Control of Stochastic Gene Expression by Host Factors at the HIV Promoter

Abstract

The HIV promoter within the viral long terminal repeat (LTR) orchestrates many aspects of the viral life cycle, from the dynamics of viral gene expression and replication to the establishment of a latent state. In particular, after viral integration into the host genome, stochastic fluctuations in viral gene expression amplified by the Tat positive feedback loop can contribute to the formation of either a productive, transactivated state or an inactive state. In a significant fraction of cells harboring an integrated copy of the HIV-1 model provirus (LTR-GFP-IRES-Tat), this bimodal gene expression profile is dynamic, as cells spontaneously and continuously flip between active (Bright) and inactive (Off) expression modes. Furthermore, these switching dynamics may contribute to the establishment and maintenance of proviral latency, because after viral integration long delays in gene expression can occur before viral transactivation. The HIV-1 promoter contains cis-acting Sp1 and NF-κB elements that regulate gene expression via the recruitment of both activating and repressing complexes. We hypothesized that interplay in the recruitment of such positive and negative factors could modulate the stability of the Bright and Off modes and thereby alter the sensitivity of viral gene expression to stochastic fluctuations in the Tat feedback loop. Using model lentivirus variants with mutations introduced in the Sp1 and NF-κB elements, we employed flow cytometry, mRNA quantification, pharmacological perturbations, and chromatin immunoprecipitation to reveal significant functional differences in contributions of each site to viral gene regulation. Specifically, the Sp1 sites apparently stabilize both the Bright and the Off states, such that their mutation promotes noisy gene expression and reduction in the regulation of histone acetylation and deacetylation. Furthermore, the NF-κB sites exhibit distinct properties, with κB site I serving a stronger activating role than κB site II. Moreover, Sp1 site III plays a particularly important role in the recruitment of both p300 and RelA to the promoter. Finally, analysis of 362 clonal cell populations infected with the viral variants revealed that mutations in any of the Sp1 sites yield a 6-fold higher frequency of clonal bifurcation compared to that of the wild-type promoter. Thus, each Sp1 and NF-κB site differentially contributes to the regulation of viral gene expression, and Sp1 sites functionally “dampen” transcriptional noise and thereby modulate the frequency and maintenance of this model of viral latency. These results may have biomedical implications for the treatment of HIV latency.

Functional Incompatibility between the Generic NF-κB Motif and a Subtype-Specific Sp1III Element Drives the Formation of the HIV-1 Subtype C Viral Promoter

“…1B) (31). Importantly, experimental evidence is suggestive that the Sp1III site is functionally more important for the viral promoter than the other two (18,19,32). Given the biological significance of the centrally located Sp1III motif and the subtype-associated variations within this motif among HIV-1 subtypes, we asked if the heterologous Sp1III sequences derived from the other HIV-1 subtypes can substitute for the original Sp1III sequence in C-LTR.…”

“…The infectious titer of each viral stock was determined by multiplying the cell count by the dilution factor. Pseudotyped lentiviral vectors used for the chromatin immunoprecipitation (ChIP) analysis were packaged in HEK 293T cells (18). Briefly, cells were seeded in 100-mm culture dishes and transfected using the calcium phosphate transfection protocol with a total of 20 g of plasmid DNA pool.…”

“…Of the three motifs, the Sp1III site plays a more deterministic role in gene expression from the LTR, as Sp1 protein binding at this site must physically interact with the p65 protein of NF-B recruited to the H-B site located immediately upstream (17). Thus, the Sp1III site critical for the regulation of gene expression for the viral promoter demonstrates subtype-specific sequence variations, and the influence of such variations has not been adequately examined (18,19). Third, in contrast to the large genetic variation of the Sp1 sites, the sequence context of the NF-B binding sites is invariable in the viral strains regardless of the copy number difference.…”

“…In previous studies, an active functional interaction in the B-LTR was demonstrated between the centrally positioned NF-B and Sp1III sites in such a way that not only the spacing between the sites but also the relative orientation of each site with respect to the other cannot be altered, suggesting that the host factors must bind the specific elements in a specific orientation (17,21). Furthermore, the centrally positioned NF-B and Sp1III motifs play a role, more critical than that of the flanking sites (one upstream H-B site and two downstream Sp1 motifs), in controlling viral gene expression (18,19). Given the special significance attached to the central NF-B and the Sp1III motifs in the HIV-1 LTR, it is enigmatic that a genetically different NF-B site (the C-B motif) is inserted in the subtype C-LTR, disrupting the original association between the H-B and Sp1III motifs.…”

Potential Implication of Residual Viremia in Patients on Effective Antiretroviral Therapy

“…However, these infected cell types must have avoided virus-specific immune responses and viral cytopathicity for their long-term survival as well as for the persistent release of viruses in the body. Whereas viral loads quickly decline after ART initiation 1,2 and eventually become undetectable after several months of treatment, 2 residual virus-infected cells still continue to produce the virus at low but perhaps fluctuating levels, 83,84 giving rise to residual viremia during suppressive ART.…”