SUMMARY Eradication of the latent HIV reservoir remains a major barrier to curing AIDS. However, the mechanisms that direct viral persistence in the host are not well understood. Studying a model system of post-integration latency, we found that proviral integration into the actively transcribed host genes led to transcriptional interference caused by the elongating RNA polymerase II transcribing through the viral promoter. As a result, physical exclusion of the pre-initiation complex formation on the 5’LTR promoted the silencing of HIV transcription. Furthermore, we demonstrated that this block could be counteracted through inhibiting the upstream transcription or cooperatively activating transcription initiation and elongation from the 5’LTR. Importantly, using a novel PCR-based method, we detected significant levels of host transcription through the 5’LTR in HIV-infected primary CD4+ T cells. Collectively, our findings suggest that transcriptional interference contributes greatly to HIV latency and has to be considered when attempting to purge the latent reservoir.

Human immunodeficiency virus (HIV) persists in a latent form in infected individuals treated effectively with highly active antiretroviral therapy (HAART). In part, these latent proviruses account for the rebound in viral replication observed after treatment interruption. A major therapeutic challenge is to purge this reservoir. In this study, we demonstrate that suberoylanilide hydroxamic acid (SAHA) reactivates HIV from latency in chronically infected cell lines and primary cells. Indeed, P-TEFb, a critical transcription cofactor for HIV, is released and then recruited to the viral promoter upon stimulation with SAHA. The phosphatidylinositol 3-kinase/Akt pathway is involved in the initiation of these events. Using flow cytometrybased single cell analysis of protein phosphorylation, we demonstrate that SAHA activates this pathway in several subpopulations of T cells, including memory T cells that are the major viral reservoir in peripheral blood. Importantly, SAHA activates HIV replication in peripheral blood mononuclear cells from individuals treated effectively with HAART. Thus SAHA, which is a Food and Drug Administration-approved drug, might be considered to accelerate the decay of the latent reservoir in HAART-treated infected humans. Highly active antiretroviral therapy (HAART)5 can reduce plasma human immunodeficiency virus (HIV) to undetectable levels indefinitely. Interruption of HAART, however, inevitably results in a rapid rebound of viremia, indicating that antiretroviral therapy-mediated viral suppression alone is unlikely to eradicate HIV. HIV DNA can integrate stably into the DNA of resting memory T cells where it will persist until these cells die (1). This major reservoir has an estimated half-life of 44 months, suggesting that 70 years of HAART treatment would be required for its eradication (2). Thus, designing new approaches to purge the reservoir of HIV represents a critical therapeutic challenge.The establishment of viral latency in the pool of memory T cells is multifactorial (3, 4). The transcriptional repression of HIV plays a critical role. Several mechanisms have been identified, such as transcriptional interference (5), chromatin remodeling via acetylation or methylation (6 -9), and lack of nuclear factor-B (NF-B) or other activators (10). Various approaches antagonizing these distinct transcription blocks have been used to reactivate HIV from latently infected cells (11). This reactivation should contribute to the clearance of otherwise hidden pro-viruses by the immune system and HAART. Ideally, because generalized immune activation also increases the number of targets for new infections and has significant toxic side effects, these strategies should induce HIV transcription without activating cells of the immune system. Indeed, administrations of IL-2 and anti-CD3 antibodies have resulted in significant side effects without having any apparent positive effect on the latent reservoir (12, 13). Various new activators have also been described, including prostratin (11,17,18), valpr...

Hexamethylene bisacetamide (HMBA) is a potent inducer of cell differentiation and HIV production in chronically infected cells. However, its mechanism of action remains poorly defined. In this study, we demonstrate that HMBA activates transiently the PI3K/Akt pathway, which leads to the phosphorylation of HEXIM1 and the subsequent release of active positive transcription elongation factor b (P-TEFb) from its transcriptionally inactive complex with HEXIM1 and 7SK small nuclear RNA (snRNA). As a result, P-TEFb is recruited to the HIV promoter to stimulate transcription elongation and viral production. Despite the continuous presence of HMBA, the released P-TEFb reassembles rapidly with 7SK snRNA and HEXIM1. In contrast, a mutant HEXIM1 protein that cannot be phosphorylated and released from P-TEFb and 7SK snRNA via the PI3K/Akt pathway antagonizes this HMBA-mediated induction of viral production. Thus, our studies reveal how HIV transcription is induced by HMBA and suggest how modifications in the equilibrium between active and inactive P-TEFb could contribute to cell differentiation.

Human immunodeficiency virus type 1 (HIV-1) transcriptional transactivator (Tat) recruits the positive transcription elongation factor b (P-TEFb) to the viral promoter. Consisting of cyclin dependent kinase 9 (Cdk9) and cyclin T1, P-TEFb phosphorylates RNA polymerase II and the negative transcription elongation factor to stimulate the elongation of HIV-1 genes. A major fraction of nuclear P-TEFb is sequestered into a transcriptionally inactive 7SK small nuclear ribonucleoprotein (snRNP) by the coordinated actions of the 7SK small nuclear RNA (snRNA) and hexamethylene bisacetamide (HMBA) induced protein 1 (HEXIM1). In this study, we demonstrate that Tat prevents the formation of and also releases P-TEFb from the 7SK snRNP in vitro and in vivo. This ability of Tat depends on the integrity of its N-terminal activation domain and stems from the high affinity interaction between Tat and cyclin T1, which allows Tat to directly displace HEXIM1 from cyclin T1. Furthermore, we find that in contrast to the Tat-independent activation of the HIV-1 promoter, Tat-dependent HIV-1 transcription is largely insensitive to the inhibition by HEXIM1. Finally, primary blood lymphocytes display a reduced amount of the endogenous 7SK snRNP upon HIV-1 infection. All these data are consistent with the model that Tat not only recruits but also increases the active pool of P-TEFb for efficient HIV-1 transcription.