional regulation and the contribution thereof to the regulation of HIV-1 gene expression during the lytic and latent stages of HIV-1 infection. Likewise, we discuss the mechanisms by which periodontal diseases may accelerate AIDS progression in infected individuals as a new systemic disease caused by periodontitis and describe potential therapeutic interventions based on epigenetic mechanisms.

Involvement of Histone H3 Lysine 9 (H3K9) Methyltransferase G9a in the Maintenance of HIV-1 Latency and Its Reactivation by BIX01294

Elucidating the mechanism of human immunodeficiency virus, type 1 (HIV-1) provirus transcriptional silencing in latently infected cells is crucial for understanding the pathophysiological process of HIV-1 infection. It is well established that hypoacetylation of histone proteins by histone deacetylases is involved in the maintenance of HIV-1 latency by repressing viral transcription. Although histone methylation is involved in the organization of chromatin domains and plays a central epigenetic role in gene expression, the role of histone methylation in the maintenance of HIV-1 latency has not been clarified. Here we present evidence that histone H3 Lys(9) (H3K9) methyltransferase G9a is responsible for transcriptional repression of HIV-1 by promoting repressive dimethylation at H3K9 and for the maintenance of viral latency. We observed that G9a significantly inhibited basal, as well as, the induced HIV-1 gene expression by tumor necrosis factor-alpha or Tat. Mutant G9a, however, lacking the SET domain responsible for the catalytic activity of histone methyltransferase, did not show such an effect. When G9a expression was knocked down by small interfering RNA, HIV-1 replication was augmented from cells transiently transfected with a full-length HIV-1 clone. Moreover, a specific inhibitor of G9a, BIX01294, could reactivate expression of HIV-1 from latently infected cells such as ACH-2 and OM10.1. Furthermore, chromatin immunoprecipitation assays revealed the presence of G9a and H3K9 dimethylation on nucleosome histones in the vicinity of the HIV-1 long terminal repeat promoter. These results suggest that G9a is responsible for the transcriptional quiescence of latent HIV-1 provirus and provide a molecular basis for understanding the mechanism by which HIV-1 latency is maintained.

Establishment and Maintenance of HIV Latency: Model Systems and Opportunities for Intervention

HAART has succeeded in reducing morbidity and mortality rates in patients infected with HIV. However, a small amount of replication-competent HIV can persist during HAART, allowing the virus to re-emerge if therapy is ceased. One significant source of this persistent virus is a pool of long-lived, latently infected CD4(+) T cells. This article outlines what is known about how this reservoir is established and maintained, and describes the model systems that have provided insights into the molecular mechanisms governing HIV latency. The therapeutic approaches for eliminating latent cells that have been attempted are also discussed, including how improvements in understanding of these persistent HIV reservoirs are being used to develop enhanced methods for their depletion.

Molecular control of HIV-1 postintegration latency: implications for the development of new therapeutic strategies

The persistence of HIV-1 latent reservoirs represents a major barrier to virus eradication in infected patients under HAART since interruption of the treatment inevitably leads to a rebound of plasma viremia. Latency establishes early after infection notably (but not only) in resting memory CD4+ T cells and involves numerous host and viral trans-acting proteins, as well as processes such as transcriptional interference, RNA silencing, epigenetic modifications and chromatin organization. In order to eliminate latent reservoirs, new strategies are envisaged and consist of reactivating HIV-1 transcription in latently-infected cells, while maintaining HAART in order to prevent de novo infection. The difficulty lies in the fact that a single residual latently-infected cell can in theory rekindle the infection. Here, we review our current understanding of the molecular mechanisms involved in the establishment and maintenance of HIV-1 latency and in the transcriptional reactivation from latency. We highlight the potential of new therapeutic strategies based on this understanding of latency. Combinations of various compounds used simultaneously allow for the targeting of transcriptional repression at multiple levels and can facilitate the escape from latency and the clearance of viral reservoirs. We describe the current advantages and limitations of immune T-cell activators, inducers of the NF-κB signaling pathway, and inhibitors of deacetylases and histone- and DNA- methyltransferases, used alone or in combinations. While a solution will not be achieved by tomorrow, the battle against HIV-1 latent reservoirs is well- underway.

