Experimental Systems for Measuring HIV Latency and Reactivation

Abstract

The final obstacle to achieving a cure to HIV/AIDS is the presence of latent HIV reservoirs scattered throughout the body. Although antiretroviral therapy maintains plasma viral loads below the levels of detection, upon cessation of therapy, the latent reservoir immediately produces infectious progeny viruses. This results in elevated plasma viremia, which leads to clinical progression to AIDS. Thus, if a HIV cure is ever to become a reality, it will be necessary to target and eliminate the latent reservoir. To this end, tremendous effort has been dedicated to locate the viral reservoir, understand the mechanisms contributing to latency, find optimal methods to reactivate HIV, and specifically kill latently infected cells. Although we have not yet identified a therapeutic approach to completely eliminate HIV from patients, these efforts have provided many technological breakthroughs in understanding the underlying mechanisms that regulate HIV latency and reactivation in vitro. In this review, we summarize and compare experimental systems which are frequently used to study HIV latency. While none of these models are a perfect proxy for the complex systems at work in HIV+ patients, each aim to replicate HIV latency in vitro.

Keywords: HIV; NFkB; P-TEFb; latency; latency reversing agents; silencing; transcription; transcription interference.

Figure 1

Two major cellular cyclin/Cdk complexes play key roles in HIV transcription. P-TEFb (CycT1:CDK9, green) is recruited to the transcription machinery on HIV LTR by various factors. Epigenetic factor Brd4, DNA-bound transactivator NFκB, and viral Tat (associated with cellular super elongation complex) bind to CycT1 and recruit P-TEFb to HIV provirus in an active chromatin environment. P-TEFb then phophorylates RNAPII and negative transcription elongation factors NELF and DSIF, which augments transcriptional elongation. CycL:Cdk11 complex (yellow), in turn, associated with transcription/export and THO complex (TREX/THOC) is recruited to RNAPII transcribing HIV genes. Cdk11 phosphorylates the CTD of RNAPII and promotes the assembly of cleavage and polyadenylation (CPA) factors at the 3′ end of genes, which ensures optimal expression of HIV mRNAs.

Figure 2

Host–viral hybrid RNAs are expressed by TI in latently infected cells. (A) When HIV integrates in the sense orientation within a host cell gene, the transcribing RNAPII reads through the boundary between the host gene and the 5’LTR or the HIV proviral DNA, and terminates in the 5′-LTR (past TAR near the poly A site), resulting in the expression of host–viral hybrid (HVH) RNAs. (B) When HIV integrates in the antisense orientation within a host gene, RNAPII reads through without stopping and HVH RNAs containing HIV antisense RNA are produced.