调控HIV-1转录和潜伏激活的机制及相关因子

Mechanism and factors that control HIV-1 transcription and latency activation 调控HIV-1转录和潜伏激活的机制及相关因子 first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:xueyuhua@xmu.edu.cn †E-mail:xueyuhua@xmu.edu.cn xueyuhua@xmu.edu.cn School of Pharmaceutical Sciences, Xiamen University, Xiamen 361102, China School of Pharmaceutical Sciences, Xiamen University, Xiamen 361102, China After reverse transcription, the HIV-1 proviral DNA is integrated into the host genome and thus subjected to transcription by the host RNA polymerase II (Pol II). With the identification and characterization of human P-TEFb in the late 1990s as a specific host cofactor required for HIV-1 transcription, it is now believed that the elongation stage of Pol II transcription plays a particularly important role in regulating HIV-1 gene expression. HIV-1 uses a sophisticated scheme to recruit human P-TEFb and other cofactors to the viral long terminal repeat (LTR) to produce full-length HIV-1 transcripts. In this process, P-TEFb is regulated by the reversible association with various transcription factors/cofactors to form several multi-subunit complexes (e.g., 7SK snRNP, super elongation complexes (SECs), and the Brd4-P-TEFb complex) that collectively constitute a P-TEFb network for controlling cellular and HIV-1 transcription. Recent progresses in HIV-1 transcription were reviewed in the paper, with the emphasis on the mechanism and factors that control HIV-1 transcription and latency activation. （1）反式激活蛋白（Tat）是病毒复制的重要因子，正性转录延伸因子b（P-TEFb）是Tat反式激活HIV-1转录所必需的特异性宿主细胞因子，其活性与HIV-1的转录密切相关。（2）在细胞内，无活性7SK snRNP复合体是功能性有活性P-TEFb的贮存和来源。在特定条件下，7SK snRNP复合体发生解离并释放出P-TEFb，从而刺激转录延伸。可以说，P-TEFb的活性受到严格的调控，维持着一种动态的平衡。（3）同时，P-TEFb还存在于一个具有超高转录活性的超级延伸复合体（SEC）中。Tat能将含有两个延伸因子P-TEFb和ELL2的SEC复合体募集至HIV-1长末端结构域（LTR），然后众多因子协同作用有效地增强HIV-1的转录作用。（4）溴区包含蛋白4（Brd4）像Tat一样，将P-TEFb募集至众多细胞基因的启动子区域，促进转录。Brd4也可激活基础水平的HIV-1转录，却对依赖Tat的HIV-1转录具有抑制作用，因为Brd4和Tat竞争性地与P-TEFb结合。（5）鉴于Brd4对Tat依赖性HIV-1转录的抑制作用，寻找能够抑制Brd4的小分子药物，激活HIV-1潜伏，结合高效抗逆转录病毒治疗（HAART），使得彻底根除HIV-1变成可能。Brd4的抑制剂JQ1就是这样的一种小分子，并且已被证明可以在多种细胞模型中激活HIV-1潜伏。 HIV-1 Transcriptional elongation RNA polymerase II Tat P-TEFb 人类免疫缺陷病毒（HIV-1） 转录延伸 RNA聚合酶II Tat P-TEFb Introduction 1 Shortly after the entry of human immunodeficiency virus type I (HIV-1) into a target cell, the single-stranded viral RNA genome is reverse-transcribed into a complementary DNA (cDNA) by the virally encoded reverse transcriptase that is transported along with the viral genome in the virus particle (Roberts et al., 1988 1988 ). The reverse transcriptase also has ribonuclease H activity that degrades the viral RNA during the synthesis of the cDNA, as well as DNA polymerase activity that creates the sense DNA from the antisense cDNA. Together, the cDNA and its complement form the double-stranded proviral DNA that is then transported into the nucleus, where the proviral DNA is integrated into the host chromosome by the virally encoded integrase and specific host co-factors (Zheng et al., 2005 2005 ). After the HIV-1 provirus is integrated into the host chromosome, it depends on host RNA polymerase II (Pol II) for transcription. The Pol II-mediated transcription can be subdivided into several interconnected stages that include pre-initiation, initiation, promoter clearance, elongation, and termination (Fuda et al., 2009 2009 ). During most of the past three decades, major efforts had been devoted to the study of the pre-initiation and initiation stages of the transcription cycle because they were believed to be the principal point where transcription is regulated (Kuras and Struhl, 1999 1999 ; Ptashne, 2005 2005 ). In contrast, elongation was considered as an unregulated process that merely involves the repetitive addition of ribonucleotides to the growing RNA chains (Saunders et al., 2006 2006 ). The first hint that this view of transcriptional elongation is questionable or at least not tenable for all genes came from studies of HIV-1, which showed that the elongation phase of Pol II transcription on the integrated proviral DNA is a key rate-limiting