Molecular mechanisms of HIV latency

Abstract

HIV seeds reservoirs of latent proviruses in the earliest phases of infection. These reservoirs are found in many sites, including circulating cells, the lymphoid system, the brain, and other tissues. The "shock and kill" strategy, where HIV transcription is reactivated so that antiretroviral therapy and the immune system clear the infection, has been proposed as one approach to curing AIDS. In addition to many defective viruses, resting hematopoietic cells harbor transcriptionally latent HIV. Understanding basic mechanisms of HIV gene expression provides a road map for this strategy, allowing for manipulation of critical cellular and viral transcription factors in such a way as to maximize HIV gene expression while avoiding global T cell activation. These transcription factors include NF-κB and the HIV transactivator of transcription (Tat) as well as the cyclin-dependent kinases CDK13 and CDK11 and positive transcription elongation factor b (P-TEFb). Possible therapies involve agents that activate these proteins or release P-TEFb from the inactive 7SK small nuclear ribonucleoprotein (snRNP). These proposed therapies include PKC and MAPK agonists as well as histone deacetylase inhibitors (HDACis) and bromodomain and extraterminal (BET) bromodomain inhibitors (BETis), which act synergistically to reactivate HIV in latently infected cells.

Figure 3. Combinatorial activation of TFs for HIV reactivation from latency.

Mechanisms of action of PKC agonists and LRAs for increased HIV gene expression as well as the V-PAC assay are shown. PKC agonists activate NF-κB and increase levels of transcriptional CDKs, P-TEFb, CycK/CDK13, and CycL/CDK11. LRAs release P-TEFb from the 7SK snRNP. This rapid release is followed by the reassembly of the 7SK snRNP, which returns activated cells to their resting states. Tat interrupts this P-TEFb cycle (see Figure 1). V-PAC measures the release of P-TEFb from the 7SK snRNP in living cells; thus, it measures the strength and kinetics of LRA effects. In this BiFC, the two halves of YFP are linked to the CTD of RNAPII and P-TEFb. When P-TEFb is released and phosphorylates the CTD, cells turn green, especially in nuclear speckles, which are sites of active transcription (inset).

Figure 2. Transcriptional interference.

(A) HIV utilizes transcriptional interference for its own replication. The 5′ LTR initiates and elongates transcription. The 3′ LTR is occluded because RNAPII displaces TFs from its DNA. Thus, RNAPII terminates past TAR near the polyA site in the 3′ LTR, where CPA of HIV transcripts takes place. However, the same RNAPII transcribes HIV sequences and host cell genes. Thus, when HIV integrates in the sense orientation within a host cell gene, the transcribing RNAPII terminates in the 5′ LTR (B). The 3′ LTR now initiates transcription, which, in the absence of Tat, does not elongate efficiently. When HIV integrates in the antisense orientation within a host gene, RNAPII reads through without stopping (C), and HIV antisense transcripts are released. When integration occurs in introns, HIV RNAs are degraded rapidly. The strength of transcriptional interference depends on rates of transcription of host cell genes. High levels of NF-κB, which bind to DNA tightly, counteract transcriptional interference. Blue and red circles represent 5′ caps; rising blue and red lines represent mRNA; squiggles represent polyA tails of host cell and HIV mRNAs.

Figure 1. HIV LTR, TFs, and their activation.

HIV transcription starts at the TSS, where the initiator element binds. The compact promoter contains a TATA box and 3 SP1 sites. The enhancer contains overlapping NF-κB and nuclear factor of activated T cells (NF-AT) binding sites (NF-AT sites not shown). The promoter recruits RNAPII. During promoter clearance, CTD Ser5 (red circles) is phosphorylated by CycH/CDK7. P-TEFb is recruited to the HIV LTR via NF-κB (p50/p65 heterodimer), the super elongation complex (SEC), and Tat. Upon recruitment, P-TEFb phosphorylates Ser2. Tat binds to TAR RNA from positions +1 to +60 in all HIV transcripts. Tat and P-TEFb bind to the 5′ bulge and central loop in TAR, respectively. P-TEFb also phosphorylates Spt5 in DSIF and NELF-E, which releases the arrested RNAPII for elongation. PKC or MAPK agonists promote the translocation of NF-κB to the nucleus and increase the synthesis of P-TEFb. LRAs as well as stress (apoptosis, UV light, transcriptional blockers) release P-TEFb from the 7SK snRNP, where P-TEFb is inactivated by HEXIM1. Tyr271 in HEXIM1 binds and occludes the ATP pocket in CDK9. Active CDK9 is phosphorylated on Thr186 in the T loop and Ser175. Note the similarity between TAR and the first stem loop in 7SK snRNA, which allows Tat and HEXIM1 to bind to both structures. Tat also competes with HEXIM1 for P-TEFb. Thus, when sufficient amounts of Tat are made, HIV transcription continues despite increased levels of 7SK snRNP.