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2. P-TEFb : a key factor in the regulation of cellular processes and HIV-1 replication

2.6. P-TEFb and human disease

2.6.3. Role of P-TEFb in HIV transcription

The human immunodeficiency virus HIV-1 and HIV-2 (HIV) are lentivirus that can lead to acquired immunodeficiency syndrome (AIDS). Many viruses depend on the infected host for transcription of their genome and, in the case of HIV, it has long been recognized that P-TEFb represents an essential cofactor for HIV replication (reviewed by Garriga and Grana, 2004 ; Stevens et al, 2006). In fact, the understanding of the mechanisms by which P-TEFb stimulates transcription elongation in eukaryotic genes has largely benefited from studies of the role of P-TEFb in regulating HIV transcription.

Transcription from the HIV-1 long terminal repeat (LTR) is tightly linked to T-cell activation through the function of factors responsive to signaling by the T-cell receptor and cytokines. Most of these factors binds to the regulatory or enhancer region of the LTR, and include NF-kB, NFAT (nuclear factor of activated T-cells), RBFs (Ras-response binding factors) and AP-1 (activator protein 1) (reviewed by Sadowski and Mitchell, 2005) (Fig.

10A). The LTR core promoter region resembles to that of many eukaryotic genes transcribed by RNAPII, and contains three Sp1 binding sites, a TATA box and an initiator (Inr) region.

The transactivating region (TAR) represents a key element in the Tat-mediated transcription activation, and forms a stable stem-loop RNA structure during the early elongation phase.

Similarly to the host genes, the integrated HIV provirus is packaged into chromatin and transcription activation requires both nucleosome acetylation at the LTR promoter followed by nucleosome remodeling to facilitate PIC assembly. In this aspect, the transcription factor NF-kB plays an important role. In unstimulated cells, HIV transcription is notably repressed by the NF-kB p50 homodimer, which was shown to interact with and to

Figure 10. Transcription activation of the HIV-1 provirus. (A) Scheme of the HIV-1 LTR region. The DNA binding sites for most of the HIV-1 transcriptional regulators identified to date are indicated. STR : steroid/thyroid hormone receptor ; GR : glucocorticoid receptor ; C/EBP : CCAAT/enhancer binding protein ; USF : upstream stimulating protein ; LEF : lymphocyte enhancer factor-1 ; RBF1/2 : Ras-response binding factor 1 and 2 ; Sp1 : SP1 binding protein ; LSF : late SV40 transcription factor ; CBF : C-promoter binding factor-1 ; NFAT : nuclear factor of activated T cells. (B) Activation of HIV-1 transcription. In latently infected cells, transcription is blocked by a repressive hypoacetylated chromatin caused by the recruitment of HDACs to the LTR via NF-kB p50 homodimers, CBF-1 and LSF/YY1. Upon cell activation, the NF-kB p50/p65 heterodimer is stimulated and recruits the p300/CBP HAT to the LTR.

Nucleosome acetylation then facilitates the recruitment of SWI/SNF chromatin remodeler such as Brahma (Brm) and the subsequent assembly of the PIC.

recruit the histone deacetylase HDAC-1 to the viral promoter. Other cellular regulators such as CBF-1 (C-promoter binding factor-1) and YY1 (Yin Yang protein 1) however were also found to accomplish this task (He and Margolis, 2002 ; Tyagi and Karn, 2008). When T-cells become activated, the NF-kB p50/p65 heterodimer associates with the histone acetyltransferase p300/CBP and displaces p50-HDAC-1 from the HIV promoter, resulting in transcriptional activation (Hiscott et al., 2001 ; Zhong et al., 2002) (Fig. 10B). As discussed before, the acetylation marks are thought to facilitate chromatin remodeling by SWI/SNF complexes, enabling the subsequent assembly of the PIC and transcription initiation.