step in controlling HIV-1 gene expression (Kao et al., 1987 1987 ). Moreover, the independent studies of Bentley and Groudine ( 1986 1986 ) and Rougvie and Lis ( 1988 1988 ) on c-myc or hsp70 also identified the promoter proximal pausing as a key regulating step in transcription. A paradigm shift in the transcription field occurred in 2007 on a much larger scale, when global analyses revealed that Pol II pauses at a promoter-proximal region of a large number of genes in both Drosophila and human stem cells before these genes are activated (Guenther et al., 2007 2007 ; Muse et al., 2007 2007 ). Subsequent data indicate that the pausing of Pol II soon after initiation is largely due to the actions of negative transcription elongation regulators that include the DRB sensitivity inducing factor (DSIF) and the negative elongation factor (NELF) (Wu et al., 2003 2003 ; Yamaguchi et al., 2013 2013 ). For the genes containing promoter-proximally paused Pol II, the time-consuming step of setting up a pre-initiation complex has been finished and they are in a state of suspended transcription. The subsequent induction of Pol II elongation can proceed very rapidly and is highly sensitive in response to transcriptional signals. The widespread existence of paused Pol II suggests that elongation plays a much more significant role in regulating gene expression and thus deserves more attention in the field (Guo and Price, 2013 2013 ). P-TEFb is a key host factor for Tat-dependent HIV-1 transcription 1 Transcription of the HIV-1 proviral DNA by host Pol II plays a critical role in the viral life cycle because the mRNA is both the template for the synthesis of viral proteins and the genome for progeny viruses. Like all viruses, HIV-1 must replicate by hijacking the cellular machinery to synthesize new virions. However, discrepant with other simpler retroviruses that rely almost entirely on the host, HIV-1 encodes additional accessory proteins that further control the viral life cycle. As a prime example, the HIV-1-encoded Tat protein is absolutely essential for activating transcriptional elongation from the viral long terminal repeat (LTR) (Jones, 1997 1997 ). In the absence of Tat, transcription by Pol II is initiated efficiently but generates only short abortive transcripts (Kao et al., 1987 1987 ). However, after Tat becomes available, it dramatically enhances the efficiency of Pol II elongation to produce the full-length viral transcripts. Tat accomplishes this task through interacting with the transactivation response (TAR) RNA element, which is a stem-loop structure located at the 5′ end of all nascent viral transcripts and synthesized by Pol II just before pausing. Almost immediately after the discovery of the Tat-TAR interaction, it became apparently clear that Tat and TAR alone are not sufficient to stimulate HIV-1 transcription. Mounting evidence suggested that specific host cellular cofactors are required in this process (Jones and Peterlin, 1994 1994 ). After numerous trials and errors, a significant achievement was obtained in the late 1990s with the identification and characterization of the human positive transcription elongation factor b (P-TEFb) as such a cofactor (Marshall and Price, 1995 1995 ; Marshall et al., 1996 1996 ; Mancebo et al., 1997 1997 ; Zhu et al., 1997 1997 ; Garber et al., 1998 1998 ). P-TEFb is a heterodimer composed of the cyclin-dependent kinase 9 (CDK9) and its regulatory partner cyclin T1 (CycT1). There are also other minor cyclin forms such as cyclin T2a and T2b due to alternative splicing, but they do not interact with Tat (Peng et al., 1998 1998 ; Fu et al., 1999 1999 ; Ivanov et al., 1999 1999 ). P-TEFb is recruited to the HIV-1 LTR through interacting with Tat and TAR. Once positioned next to the paused Pol II, P-TEFb is able to phosphorylate, either directly or indirectly, the C-terminal domain (CTD) of the largest subunit (RPB1) of Pol II on serines at position 2 (Ser2) of the 52 heptapeptide (YSPTSPS) repeats that make up the human Pol II CTD (Fig. 1 1 ). Two different kinases are believed