HIV-1 regulation of latency in the monocyte-macrophage lineage and in CD4+ T lymphocytes

The introduction in 1996 of the HAART raised hopes for the eradication of HIV-1. Unfortunately, the discovery of latent HIV-1 reservoirs in CD4+ T cells and in the monocyte-macrophage lineage proved the optimism to be premature. The long-lived HIV-1 reservoirs constitute a major obstacle to the eradication of HIV-1. In this review, we focus on the establishment and maintenance of HIV-1 latency in the two major targets for HIV-1: the CD4+ T cells and the monocyte-macrophage lineage. Understanding the cell-type molecular mechanisms of establishment, maintenance, and reactivation of HIV-1 latency in these reservoirs is crucial for efficient therapeutic intervention. A complete viral eradication, the holy graal for clinicians, might be achieved by strategic interventions targeting latently and productively infected cells. We suggest that new approaches, such as the combination of different kinds of proviral activators, may help to reduce dramatically the size of latent HIV-1 reservoirs in patients on HAART.

Reactivation of Latent HIV-1 Infection by the Periodontopathic Bacterium Porphyromonas gingivalis Involves Histone Modification

Latently infected cells harbor the HIV-1 proviral DNA genome primarily integrated into heterochromatin, allowing the persistence of transcriptionally silent proviruses. Hypoacetylation of histone proteins by histone deacetylases (HDAC) is involved in the maintenance of HIV-1 latency by repressing viral transcription. In addition, periodontal diseases, caused by polymicrobial subgingival bacteria including Porphyromonas gingivalis, are among the most prevalent infections of mankind. Here we demonstrate the effects of P. gingivalis on HIV-1 replication. This activity could be ascribable to the bacterial culture supernatant but not to other bacterial components such as fimbriae or LPS. We found that this HIV-1-inducing activity was recovered in the lower molecular mass (<3 kDa) fraction of the culture supernatant. We also demonstrated that P. gingivalis produces high concentrations of butyric acid, acting as a potent inhibitor of HDACs and causing histone acetylation. Chromatin immunoprecipitation assays revealed that the corepressor complex containing HDAC1 and AP-4 was dissociated from the HIV-1 long terminal repeat promoter upon stimulation with bacterial culture supernatant concomitantly with the association of acetylated histone and RNA polymerase II. We thus found that P. gingivalis could induce HIV-1 reactivation via chromatin modification and that butyric acid, one of the bacterial metabolites, is responsible for this effect. These results suggest that periodontal diseases could act as a risk factor for HIV-1 reactivation in infected individuals and might contribute to the systemic dissemination of the virus.

Factors Controlling Chromatin Organization and Nucleosome Positioning for Establishment and Maintenance of HIV Latency

Transcription of the integrated HIV provirus is subject to regulation by chromatin organization and must employ host cell transcription factors and chromatin modifying complexes to promote the formation of latency, and then reverse this process to replicate in response to T cell activation. The repressed latent HIV-1 proviral 5' LTR is organized into a defined structure where two de-acetylated and positioned nucleosomes flank the enhancer region, presumably imposing a block to transcriptional initiation and elongation. LTR-associated nucleosomes undergo further histone H3 K9 trimethylation, to cause silencing by recruitment of HP1. In this article, we review current understanding of how the transcriptionally silenced provirus might be established through the function of transcription factors that bind conserved cis-elements, including SP1, YY1, NF-kappaB, CBF-1 and RBF-2 (USF/TFII-I), and propose mechanisms by which factors bound to the repressed LTR can enable reactivation in response to cell signaling.

Biology of Retroviruses: Detection, Molecular Biology, and Treatment of Retroviral Infection

Although human retrovirology can trace its roots to the early part of this century, it has been within the last decade that the field has seen its greatest advances, including the description of the first human retroviruses, HTLV-I, and the subsequent discovery of HTLV-II and the AIDS retroviruses, HIV-1 and HIV-2. The growth this field has undergone in recent years would not have been possible had it not been for key advances in the fields of cell biology, immunology, biochemistry, and molecular biology. Nowhere has this been more evident than in the description of HIV as the etiologic agent of AIDS. In the relatively short time since the disease was initially described, the agent responsible has been isolated, much of its complex biology has been defined, and a drug that helps combat it has been licensed for use. The rapidity with which these events have transpired is largely unprecedented. There remain many unanswered questions regarding HIV and AIDS, and there still is no effective vaccine or cure. Significant questions regarding the immune response to HIV and the HIV response to immune or chemotherapeutic challenge, the establishment and maintenance of HIV latency, and the all-important mechanisms of HIV cytopathicity remain unanswered. It is clear that HIV offers no easy answers, and that significant work remains to be done before death from AIDS can be halted.