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Review
. 2024 Apr 5;21(1):6.
doi: 10.1186/s12977-024-00639-w.

The cell biology of HIV-1 latency and rebound

Uri Mbonye  1 , Jonathan Karn  2
Affiliations
Review

The cell biology of HIV-1 latency and rebound

Uri Mbonye et al. Retrovirology. .

Abstract

Transcriptionally latent forms of replication-competent proviruses, present primarily in a small subset of memory CD4+ T cells, pose the primary barrier to a cure for HIV-1 infection because they are the source of the viral rebound that almost inevitably follows the interruption of antiretroviral therapy. Over the last 30 years, many of the factors essential for initiating HIV-1 transcription have been identified in studies performed using transformed cell lines, such as the Jurkat T-cell model. However, as highlighted in this review, several poorly understood mechanisms still need to be elucidated, including the molecular basis for promoter-proximal pausing of the transcribing complex and the detailed mechanism of the delivery of P-TEFb from 7SK snRNP. Furthermore, the central paradox of HIV-1 transcription remains unsolved: how are the initial rounds of transcription achieved in the absence of Tat? A critical limitation of the transformed cell models is that they do not recapitulate the transitions between active effector cells and quiescent memory T cells. Therefore, investigation of the molecular mechanisms of HIV-1 latency reversal and LRA efficacy in a proper physiological context requires the utilization of primary cell models. Recent mechanistic studies of HIV-1 transcription using latently infected cells recovered from donors and ex vivo cellular models of viral latency have demonstrated that the primary blocks to HIV-1 transcription in memory CD4+ T cells are restrictive epigenetic features at the proviral promoter, the cytoplasmic sequestration of key transcription initiation factors such as NFAT and NF-κB, and the vanishingly low expression of the cellular transcription elongation factor P-TEFb. One of the foremost schemes to eliminate the residual reservoir is to deliberately reactivate latent HIV-1 proviruses to enable clearance of persisting latently infected cells-the "Shock and Kill" strategy. For "Shock and Kill" to become efficient, effective, non-toxic latency-reversing agents (LRAs) must be discovered. Since multiple restrictions limit viral reactivation in primary cells, understanding the T-cell signaling mechanisms that are essential for stimulating P-TEFb biogenesis, initiation factor activation, and reversing the proviral epigenetic restrictions have become a prerequisite for the development of more effective LRAs.

