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...

Latent HIV proviruses are thought to be primarily reactivated in vivo through stimulation of the T-cell receptor (TCR). Activation of the T-cell receptor (TCR) induces multiple signal transduction pathways leading to the ordered nuclear migration of the HIV transcription initiation factors NF-κB and NFAT as well as potential effects on HIV transcriptional elongation. We have monitored the kinetics of proviral reactivation using chromatin immunoprecipitation (ChIP) assays to measure changes in the distribution of RNA polymerase II (RNAP II) on the HIV provirus. Surprisingly, in contrast to TNF-α activation, where early transcription elongation is highly restricted due to rate-limiting concentrations of Tat, efficient and sustained HIV elongation and P-TEFb recruitment is detected immediately after activation of latent proviruses through the T-cell receptor. Inhibition of NFAT activation by cyclosporine had no effect on either HIV transcription initiation or elongation. However, examination of P-TEFb complexes by gel filtration chromatography showed that TCR signaling led to the rapid dissociation of the large inactive 7SK RNP complex and release of active low molecular weight P-TEFb complexes. Both P-TEFb recruitment to the HIV LTR and enhanced HIV processivity were blocked by the ERK kinase inhibitor U0126 but not by AKT and PI3 kinase inhibitors. In contrast to treatment by HMBA and DRB, which disrupt the large 7SK RNP complex but do not stimulate early HIV elongation, TCR signaling provides the first example of a physiological pathway that can shift the balance between the inactive and active P-TEFb pools and thereby stimulate proviral reactivation.

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.

Stimulation of transcriptional elongation by the human immunodeficiency virus type 1 Tat protein is mediated by CDK9, a kinase that phosphorylates the RNA polymerase II carboxyl-terminal domain (CTD). In order to obtain direct evidence that this phosphorylation event can alter RNA polymerase processivity, we prepared transcription elongation complexes that were arrested by the lac repressor. The CTD was then dephosphorylated by treatment with protein phosphatase 1. The dephosphorylated transcription complexes were able to resume the transcription elongation when IPTG (isopropyl-␤-D-thiogalactopyranoside) and nucleotides were added to the reaction. Under these chase conditions, efficient rephosphorylation of the CTD was observed in complexes containing the Tat protein but not in transcription complexes prepared in the absence of Tat protein. Immunoblots and kinase assays with synthetic peptides showed that Tat activated CDK9 directly since the enzyme and its cyclin partner, cyclin T1, were present at equivalent levels in transcription complexes prepared in the presence or absence of Tat. Chase experiments with the dephosphorylated elongation transcription complexes were performed in the presence of the CDK9 kinase inhibitor DRB (5,6-dichloro-1-␤-D-ribofuranosyl-benzimidazole). Under these conditions there was no rephosphorylation of the CTD during elongation, and transcription through either a stem-loop terminator or bent DNA arrest sequence was strongly inhibited. In experiments in which the CTD was phosphorylated prior to elongation, the amount of readthrough of the terminator sequences was proportional to the extent of the CTD modification. The change in processivity is due to CTD phosphorylation alone, since even after the removal of Spt5, the second substrate for CDK9, RNA polymerase elongation is enhanced by Tat-activated CDK9 activity. We conclude that phosphorylation of the RNA polymerase II CTD by CDK9 enhances transcription elongation directly.Transcription in eukaryotic cells is regulated by a series of phosphorylation events involving the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) (for a review, see reference 55). Initially, Pol II with a hypophosphorylated CTD (Pol II a ) is recruited to the promoter. The hypophosphorylated CTD is able to form a tightly packed complex at the promoter, together with general initiation factors and the mediator complex. During initiation, the CTD becomes highly phosphorylated, creating a form of the enzyme called Pol II o . The large conformational change induced in the enzyme during the transition from initiation to early elongation releases the phosphorylated CTD and permits it to interact with a variety of elongation factors, as well as with proteins involved in mRNA processing (28).The CTD comprises 52 tandem repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Three cyclindependent kinases-CDK7, CDK8, and CDK9-are known to phosphorylate the CTD during transcription (55). The three CTD kinases primarily pho...