to be required for phosphorylation of the CTD (Prelich, 2002 2002 ). First, CDK7, a component of the multi-subunit general transcription factor TFIIH, phosphorylates serines at position 5 (Ser5) within the heptapeptide repeats. Subsequently, the Ser5-phosphorylated CTD is further phosphorylated by CDK9 at Ser2. Through interacting with TAR RNA, Tat specifically delivers SEC to the HIV-1 LTR and releases Pol II from promoter-proximal pausing. SEC is a multi-subunit complex that contains P-TEFb, AFF1/4, ELL1/2, and AF9/ENL. Within the SEC, AFF1 or its homolog AFF4 serves as a molecular scaffold, whereas ENL/AF9 uses their N-terminal YEATS domain to target the SEC to Pol II on chromatin. P-TEFb phosphorylates Pol II CTD as well as negative elongation regulators DSIF and NELF. In addition, SEC also contains another well-characterized elongation factor, ELL1/2, which increases the catalytic rate by preventing Pol II backtracking. Thus, Tat significantly activates HIV-1 transcription by recruiting two totally different elongation factors in a single SEC complex In addition to CTD, both the Spt5 subunit of DSIF (Ivanov et al., 2000 2000 ) and the RD subunit of NELF (Fujinaga et al., 2004 2004 ) are also substrates for P-TEFb-mediated phosphorylation (Fig. 1 1 ). These phosphorylation events antagonize the inhibitory effects of these two negative elongation regulators and release Pol II from promoter-proximal pausing, leading to the production of full-length HIV-1 transcripts. P-TEFb is maintained in a functional equilibrium 1 P-TEFb is not only required for HIV-1 transcription but also efficient expression of numerous cellular genes (Rahl et al., 2010 2010 ). Because of this general dependence on P-TEFb for cellular and viral transcription, the activity of P-TEFb has to be tightly controlled in response to changing cellular requirements for P-TEFb-dependent transcription. It was found that not every CDK9-CycT1 heterodimer in the cell displays the signature P-TEFb kinase and transcriptional activity. Two independent groups subsequently reported that about half of the nuclear P-TEFbs in HeLa cells are sequestered in a kinase-inactive complex that contains the 7SK small nuclear RNA (snRNA) (Nguyen et al., 2001 2001 ; Yang et al., 2001 2001 ). Produced by RNA Pol III, 7SK is an abundant 331-nucleotide long noncoding RNA that is highly conserved in higher eukaryotes (Wassarman and Steitz, 1991 1991 ). The 7SK-bound P-TEFb was found to lack the ability to phosphorylate the Pol II CTD on Ser2 and mediate Tat-activation of HIV-1 transcription (Nguyen et al., 2001 2001 ; Yang et al., 2001 2001 ). Soon after this discovery, it was found that the 7SK-P-TEFb interaction alone was not enough to inactivate P-TEFb and that the presence of additional factor(s) in the 7SK-P-TEFb complex could be required for the inactivation (Yik et al., 2003 2003 ). Indeed, this hypothesis was soon proven to be true by the identification of a nuclear protein called hexamethylene bis-acetamide inducible 1 (HEXIM1) as a P-TEFb/7SK snRNA-associated factor (Michels et al., 2004 2004 ; Yik et al., 2004 2004 ). It was shown that HEXIM1’s inhibitory effect on P-TEFb is a 7SK snRNA-dependent process that requires the RNA to serve as a molecular scaffold to mediate the interaction between HEXIM1 and P-TEFb. The identification of novel components of the 7SK snRNA did not just stop at HEXIM1. Subsequent studies reveal that, in addition to HEXIM1, 7SK snRNP also contains the Lupus antigen (La)-related protein 7 (LARP7) and a 7SK snRNA-specific methylphosphate capping enzyme called MePCE (Jeronimo et al., 2007 2007 ; He et al., 2008 2008 ; Xue et al., 2010 2010 ). While MePCE helps protect 7SK snRNA against exonuclease cleavage from the 5′ end (Jeronimo et al., 2007 2007 ), the 3′ end of 7SK is occupied by LARP7 that binds to the poly(U) sequence (He et al., 2008 2008 ; Markert et al., 2008 2008 ). Intriguingly, when bound by LARP7 in the 7SK snRNP, MePCE loses its capping activity and exerts a capping-independent function to promote the LARP7-7SK interaction (Xue