No abstract

The molecular mechanisms utilized by human immunodeficiency virus (HIV) to enter latency are poorly understood. Following the infection of Jurkat T cells with lentiviral vectors that express Tat in cis, gene expression is progressively silenced. Silencing is greatly enhanced when the lentiviral vectors carry an attenuated Tat gene with the H13L mutation. Individual clones of lentivirus-infected cells showed a wide range of shutdown rates, with the majority showing a 50% silencing frequency between 30 to 80 days. The silenced clones characteristically contained a small fraction (0 to 15%) of activated cells that continued to express d2EGFP. When d2EGFP؉ and d2EGFP ؊ cell populations were isolated from the shutdown clones, they quickly reverted to the original distribution of inactive and active cells, suggesting that the d2EGFP ؉ cells arise from stochastic fluctuations in gene expression. The detailed analysis of transcription initiation and elongation using chromatin immunoprecipitation (ChIP) assays confirms that Tat levels are restricted in the latently infected cells but gradually rise during proviral reactivation. ChIP assays using clones of latently infected cells demonstrate that the latent proviruses carry high levels of deacetylated histones and trimethylated histones. In contrast, the cellular genes IB␣ and GAPDH had high levels of acetylated histones and no trimethylated histones. The levels of trimethylated histone H3 and HP1-␣ associated with HIV proviruses fell rapidly after tumor necrosis factor alpha activation. The progressive shutdown of HIV transcription following infection suggests that epigenetic mechanisms targeting chromatin structures selectively restrict HIV transcription initiation. This decreases Tat production below the levels that are required to sustain HIV gene expression.The vast majority of human immunodeficiency virus (HIV) infections result in active viral transcription and replication; however, in a few rare cases the virus can enter a long-lived latent state in which viral gene expression is silenced. Although the pool of latently infected cells is very small (approximately 1 in 10 6 resting CD4 ϩ T cells in the peripheral circulation), the ability to latently infect cells helps HIV to establish chronic infections despite strong humoral and cellular immune responses and to evade intensive antiretroviral therapy. Siliciano and his colleagues (29, 41) have proposed that the generation of latently infected cells is a consequence of normal maturation and cellular differentiation events leading to the formation of quiescent T-cell populations. For example, if an activated effector T cell becomes infected by HIV when it is in the process of reverting to being a resting memory T cell, a stably integrated but transcriptionally silent form of the provirus can be generated. Similarly, Zack and colleagues have effectively used the HIV SCID-hu (Thy/Liv) mouse model to recapitulate the generation of latent HIV during thymopoiesis (1, 4, 5). As infected CD4 ϩ CD8 ϩ thymocytes differentiate...

The development of suitable experimental systems for studying HIV latency in primary cells that permit detailed biochemical analyses and the screening of drugs is a critical step in the effort to develop viral eradication strategies. Primary CD4 ؉ T cells were isolated from peripheral blood and amplified by antibodies to the T-cell receptor (TCR). The cells were then infected by lentiviral vectors carrying fluorescent reporters and either the wild-type Tat gene or the attenuated H13L Tat gene. After sorting for the positive cells and reamplification, the infected cells were allowed to spontaneously enter latency by long-term cultivation on the H80 feeder cell line in the absence of TCR stimulation. By 6 weeks almost all of the cells lost fluorescent protein marker expression; however, more than 95% of these latently infected cells could be reactivated after stimulation of the TCR by ␣-CD3/CD28 antibodies. Chromatin immunoprecipitation assays showed that, analogously to Jurkat T cells, latent proviruses in primary CD4؉ T cells are enriched in heterochromatic markers, including high levels of CBF-1, histone deacetylases, and methylated histones. Upon TCR activation, there was recruitment of NF-B to the promoter and conversion of heterochromatin structures present on the latent provirus to active euchromatin structures containing acetylated histones. Surprisingly, latently infected primary cells cannot be induced by tumor necrosis factor alpha because of a restriction in P-TEFb levels, which can be overcome by activation of the TCR. Thus, a combination of restrictive chromatin structures at the HIV long terminal repeat and limiting P-TEFb levels contribute to transcriptional silencing leading to latency in primary CD4؉ T cells.

The establishment of HIV proviral latency requires the creation of repressive chromatin structures that impair the initiation of transcription and restrict RNAP II elongation. We have found that C‐promoter binding factor‐1 (CBF‐1), a CSL (CBF‐1, Su(H) and Lag‐1)‐type transcription factor and key effector of the Notch signaling pathway, is a remarkably potent and specific inhibitor of the HIV‐1 LTR promoter. Knockdown of endogenous CBF‐1 using specific small hairpin RNAs expressed on lentiviral vectors results in the partial reactivation of latent HIV proviruses, recruitment of RNAP II, loss of histone deacetylases and the concomitant acetylation of histones. An important property of any repressor utilized to establish HIV latency is that it must become displaced or deactivated upon T‐cell activation. Consistent with this hypothesis, CBF‐1 mRNA and protein levels are highest in quiescent or unstimulated T cells but decline rapidly in response to proliferative stimulation such as activation of the T‐cell receptor or treatment with TNF‐α. We conclude that CBF‐1 is a previously overlooked factor that induces transcriptional silencing during the establishment of HIV latency.

