MicrobiologyBytes

When HIV infects a T lymphocyte, it first inserts a copy of its genome into the cell’s DNA. This inserted virus, called a provirus, then races to make as many new viruses as possible before its host cell dies. But in a few infected cells, HIV does not immediately turn its host into a viral factory. Instead, the provirus is carried around in the DNA of the cell as a transcriptionally silent (“latent”) passenger, only to explode back into action at a later time, when its host cell attempts to participate in an immune response to infection by other pathogens. Because they target the products of HIV transcription, current antiviral therapies like HAART can’t kill latent HIV. And because a full-blown infection can be re-established from a tiny reservoir of latently infected cells, viral latency is an important contributor to our struggle against HIV (Dissecting HIV’s Latent Menace. (2011) PLoS Biol 9(11): e1001209).

The following article examines the molecular mechanism responsible for the establishment and maintenance of HIV latency and its re-activation, and uncovers the role played in this process by the SWI/SNF class of chromatin remodeling complexes, which use energy from ATP to alter the structure of chromatin. Two distinct sub-classes of SWI/SNF, BAF and PBAF, play functionally opposing roles in distinct steps of the HIV promoter (or long terminal repeat, LTR) transcription cycle. The PBAF complex augments transcription of the LTR by the viral transactivator Tat. In contrast, the distinct BAF complex generates a chromatin structure at the LTR that is energetically unfavorable with respect to the intrinsic histone-DNA sequence preferences. Specifically, 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. The 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.

Repressive LTR Nucleosome Positioning by the BAF Complex Is Required for HIV Latency. (2011) PLoS Biol 9(11): e1001206
Persistence of a reservoir of latently infected memory T cells provides a barrier to HIV eradication in treated patients. Several reports have implicated the involvement of SWI/SNF chromatin remodeling complexes in restricting early steps in HIV infection, in coupling the processes of integration and remodeling, and in promoter/LTR transcription activation and repression. However, the mechanism behind the seemingly contradictory involvement of SWI/SNF in the HIV life cycle remains unclear. Here we addressed the role of SWI/SNF in regulation of the latent HIV LTR before and after transcriptional activation. We determined the predicted nucleosome affinity of the LTR sequence and found a striking reverse correlation when compared to the strictly positioned in vivo LTR nucleosomal structure; sequences encompassing the DNase hypersensitive regions displayed the highest nucleosome affinity, while the strictly positioned nucleosomes displayed lower affinity for nucleosome formation. To examine the mechanism behind this reverse correlation, we used a combinatorial approach to determine DNA accessibility, histone occupancy, and the unique recruitment and requirement of BAF and PBAF, two functionally distinct subclasses of SWI/SNF at the LTR of HIV-infected cells before and after activation. We find that establishment and maintenance of HIV latency requires BAF, which removes a preferred nucleosome from DHS1 to position the repressive nucleosome-1 over energetically sub-optimal sequences. Depletion of BAF resulted in de-repression of HIV latency concomitant with a dramatic alteration in the LTR nucleosome profile as determined by high resolution MNase nucleosomal mapping. Upon activation, BAF was lost from the HIV promoter, while PBAF was selectively recruited by acetylated Tat to facilitate LTR transcription. Thus BAF and PBAF, recruited during different stages of the HIV life cycle, display opposing function on the HIV promoter. Our data point to the ATP-dependent BRG1 component of BAF as a putative therapeutic target to deplete the latent reservoir in patients.

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The source of persistent HIV in patients on suppressive therapy is debated. Recent studies of treatment intensification have produced varied results: no reduction in low-level plasma viremia indicating the source of persistent viremia is long-lived HIV-infected cells that release HIV when activated and increase in episomal HIV DNA indicating active replication persists in some infected individuals on suppressive therapy. In addition, clonal HIV sequences found in plasma from patients on long-term suppressive therapy are rarely found in CD4 memory T cells. These results indicate that persistent viremia may arise from several different sources. Recent studies emphasize the complexity of HIV latency. Current strategies for HIV eradication focus on compounds that activate viral transcription in memory CD4 T cells by many routes, including inhibiting histone deacetylation and activating nuclear factor kappa B. Several compounds and combinations of these compounds appear to induce the expression of integrated HIV in different latency models. This review focuses on recent advances in HIV research and therapy that seek to eradicate persistent HIV in patients on suppressive therapy.

Many researchers currently investigate the source and dynamics of residual viremia. A well defined latent reservoir of HIV is memory CD4 T cells. However, other cell types such as hematopoietic stem cells and cells of the monocyte/macrophage lineage may also serve as long-term reservoirs of HIV. Several mechanisms appear to play a role in maintaining HIV latency including viral integration sites, chromatin environment, and downregulated transcription factors. Further studies are necessary to better understand the mechanisms that promote HIV latency in vivo. Recent studies shed new light on persistent HIV reservoirs and the mechanisms of latency. These studies highlight an important conclusion: any long-term strategy for HIV eradication must take HIV latency and its implications into account. Importantly, approaches to eradication of latent HIV reservoirs should not lead to new HIV infection as a result of activating latently infected cells or cause global T-cell activation. Although many challenges remain, it is encouraging to note that new research and debate have begun to seriously address HIV eradication and/or remission.

Can HIV infection be eradicated through use of potent antiviral agents? Curr Opin Infect Dis. Sep 16 2010

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• HIV-1 latency and the eradication of long-term virus reservoirs
• HIV “can never be cured”

Human cytomegalovirus (HCMV) is an opportunistic human pathogen that establishes a lifelong latent infection that can periodically reactivate which, if unchecked by a robust immune response, can result in severe disease in immuno-compromised patients. Following primary infection of healthy individuals, human cytomegalovirus (HCMV) is met with a robust immune response resulting in an asymptomatic infection and subsequent establishment of latency. To date, HCMV remains one of the leading viral agents of disease in immuno-suppressed transplant patients and is a cause of severe morbidity in late stage AIDS sufferers and cancer patients.

The threat from HCMV is exacerbated by the dual threat of primary infection/re-infection, and virus reactivation within the host. Since many instances of virus disease result from HCMV reactivation, many studies have analysed the regulation of latency and reactivation using experimental models. An informed consensus supports the myeloid lineage as an important site of HCMV latency and carriage and that terminal myeloid differentiation is needed for HCMV reactivation. A recurrent theme in HCMV latency/reactivation, this differentiation-dependent permissiveness also applies to lytic infection. Latently infected cells are, by definition, unable to support the viral lytic transcription program – pivotal to this is failure of IE gene expression.

Reactivation of latent virus in myeloid progenitor cells is concomitant with cellular differentiation through regulation of the MIEP by chromatin remodelling. This study analyses the expression of the latent gene transcript UL81-82as (LUNA). LUNA is expressed in latently infected CD34+ cells and expression decreases as CD34+ cells differentiate to immature dendritic cells. Upon maturation (and HCMV reactivation) a second wave of transcription occurs consistent with expression during lytic infection also. Furthermore, it shows that the LUNA promoter is associated with acetylated histones during HCMV latency in experimentally and naturally infected CD34+ cells thus suggesting that latent gene promoters are, like the MIEP, regulated by post-translational modifications of their associated histone proteins.

Analysis of latent viral gene expression in natural and experimental latency models of human cytomegalovirus and its correlation with histone modifications at a latent promoter. J Gen Virol. Nov 11 2009

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• Human Cytomegalovirus-encoded microRNA regulates expression of multiple genes involved in replication
• Human cytomegalovirus and the cell cycle