et al., 2010 2010 ). Thus, the integrity of 7SK snRNP is maintained by the cooperative action of MePCE and LARP7 on 7SK snRNA, which in turn sequesters the excess, unused cellular P-TEFb in an inactive state. It is interesting to note that although CDK9 lacks kinase activity when sequestered in the 7SK snRNP, it nevertheless has its T-loop already in the phosphorylated state (Chen et al., 2004 2004 ; Li et al., 2005 2005 ), which is typically associated with an activated CDK with accessible catalytic site (Baumli et al., 2008 2008 ). These observations suggest that P-TEFb is only temporarily sequestered in the inactive 7SK snRNP when cellular transcriptional demand is low. Since CDK9 is already phosphorylated on the T-loop and thus poised to become active again, transcription can begin almost immediately once 7SK snRNP is disrupted to release active P-TEFb in response to stimulatory signals (Zhou and Yik, 2006 2006 ). Indeed, sequestration of P-TEFb in the 7SK snRNP is completely reversible, and thus the snRNP can be viewed as a major cellular reservoir of unused P-TEFb activity, which can be withdrawn to promote P-TEFb-dependent gene expression (Fig. 2 2 ). Consistent with this notion, it has been shown that the exposure of cells to hypertrophic or stress signals, such as flavopiridol, 5,6-dichloro-1-β-D-ribofurano-sylbenzimidazole (DRB), actinomycin D, or UV irradiation, leads to a rapid release of P-TEFb from 7SK snRNP and activation of transcription (Chen et al., 2004 2004 ; He et al., 2006 2006 ; Biglione et al., 2007 2007 ). In addition, activation of TCR pathway in Jurkat T cells by anti-CD3/anti-CD28 antibodies also mediates the disruption of 7SK snRNP and liberation of P-TEFb (Natarajan et al., 2010 2010 ). Moreover, the exposure of murine erythroleukemia cells to hexamethylene bisacetamide (HMBA) results in a transient disruption of the complex, which is then followed by a permanent increase in the levels of HEXIM1 and 7SK snRNP after a prolonged treatment (He et al., 2006 2006 ). P-TEFb is regulated by a reversible association with various transcription factors/cofactors to form several multi-subunit complexes (e.g., 7SK snRNP, super elongation complexes (SECs), and the Brd4-P-TEFb complex) that collectively constitute a P-TEFb network for controlling cellular and HIV transcription. The 7SK snRNP is a cellular reservoir of inactive P-TEFb that requires the 7SK snRNA to serve as a molecular scaffold. The integrity of 7SK snRNP is maintained by LARP7 and MePCE, whereas HEXIM1 inhibits P-TEFb kinase activity. Under specific conditions including TCR activation or the exposure of cells to hypertrophic or stress signals, 7SK snRNP tends to be disrupted to release P-TEFb. Generally, the free P-TEFb is picked up by Brd4 which interacts with acetylated histones and consequently recruits P-TEFb to chromatin to stimulate elongation. Inversely, prolonged treatment with HMBA pushes the P-TEFb equilibrium and leads to reformation of the 7SK snRNP. During HIV-1 infection, Tat competes with Brd4 for binding of free P-TEFb and extracts P-TEFb out of 7SK snRNP to assemble SEC together with several other host factors. The BET bromodomain inhibitor JQ1 efficiently displaces Brd4 from the LTR region of HIV-1 chromatin and increases the association of P-TEFb/SEC with Tat It is important to point out that there are virtually no free P-TEFb heterodimers in the cell. Once liberated from the 7SK snRNP, most P-TEFbs are found to bind to the bromodomain-containing protein 4 (Brd4), which can interact with the acetylated histones and the transcriptional mediator complex (Jiang et al., 1998 1998 ; Dey et al., 2003 2003 ). Through the Brd4-P-TEFb interaction, P-TEFb is thus recruited to the promoters of many cellular genes to stimulate transcription (Wu and Chiang, 2007 2007 ; Dey et al., 2009 2009 ). Although the Brd4-P-TEFb interaction appears to be also important for basal HIV-1 transcription, it is found to be inhibitory to Tat-transactivation (Yang et al., 2005 2005 ). This is because Brd4 can compete with Tat for binding to