The human immunodeficiency virus type 1 (HIV-1) Tat protein activates transcription elongation by stimulating the Tat-activated kinase (TAK/p-TEFb), a protein kinase composed of CDK9 and its cyclin partner, cyclin T1. CDK9 is able to hyperphosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase during elongation. In addition to TAK, the transcription elongation factor Spt5 is required for the efficient activation of transcriptional elongation by Tat. To study the role of Spt5 in HIV transcription in more detail, we have developed a three-stage Tat-dependent transcription assay that permits the isolation of active preinitiation complexes, early-stage elongation complexes, and Tat-activated elongation complexes. Spt5 is recruited in the transcription complex shortly after initiation. After recruitment of Tat during elongation through the transactivation response element RNA, CDK9 is activated and induces hyperphosphorylation of Spt5 in parallel to the hyperphosphorylation of the CTD of RNA polymerase II. However, immunodepletion experiments demonstrate that Spt5 is not required for Tat-dependent activation of the kinase. Chase experiments using the Spt5-depleted extracts demonstrate that Spt5 is not required for early elongation. However, Spt5 plays an important role in late elongation by preventing the premature dissociation of RNA from the transcription complex at terminator sequences and reducing the amount of polymerase pausing at arrest sites, including bent DNA sequences. This novel biochemical function of Spt5 is analogous to the function of NusG, an elongation factor found in Escherichia coli that enhances RNA polymerase stability on templates and shows sequence similarity to Spt5.Transcription from the human immunodeficiency virus (HIV) promoter is regulated both at the level of initiation and elongation (for recent reviews, see references 14, 26, 48, and 54). In activated T cells and other permissive cell types, initiation of HIV transcription is very efficient, but production of full-length transcripts requires the viral regulatory protein Tat. In the absence of Tat, the majority of RNA polymerase II complexes that initiate transcription at the HIV promoter disengage from the template near to the promoter (24, 35). Tat strongly activates RNA polymerase processivity and permits the synthesis of full-length HIV transcripts both in vivo and in cell-free transcription systems (11,17,27,28,39,40,50,53).Tat is an RNA-binding protein that is recruited to early HIV transcription complexes by binding to the transactivation response element (TAR), an RNA stem-loop structure that is located at the 5Ј end of HIV transcripts (5, 12, 28, 51, 52). The interaction between Tat and TAR involves not only the binding of Tat to TAR RNA but also its association with the Tat-activated kinase (TAK) (20, 38, 69), a protein kinase that is able to phosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (21,43,65).TAK is composed of a kinase subunit, CDK9, and its c...

Natural killer (NK) cells are innate immune effector cells that protect against cancer and some viral infections. Until recently, most studies have investigated the molecular signatures of human or mouse NK cells to identify genes that are specifically expressed during NK cell development. However, the mechanism regulating NK cell development remains unclear. Here, we report a regulatory network of potential interactions during in vitro differentiation of human NK cells, identified using genome-wide mRNA and miRNA databases through hierarchical clustering analysis, gene ontology analysis and a miRNA target prediction program. The microRNA (miR)-583, which demonstrated the largest ratio change in mature NK cells, was highly correlated with IL2 receptor gamma (IL2Rγ) expression. The overexpression of miR-583 had an inhibitory effect on NK cell differentiation. In a reporter assay, the suppressive effect of miR-583 was ablated by mutating the putative miR-583 binding site of the IL2Rγ 3′ UTR. Therefore, we show that miR-583 acts as a negative regulator of NK cell differentiation by silencing IL2Rγ. Additionally, we provide a comprehensive database of genome-wide mRNA and miRNA expression during human NK cell differentiation, offering a better understanding of basic human NK cell biology for the application of human NK cells in immunotherapy.