Molecular control of HIV-1 postintegration latency: implications for the development of new therapeutic strategies

Abstract

The persistence of HIV-1 latent reservoirs represents a major barrier to virus eradication in infected patients under HAART since interruption of the treatment inevitably leads to a rebound of plasma viremia. Latency establishes early after infection notably (but not only) in resting memory CD4+ T cells and involves numerous host and viral trans-acting proteins, as well as processes such as transcriptional interference, RNA silencing, epigenetic modifications and chromatin organization. In order to eliminate latent reservoirs, new strategies are envisaged and consist of reactivating HIV-1 transcription in latently-infected cells, while maintaining HAART in order to prevent de novo infection. The difficulty lies in the fact that a single residual latently-infected cell can in theory rekindle the infection. Here, we review our current understanding of the molecular mechanisms involved in the establishment and maintenance of HIV-1 latency and in the transcriptional reactivation from latency. We highlight the potential of new therapeutic strategies based on this understanding of latency. Combinations of various compounds used simultaneously allow for the targeting of transcriptional repression at multiple levels and can facilitate the escape from latency and the clearance of viral reservoirs. We describe the current advantages and limitations of immune T-cell activators, inducers of the NF-kappaB signaling pathway, and inhibitors of deacetylases and histone- and DNA- methyltransferases, used alone or in combinations. While a solution will not be achieved by tomorrow, the battle against HIV-1 latent reservoirs is well- underway.

Figure 1

A simplified view of the multiple mechanisms of transcriptional interference implicated in HIV-1 postintegration latency. (a) HIV-1 integrates into the host cell genome predominantly in intronic regions of actively transcribed genes [55-57]. Transcriptional interference may lead to the establishment of latency by different mechanisms depending at least on the orientation of viral integration compared to the host gene. (b) steric hindrance: when proviral integration occurs in the same transcriptional orientation as the cellular host gene, "read-through" RNA polymerase II (RNAPII) transcription from the upstream promoter displaces key transcription factors (TFs) from the HIV-1 promoter [60] and prevents assembly of the pre-initiation complex on the viral promoter. The integrated virus is thought to be transcribed along with the other intronic regions of the cellular gene, but is then merely spliced out. HIV-1 transcription inhibition could be reversed by hindering the upstream transcription or by cooperatively activating viral transcription initiation and elongation; certain host transcription factors and/or viral activators, which bind strongly to their cognate site, could resist the "read-through" RNAPII passage [61]. This phenomenon was also observed following Tat-mediated transactivation of HIV-1 transcription [67]. (c) promoter occlusion: provirus integration in the opposite orientation compared to the host gene may lead to collisions of the elongating RNA polymerases from each promoter, resulting in a premature termination of transcription from the weaker or from both promoters. (d) enhancer trapping: an enhancer of one gene (the 5'LTR enhancer of HIV-1 in this case) is placed out of context near the promoter of a second gene (a cellular gene in this case) and acts on the transcriptional activity of this cellular promoter, thereby preventing the enhancer action on the 5'LTR.

Figure 2

Transcription factor binding sites and chromatin organization in the 5'LTR and leader region of HIV-1. (A) Representation of the HIV-1 genome. The intragenic hypersensitive site HS7 located in the pol gene is indicated. (B) Schematic representation of the main transcription factor binding sites located in the 5'LTR and in the beginning of the leader region of HIV-1. The U3, R, U5 and leader regions are indicated. Nucleotide 1 (nt1) is the start of U3 in the 5'LTR. The transcription start site corresponds to the junction of U3 and R. (C) Schematic representation of the nucleosomal organization of the HIV-1 genome 5' region. Hypersensitive sites HS2, HS3 and HS4 are indicated. The assignment of nucleosome position in this region is based on DNase I, micrococcal nuclease and restriction enzyme digestion profiles [72,73]. During transcriptional activation, a single nucleosome, named nuc-1 and located immediately downstream of the transcription start site, is specifically and rapidly remodeled [73].