In cells infected with HIV type 1 (HIV-1), the integrated viral promoter is present in a chromatin-bound conformation and is transcriptionally silent in the absence of stimulation. The HIV-1 Tat protein binds to a stem-loop structure at the 5 end of viral mRNA and relieves this inhibition by inducing a remodeling of the nucleosome arrangement downstream of the transcription-initiation site. Here we show that Tat performs this activity by recruiting to the viral long terminal repeat (LTR) the transcriptional coactivator p300 and the closely related CREB-binding protein (CBP), having histone acetyltransferase (HAT) activity. Tat associates with HAT activity in human nuclear extracts and binds to p300 and CBP both in vitro and in vivo. Integrity of the basic domain of Tat is essential for this interaction. By a quantitative chromatin immunoprecipitation assay we show that the delivery of recombinant Tat induces the association of p300 and CBP with the chromosomally integrated LTR promoter. Expression of human p300 in both human and rodent cells increases the levels of Tat transactivation of the integrated LTR. These results reinforce the evidence that p300 and CBP have a pivotal function at both cellular and viral promoters and demonstrate that they also can be recruited by an RNA-targeted activator. Additionally, these findings have important implications for the understanding of the mechanisms of HIV-1 latency and reactivation.

Translocation through the plasma membrane is a major limiting step for the cellular delivery of macromolecules. A promising strategy to overcome this problem consists in the chemical conjugation (or fusion) to cell penetrating peptides (CPP) derived from proteins able to cross the plasma membrane. A large number of dierent cargo molecules such as oligonucleotides, peptides, peptide nucleic acids, proteins or even nanoparticles have been internalized in cells by this strategy. One of these translocating peptides was derived from the HIV-1 Tat protein. The mechanisms by which CPP enter cells remain unknown. Recently, convincing biochemical and genetic ®ndings has established that the full-length Tat protein was internalized in cells via the ubiquitous heparan sulfate (HS) proteoglycans. We demonstrate here that the short Tat CPP is taken up by a route that does not involve the HS proteoglycans.

Latently infected cells rapidly initiate HIV transcription after exposure to signals that induce NF-jB. To investigate the role of TFIIH during HIV reactivation in vivo, we developed a population of Jurkat cells containing integrated, but transcriptionally silent, HIV proviruses. Surprisingly, the HIV promoter in unactivated Jurkat T cells is partially occupied and carries Mediator containing the CDK8 repressive module, TFIID and RNAP II that is hypophosphorylated and confined to the promoter region. Significantly, the promoter is devoid of TFIIH. Upon stimulation of the cells by TNF-a, NF-jB and TFIIH are rapidly recruited to the promoter together with additional Mediator and RNAP II, but CDK8 is lost. Detailed time courses show that the levels of TFIIH at the promoter fluctuate in parallel with NF-jB recruitment to the promoter. Similarly, recombinant p65 activates HIV transcription in vitro and stimulates phosphorylation of the RNAP II CTD by the CDK7 kinase module of TFIIH. We conclude that the recruitment and activation of TFIIH represents a rate-limiting step for the emergence of HIV from latency.

The RNA response element TAR plays a critical role in HIV replication by providing a binding site for the recruitment of the viral transactivator protein Tat. Using a structure-guided approach, we have developed a series of conformationally-constrained cyclic peptides that act as structural mimics of the Tat RNA binding region and block Tat-TAR interactions at nanomolar concentrations in vitro. Here we show that these compounds block Tat-dependent transcription in cell-free systems and in cell-based reporter assays. The compounds are also cell permeable, have low toxicity, and inhibit replication of diverse HIV-1 strains, including both CXCR4-tropic and CCR5-tropic primary HIV-1 isolates of the divergent subtypes A, B, C, D and CRF01_AE. In human peripheral blood mononuclear cells, the cyclic peptidomimetic L50 exhibited an IC50 ∼250 nM. Surprisingly, inhibition of LTR-driven HIV-1 transcription could not account for the full antiviral activity. Timed drug-addition experiments revealed that L-50 has a bi-phasic inhibition curve with the first phase occurring after HIV-1 entry into the host cell and during the initiation of HIV-1 reverse transcription. The second phase coincides with inhibition of HIV-1 transcription. Reconstituted reverse transcription assays confirm that HIV-1 (−) strand strong stop DNA synthesis is blocked by L50-TAR RNA interactions in-vitro. These findings are consistent with genetic evidence that TAR plays critical roles both during reverse transcription and during HIV gene expression. Our results suggest that antiviral drugs targeting TAR RNA might be highly effective due to a dual inhibitory mechanism.