the same surface on P-TEFb, which interferes with the more efficient Tat-dependent recruitment of P-TEFb to the HIV-1 promoter (Yang et al., 2005 2005 ). Taken together, the published data are consistent with the model that P-TEFb is normally maintained in a functional equilibrium through reversible associations with the positive (Brd4) and negative (HEXIM1/7SK/LARP7/MePCE) regulators (Fig. 2 2 ). According to this model, a shift of the P-TEFb equilibrium can produce profound physiological consequences by adjusting the nuclear concentrations of active P-TEFb in accordance to various cellular transcriptional demands and regulatory signals. Tat assembles the super elongation complex (SEC) to promote HIV-1 transcription 1 It is worth noting that the 7SK-binding motif in HEXIM1 shares high similarity with the arginine-rich TAR-binding motif in the HIV-1 Tat protein (Yik et al., 2004 2004 ). In addition, both HEXIM1 and Tat interact with P-TEFb through the same small region near the cyclin box in CycT1 (Karn, 1999 1999 ; Michels et al., 2003 2003 ). It is likely due to these similarities between Tat and HEXIM1 that Tat was found to efficiently displace HEXIM1 and trigger the release of P-TEFb from 7SK snRNP (Barboric et al., 2007 2007 ; Sedore et al., 2007 2007 ). Consistently, nuclear 7SK snRNP in primary blood lymphocytes shows a marked reduction upon HIV-1 infection (Barboric et al., 2007 2007 ). Upon its extraction of P-TEFb from 7SK snRNP, it is unclear whether Tat assembles only the Tat-TAR-P-TEFb trio on the viral LTR or additional cellular factors may be involved for Tat to fully activate HIV-1 transcription. It is interesting to point out that anecdotal evidence has been reported in the past, which appears to support the latter scenario. For example, the loss of Tat-transactivation in CDK9-depleted nuclear extracts can be rescued by addition of partially purified human P-TEFb, which may contain other associated factors, but not recombinant CDK9-CycT1 (Suñé et al., 2000 2000 ). To test the hypothesis that additional factors associated with the CDK9-CycT1 heterodimer of core P-TEFb are also required to promote efficient Tat-transactivation, tandem affinity-purifications were performed to isolate any proteins that may bind to both Flag-tagged CDK9 and HA-tagged Tat in a single complex (He et al., 2010 2010 ). This approach has led to the isolation and identification of ELL1/2, AFF1/4, ENL, and AF9 as novel components of the Tat-P-TEFb complex (Fig. 2 2 ). Notably, the same set of factors is also independently identified and confirmed through a similar purification scheme that targets Tat only (Sobhian et al., 2010 2010 ). This novel multi-subunit complex containing P-TEFb, AFF1/4, ELL1/2, and AF9/ENL is now called SEC. Within an SEC, AFF1 or its homolog AFF4 serves as a molecular scaffold and uses short interspersed hydrophobic stretches to allow dynamic and flexible assembly of multiple SEC subunits and connect them to each other (He et al., 2011 2011 ; Chou et al., 2013 2013 ). Although initially thought to be a subunit unique to only the SEC, recent data indicate that AFF1/4 also exists in a portion of 7SK snRNP in the cell (Lu et al., 2014 2014 ). Notably, AFF1/4 does not perform a scaffolding role in the 7SK snRNP. Instead, they function to strongly enhance the ability of Tat to extract P-TEFb from 7SK snRNP and form the Tat-SEC complex (Lu et al., 2014 2014 ). Besides AFF1/4, the SEC subunit ENL and its homologous AF9 also make key contributions to SEC’s function. Displaying similar but non-identical functions and existing in separate but closely related SECs, ENL/AF9 uses their N-terminal YEATS (after YNK7, ENL, AF-9, and TFIIF small subunit) domain to target the SEC to Pol II on chromatin through contacting the human PAFc complex, which allows the SEC to stimulate productive elongation (Kim et al., 2010 2010 ; He et al., 2011 2011 ). Like P-TEFb, ELL1/2 is also well-characterized transcriptional elongation factors belonging to the ELL family of Pol II elongation-stimulatory factors (Shilatifard et al., 1997 