In the absence of the viral transactivator Tat, only short transcripts encompassing the TAR region are produced, while the elongation phase is aborted by the negative elongation factors NELF and DSIF. In the presence of Tat however, full-length polyadenylated transcripts are generated and, as mentioned previously, this depends on the formation of a ternary complex between Tat, P-TEFb and the TAR stem-loop RNA (Fig. 11A). Tat specifically interacts with the TRM motif of CyclinT1, allowing P-TEFb recruitment to the LTR promoter region. Hence, this association dramatically enhances the affinity of Tat/P-TEFb to the TAR RNA (Wei et al., 1998 ; Fong and Zhou, 2000). Once recruited to the polymerase, P-TEFb phosphorylates NELF, DSIF and the RNAPII CTD at Ser-2 to alleviate transcriptional pausing and to trigger productive elongation (Ping and Rana, 2001 ; Bourgeois et al., 2002 ; Fujinaga et al., 2004). Although transcription initiation at the HIV promoter requires the CTD kinase activity of TFIIH, the substrate specificity of P-TEFb towards the CTD is modified by Tat enabling the kinase to phosphorylate both Ser-5 and Ser-2 (Ping and Rana, 1999 ; Zhou et al., 2000). Interestingly, Tat and P-TEFb were also shown to facilitate the binding of TBP to the HIV promoter, and thus to stimulate subsequent rounds of PIC assembly and transcription initiation (Raha et al., 2005). Finally, in addition to Tat, NF-kB was also found to interact with TEFb and to stimulate transcription elongation through P-TEFb recruitment at the HIV promoter (West et al., 2001 ; Barboric et al., 2001).

HIV transcription is highly dependent on the availability of active P-TEFb molecules.

This notion is notably supported by the observation that HEXIM1 overexpression potently inhibit HIV expression both in vivo and in vitro (Yik et al., 2003 ; Fraldi et al., 2005 ; Shimizu et al., 2007). As mentioned earlier, both HEXIM1 and Tat contact CyclinT1 through a region near the cyclin box, and thus compete for P-TEFb interaction (Michels et al., 2003 ; Chen et al., 2004). In addition, the 3’ stem-loop of 7SK RNA that contacts CyclinT1 displays remarkable similarity to the TAR stem-loop RNA (Egloff et al., 2006 ; Zhou and Yik, 2006) (Fig. 11B). These findings led to the hypothesis that HIV has developed a RNA-dependent

Figure 11. Transcription from the HIV-1 LTR. (A) Regulation of HIV elongation by the Tat/TAR/P-TEFb complex. In absence of Tat, only short transcripts scaffolding the TAR stem-loop RNA are produced. In presence of Tat however, P-TEFb is efficiently recruited to the HIV LTR to trigger transcription elongation. The Tat/P-TEF complex interacts with TAR allowing the kinase to phosphorylate RNAPII at Ser-2, DSIF and NELF. (B) Sequence similarities between the bulge-loop region of TAR and the apical region of the 7SK RNA 3’ stem-loop. The conserved nucleotides are shaded in red (Zhou and Yik, 2006). The CyclinT1-interacting region of TAR and 7SK are highly similar, and both RNAs interacts with the same domain within the cyclin box of CyclinT1. The region involved in the Tat/TAR interaction is also indicated.

mechanism, similar to that of HEXIM1/7SK/TEFb, to alleviate the negative regulation of P-TEFb from the host cell. Consistently, it was recently shown that Tat can promote the disruption of the 7SK snRNP complex by displacing HEXIM1, and thus increasing the active pool of cellular P-TEFb for efficient HIV transcription (Barboric et al., 2007 ; Sedore et al., 2007). It has long been accepted that HIV replication in the human primary peripheral blood lymphocytes (PBL) occurs when the cells become activated. In quiescent primary T-cells, the level of cdk9 and cyclinT1 expression is low, while both genes are upregulated during T-cell activation (Marshall et al., 2005 ; Marshall and Grana, 2006). Concomitant to the increase level of P-TEFb, the 7SK RNA is also upregulated in stimulated PBL indicating that a large pool of P-TEFb is sequestered into 7SK snRNP (Haaland et al., 2003). Therefore, it appears that this reservoir of inactive P-TEFb is actually necessary for HIV replication and that Tat is required to subvert the 7SK snRNP inhibitory circuit.

In agreement with this model, all P-TEFb inhibitors identified to date (i.e. DRB, flavopiridol, roscovitine), despite being incompletely specific, can block HIV-1 replication at concentration that are not cytotoxic (Chao et al., 2000 ; Biglione et al., 2007). Similarly, a partial depletion of P-TEFb by RNAi or the expression of a dominant-negative form of Cdk9 can inhibit HIV replication without affecting cell survival (Mancebo et al., 1997 ; Flores et al., 1999 ; Chiu et al., 2004). Finally, a recent study indicates that direct inhibition of Cdk9 in PBL using a dominant-negative kinase potently inhibits HIV-1 replication without preventing T-cell activation (Salerno et al., 2007). Taken together, these findings strongly suggest that the development of highly selective P-TEFb inhibitors is a rational strategy to therapeutically suppress HIV-1 replication and possibly other pathogenic viruses.