Keywords: 7SK snRNP; Epigenetic silencing; HIV-1 Tat; HIV-1 latency; HIV-1 reservoir; Latency reversal; P-TEFb; T-cell receptor signaling; Transcription elongation.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
HIV reservoir formation and dynamics. A The reservoir is established primarily in memory CD4+ T cells arising during the transition of infected effector cells to achieve immunological memory. Naïve cells become activated during HIV-1 infection due to HIV-1 itself and other antigenic stimuli. The resulting activated effector cells are ideal targets for productive HIV-1 infection. A large fraction of the infected effector cells will not survive, but an important subset become quiescent and transition to a memory cell phenotype, thereby silencing HIV-1. B The primary mechanism for reservoir persistence is due to the clonal expansion of partially activated latently infected cells due to homeostatic proliferation driven by IL-7 or antigen stimulation. Different viral clones reactivate and expand under different conditions, with some clones being reduced or eliminated due to viral cytopathic effects. The result is a gradual simplification of the clonal population as demonstrated by recurring integration site sequences (denoted in the figure by different colors for the proviruses)
Fig. 2
Fig. 2
Epigenetic control of HIV-1 transcription initiation. The structure of the HIV-1 LTR and flanking nucleosomes (Nuc-0, Nuc-1, and Nuc-2) is shown at the center. In the latent state, the proviral promoter is bound by repressive trans-acting factors, including CBF-1, YY1, and NF-κB p50/p50, which direct the recruitment of histone deacetylase enzymes (HDACs). Subsequent occupancy of the promoter by the polycomb repressive complex 2 (PRC2) and EHMT2 results in the methylation of the deacetylated histone H3 at Lys27 and Lys9 positions, respectively. PRC2 also functions to recruit the polycomb repressive complex 1 (PRC1), in part via the binding of CBX4 to H3K27me3. Methylation of the 5’ CpG island (5ʹ CpGI) by DNMT1 and DNMT3a promotes the repressive histone methylation status of Nuc-1 by mediating the recruitment of UHRF1 and the HDAC-containing NuRD complex through MBD2. The SWI/SNF chromatin remodeling complex BAF interacts with Nuc-1 and is required to maintain increased Nuc-1 density around the HIV-1 transcription start site. By associating with CBX4, CAF-1, and PML, latent proviruses are likely to be situated in liquid–liquid phase-separated (LLPS) nuclear condensates that may concentrate transcriptionally poised genes with repressive heterochromatic features. CAF-1 may also play a central role in the initial assembly of nucleosomes at the provirus following integration and DNA replication. Typical of bivalent promoters, the HIV-1 LTR is in a reversible epigenetically repressed state poised for rapid inducible transcription. Recruitment of histone acetyltransferases, the H3K27me3 demethylase UTX/KMDA, and the PBAF SWI/SNF remodeling complex to the HIV-1 promoter following the nuclear induction of transcription activators enables the displacement of Nuc-1 and Nuc-2 thereby stimulating viral transcription initiation
Fig. 3
Fig. 3
Stimulation of efficient HIV-1 transcription elongation through Tat-dependent P-TEFb recruitment and remodeling of the chromatin barrier. The current understanding of the regulation of processive HIV-1 transcription, as portrayed here, is based mainly on studies conducted using cell line models. A Epigenetic repressive features at the proviral promoter prevent the recruitment of RNA polymerase II (RNAP II) and assembly of the preinitiation complex, thereby restricting the expression of Tat. Latent proviruses also characteristically possess elevated acetylated histone H4 (AcH4) levels that permit their occupancy by the short isoform of BRD4, which reinforces viral latency through direct recruitment of BAF SWI/SNF complexes. Without Tat expression, an accumulation of inefficiently transcribing promoter-proximally paused RNAP II complexes due to NELF and DSIF activity may lead to abortive transcription. B Chromatin remodeling and efficient assembly of preinitiation complexes may initially allow for the onset of Tat-independent transcription elongation likely occurring through the recruitment of P-TEFb by NF-κB, BRD4, TRIM28, or HSF1. Synthesized Tat efficiently trans-activates HIV-1 transcription elongation by outcompeting BRD4 for P-TEFb binding and recruiting P-TEFb with the super elongation complex (SEC) to the TAR hairpin. P-TEFb eventually phosphorylates the RNAP II C-terminal domain (CTD), its linker region between the polymerase core and CTD, the SPT5 subunit of DSIF, and the NELF-E subunit. Phosphorylated DSIF is transformed into a positive elongation factor, while NELF-E phosphorylation leads to the dissociation of the NELF complex from RNAP II. The phosphorylated RNAP II CTD and linker provide a scaffold to anchor the histone chaperone SPT6, which, along with FACT, is essential in enabling the elongating machinery to transcribe through nucleosomal barriers. These RNAP II phospho-modifications may also anchor elongation factors, co-transcriptional processing complexes, and chromatin-modifying enzymes
Fig. 4
Fig. 4
Biogenesis of P-TEFb in primary T cells and proposed mechanism for the recruitment of Tat:P-TEFb to the HIV-1 provirus. A P-TEFb is expressed in resting memory CD4+ T cells at vanishingly low levels due to posttranscriptional mechanisms that limit CycT1 expression. This causes the CDK9 subunit to be sequestered in the cytoplasm by the kinase-specific chaperone complex Hsp90/Cdc37. TCR co-stimulation induces CycT1 protein synthesis, leading to the heterodimeric assembly of P-TEFb that is stabilized by CDK9 phosphorylation at Thr186. Upon assembly, P-TEFb then enters the nucleus where it is incorporated into the 7SK snRNP complex. BRD4 and Tat can physically engage with 7SK snRNP but compete with one another to dissociate P-TEFb from the complex. The signal-dependent modification pSer175 CDK9 preferentially enhances the binding of Tat to P-TEFb. Ser175 on the activation loop of CDK9 is an essential contact point for BRD4; mutation of Ser175 to a phosphomimetic residue or an alanine produces severe defects in the association of P-TEFb with BRD4. B Microscopic imaging of the assembly of P-TEFb in memory CD4+ T cells following TCR activation. Cells were stimulated or not with soluble anti-CD3 and anti-CD28 antibodies. After immunostaining, images were captured by deconvolution microscopy at 60x. Scale bar: 10 μm. CDK9 is sequestered in the cytoplasm in the unstimulated cells, and CycT1 is present in very low amounts. After stimulation, CDK9 and synthesized CycT1 are present in the nucleus as a complex (P-TEFb), presumably sequestered by 7SK snRNP. C Combined immunofluorescence and RNA FISH detection of CycT1 and 7SK snRNA in memory CD4+ T cells. Cells were stimulated or not with anti-CD3 and anti-CD28 Dynabeads at a 1:1 bead-to-cell ratio. Images were captured by deconvolution microscopy at 100x. Scale bar: 1 μm. Cellular stimulation results in a doughnut-shaped redistribution of 7SK snRNA within the nucleus that tightly colocalizes with induced CycT1
Fig. 5
Fig. 5
Membrane events and generation of intracellular second messengers essential for TCR-induced mobilization of activators of transcription initiation and P-TEFb. During antigen presentation, maximal activation of TCR signaling requires a costimulatory engagement that usually involves a pairwise linkage between B7 and its T-cell counterpart CD28. TCR stimulation triggers a receptor and non-receptor phospho-tyrosine cascade that results in the generation of diacylglycerol (DAG), intracellular mobilization of calcium (Ca++) from the endoplasmic reticulum (ER), and formation of phosphatidylinositol-3,4,5 triphosphate (PIP3) which serves to anchor PDK1, AKT, and mTORC2 to the membrane. PLCγ-generated DAG initiates the membrane recruitment and activation of PKC-θ and RasGRP. DAG can be phosphorylated by diacylglycerol kinase (DGK), which leads to the curtailing of DAG signaling and the generation of an additional lipid second messenger phosphatidic acid (PA)
Fig. 6
Fig. 6
TCR signaling pathways in primary CD4+ T cells contribute to the stimulation of proviral HIV-1 transcription. RasGRP1-Ras-Raf-MEK-ERK1/2 and PI3K-mTORC2-AKT-mTORC1 complement one another in stimulating the posttranscriptional synthesis of CycT1, leading to P-TEFb assembly. The exact mechanisms by which ERK and mTORC1 stimulate CycT1 translation are yet to be fully delineated. Although intracellular calcium release, the activation of PKC-θ and the JNK MAPK pathway are dispensable for the formation of P-TEFb, they are likely to mediate the recruitment of RNA polymerase II (RNAP II) to proviral HIV and thus its eventual phosphorylation by P-TEFb to stimulate processive transcription elongation
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