Figure 3

HDAC and HAT recruitment to the HIV-1 5'LTR. (A) During latency, nuc-1 blocks transcriptional initiation and/or elongation because it is maintained hypoacetylated by nearby recruited HDACs. The targeting of nuc-1 by these HDACs is mediated by their recruitment to the 5'LTR via several transcription factor binding sites. Thin arrows indicate that the implicated transcription factors were demonstrated to recruit HDACs to the 5'LTR (by ChIP experiments or following knock-down of the corresponding transcription factor). The dotted arrow indicates that the USF transcription factor could potentially recruit HDAC-3 to the nuc-1 region based on its interactome partners in the literature, but this recruitment has not been demonstrated so far in the specific context of the HIV-1 promoter. (B) Nuc-1 is a major obstacle to transcription and has to be remodeled to activate transcription. This disruption could happen following local recruitment of HATs by DNA-binding factors, and/or by the viral protein Tat, which binds to the neo-synthesized TAR element. This would result in nuc-1 hyperacetylation and remodeling, thereby eliminating the block to transcription at least for certain forms of viral latency. This acetylation-based activation model has been validated notably regarding the involvement of the transcription factors NF-κB p65 and Tat.

Figure 4

Mechanisms of transcriptional activation by the viral protein Tat. (A) In the absence of Tat, transcription from the HIV-1 5'LTR produces predominantly short mRNAs as a result of the activity of the negative elongation factor N-TEF, composed of NELF and DSIF, which binds to the hypophosphorylated RNA polymerase II and impedes transcriptional elongation. (B) Following the synthesis of the first molecules of Tat, this viral protein migrates to the nucleus. Tat then binds to the RNA hairpin TAR, located in the 5' region of all nascent HIV-1 transcripts and activates viral transcription by recruiting the positive elongation factor pTEFb, composed of Cdk9 and CyclT1. This recruitment is enhanced through Tat acetylation by PCAF on K28, located in the transactivation domain of the viral Tat protein. Cdk9 phosphorylates the CTD domain of RNAPII, leading to processive transcriptional elongation and to the dissociation of N-TEF. Acetylation of Tat on K50 by p300 and Gcn5 promotes the release of pTEFb [246], dissociation of Tat from TAR and its subsequent transfer to the elongating polymerase complex. Tat then recruits PCAF to the elongation complex. Tat also recruits the ATP-dependent remodeling complex SWI/SNF. Another model based on FRAP experiments propose that the Tat/pTEFb complex dissociates from the RNAPII complex following transcription initiation and undergoes subsequent cycles of association/dissociation [249]. (C) At the end of the elongation process, Tat deacetylation by the class III HDAC Sirtuin 1 allows its dissociation from RNAPII and from PCAF, and the recycling of Tat initiates a new cycle of transcriptional activation.

Figure 5

Model for CTIP-2-mediated establishment of a heterochromatin environment at the HIV-1 promoter region. (A) CBP recruitment occurs following HIV-1 activation with phobol esters via CTIP-2, which binds to Sp1 binding sites. (B) However, in latent conditions, the corepressor CTIP-2 interacts with the Sp1 transcription factor at three sites in the HIV-1 5'LTR and consequently recruits HDAC-1 and HDAC-2, leading to H3K9 deacetylation in the nuc-1 region. (C) CTIP-2 then recruits the HMT Suv39 h1, which trimethylates H3K9 (a repressive chromatin mark). (D) This latter epigenetic modification allows HP1 binding and polymerization, heterochromatin formation and propagation at least to the nuc-2 region, and in fine the establishment of HIV-1 silencing [202,203]. This mechanism of viral latency can be revoked by different treatment strategies: HDACIs hinder the repressive action of HDACs and specific HMTIs directed against Suv39 h1 could avoid the recruitment of this silencing machinery.