1997 ). Employing a different mechanism, ELL1/2 increases the catalytic rate of Pol II by keeping the 3′ end of nascent mRNA in alignment with the catalytic site to prevent Pol II backtracking (Shilatifard et al., 1996 1996 ). Thus, two totally different elongation factors, P-TEFb and ELL1/2, are recruited by Tat to the viral LTR in the same SEC complex, where they can work synergistically to release the paused Pol II to stimulate elongation (He et al., 2010 2010 ; Lu et al., 2014 2014 ) (Fig. 1 1 ). This explains why Tat is such a powerful transcriptional activator. Besides HIV-1 Tat, chimeric fusion proteins produced by chromosomal translocations and containing the mixed lineage leukemia (MLL) protein also take advantage of the powerful elongation stimulatory activity of the SEC and recruit the complex to many MLL-target genes to stimulate transcription and cause leukemia (Lin et al., 2010 2010 ). Despite its important role in SEC’s function, ELL2 turns out to be a short-lived protein that undergoes rapid proteasomal degradation (He et al., 2010 2010 ). Recently, ELL2 was shown to be a polyubiquitinated protein and the ubiquitination is carried out by the RING (a really interesting new gene) domain protein Siah1, which is identified as an E3 ubiquitin ligase for ELL2 (Liu et al., 2012 2012 ). The interaction of ELL2 with the scaffolding protein AFF1/4 dramatically increases the stability of ELL2 and this stabilization is further enhanced by the presence of Tat (He et al., 2010 2010 ), which in turn promotes SEC formation and SEC-dependent HIV-1 transcription. Brd4-P-TEFb interaction and its effect on HIV-1 transcription and latency 1 In cells that are free of HIV-1 infection, Brd4 serves as the cellular equivalent of Tat to recruit P-TEFb to the chromatin loci of many genes, especially the primary response genes, to promote transcriptional elongation (Jang et al., 2005 2005 ; Yang et al., 2005 2005 ; Delmore et al., 2011 2011 ). Brd4 is a ubiquitously expressed nuclear protein belonging to the bromodomain and extraterminal domain (BET) protein family. It contains two N-terminal tandem bromodomains and an extraterminal domain (Jeanmougin et al., 1997 1997 ; Dey et al., 2000 2000 ). While the two bromodomains are able to interact with acetylated histone H3 and H4 (Dey et al., 2003 2003 ), the C terminus of Brd4 is responsible for binding to P-TEFb (Bisgrove et al., 2007 2007 ). Thus, P-TEFb can be recruited to the chromatin template by Brd4 and activate gene transcription (Mochizuki et al., 2008 2008 ). Although Brd4 plays a positive role in releasing Pol II from pausing at many cellular gene promoters, it has different effects on Tat-independent and -dependent HIV-1 transcriptions (Jang et al., 2005 2005 ; Yang et al., 2005 2005 ). While Brd4 elevates the basal Tat-independent HIV-1 transcription, it is a potent inhibitor of Tat-transactivation as it competes with Tat for binding to P-TEFb (Yang et al., 2005 2005 ). Furthermore, overexpression of the P-TEFb-interacting domain of Brd4 has been shown to inhibit Tat function and reactivation of HIV-1 latency (Bisgrove et al., 2007 2007 ). As a potent suppressor of Tat-transactivation, it is not surprising that Brd4 has been shown to contribute significantly to the establishment and maintenance of HIV-1 latency (Zhu et al., 2012 2012 ; Mbonye et al., 2013 2013 ), which is a main obstacle to eradication of infection. Identification of small molecule drugs that can antagonize Brd4’s inhibition of Tat-transactivation and thereby activate latent HIV-1 proviruses may, in the presence of highly active antiretroviral therapy (HAART), permit clearance of infected cells by the immune system (Richman et al., 2009 2009 ). To this end, the BET bromodomain inhibitor JQ1, which binds competitively to the acetyl-lysine recognition motifs or bromodomains in Brd4, has been shown to efficiently reactivate latent HIV-1 in various models (Filippakopoulos et al., 2010 2010 ; Banerjee et al., 2012 2012 ). JQ1 is able to displace Brd4 from the LTR region of HIV-1 chromatin, thereby antagonizing Brd4’s inhibition of Tat-transactivation (Li et al., 2013 2013 ). In-depth analyses reveal that JQ1 significantly increases the association of P-TEFb/SEC with Tat, which results in more SECs recruited to the LTR by Tat and TAR to promote HIV-1 transcriptional elongation (Zhu et al., 2012 2012 ; Li et al., 2013 2013 ). Besides the inhibition of Brd4-chromatin interaction, JQ1 also leads to a transient release of P-TEFb from the 7SK snRNP. The released P-TEFb was found to associate with Brd4 and the SEC, which also contributes to HIV-1 transcription and latency activation (Bartholomeeusen et al., 2012 2012 ; Li et al., 2013 2013 ). It should be noted that JQ1 alone does not appear to reactivate HIV-1 latency very efficiently in infected primary T cells, probably due to the fact that the P-TEFb level in these cells is very low (Chiang and Rice, 2012 2012 ; Zhu et al., 2012 2012 ). However, JQ1 has been shown to cooperate well with other well-known latency activators such as SAHA and HMBA (Bartholomeeusen et al., 2012 2012 ), which may potentially prime the system to allow JQ1 to function properly. Finally, since JQ1 has been developed as an anti-cancer and growth-suppressive agent due to its inhibition of Brd4-dependent recruitment of P-TEFb to many primary response genes such as c-myc (Delmore et al., 2011 2011 ; Bartholomeeusen et al., 2012 2012 ), its Tat-specific effect in activating HIV-1 latency ensures that it does not induce general T cell activation, an unwanted side effect of latency reactivation. Indeed, the changes in global gene expression induced by JQ1 or αCD3/αCD28 have been compared through a microarray analysis, which indicates that JQ1 down-regulates T cell activation genes but up-regulates histone modification genes (Banerjee et al., 2012 2012 ). Besides inhibition of Tat-transactivation, several other mechanisms are also associated with the establishment of HIV-1 latency, which include transcriptional interference, limited availability of transcription factors, chromatin modifications, DNA methylation, etc. Since we are focusing on P-TEFb and related factors, other molecular mechanisms of HIV-1 latency will not be discussed in this review. For more details of HIV-1 latency, please refer to relevant recent reviews (Donahue and Wainberg, 2013 2013 ; Ruelas and Greene, 2013 2013 ). Conclusions and perspectives 1 More than thirty years have passed since HIV-1 was first identified as the causative agent of acquired immune deficiency syndrome (AIDS). Significant progress has been made toward finding effective ways to suppress viral replication. The current antiretroviral therapy (ART) can inhibit the activities of several viral enzymes, prevent virus replication, and protect the immune system of the host. However, life-long treatment is still compulsive because of the existence of long-lived latency reservoirs, and there are serious side effects associated with the therapy (Hakre et al., 2012 2012 ). To eradicate the latent reservoirs, the so-called “shock and kill” strategy has been proposed, which combines the HIV-1 latency reactivation and ART treatment to achieve a functional cure for HIV/AIDS (Richman et al., 2009 2009 ; Karn, 2011 2011 ; Siliciano and Greene, 2011 2011 ; Archin et al., 2012 2012 ). Towards this goal, high specific and efficient HIV-1 latency activators have to be developed first. Since P-TEFb, a very important human transcription factor and Tat cofactor, is not present on the HIV-1 LTR in latent cells, devising effective ways to release P-TEFb from the 7SK snRNP and promote the formation of the Tat-SEC complex on the viral promoter offers a promising strategy to activate HIV-1 transcription and latency. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1400059 Project supported by the National Natural Science Foundation of China (No. 81201276) and the Natural Science Foundation of Fujian Province (No. 2012J05067), China 2014-02-28 2014-04-18 2014-04-13 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2014 2014 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 05 15