HIV Provirus Stably Reproduces Parental Latent and Induced Transcription Phenotypes Regardless of the Chromosomal Integration Site

Abstract

Understanding the mechanisms of HIV proviral latency is essential for development of a means to eradicate infection and achieve a cure. We have previously described an in vitro latency model that reliably identifies HIV expression phenotypes of infected cells using a dual-fluorescence reporter virus. Our results have demonstrated that ∼50% of infected cells establish latency immediately upon integration of provirus, a phenomenon termed early latency, which appears to occur by mechanisms that are distinct from epigenetic silencing observed with HIV provirus that establishes productive infections. In this study, we have used a mini-dual HIV reporter virus (mdHIV) to compare the long-term stability of provirus produced as early latent or productive infections using Jurkat-Tat T cell clones. Cloned lines bearing mdHIV provirus integrated at different chromosomal locations display unique differences in responsiveness to signaling agonists and chromatin-modifying compounds, and they also produce characteristic expression patterns from the 5′ long terminal repeat (LTR) dsRed and internal EIF1α-enhanced green fluorescent protein (EIF1α-eGFP) reporters. Furthermore, reporter expression profiles of single cell sorted subcultures faithfully reproduce expression profiles identical to that of their original parental population, following prolonged growth in culture, without shifting toward expression patterns resembling that of cell subclones at the time of sorting. Comparison of population dispersion coefficient (CV) and mean fluorescence intensity (MFI) of the subcloned lines showed that both untreated and phorbol myristate acetate (PMA)-ionomycin-stimulated cultures produce expression patterns identical to those of their parental lines. These results indicate that HIV provirus expression characteristics are strongly influenced by the epigenetic landscape at the site of chromosomal integration. IMPORTANCE There is currently considerable interest in development of therapies to eliminate latently infected cells from HIV-infected patients on antiretroviral therapy. One proposed strategy, known as “shock and kill,” would involve treatment with therapies capable of inducing expression of latent provirus, with the expectation that the latently infected cells could be killed by a host immune response or virus-induced apoptosis. In clinical trials, histone deacetylase (HDAC) inhibitors were shown to cause reactivation of latent provirus but did not produce a significant effect toward eliminating the latently infected population. Results shown here indicate that integration of HIV provirus at different chromosomal locations produces significant effects on the responsiveness of virus expression to T cell signaling agonists and chromatin-modifying compounds. Given the variety of phenotypes produced by integrated provirus, it is unlikely that any single potential shock-and-kill therapy will be effective toward purging the latently infected population.

Compounds producing an effective combinatorial regimen for disruption of HIV ‐1 latency

“…HIV‐1 tends to integrate into actively transcribed regions of the host genome (Jordan et al , ; Lewinski et al , ; Ikeda et al , ). Each HIV‐1‐infected patient carries viral DNA integrated at different chromosomal locations, and as we have previously shown, the site of viral integration influences how latently infected T cells respond to different stimuli (Hashemi et al , ). Therefore, an ideal LRA should be capable of disrupting latency in cell models carrying integrated viral reporters at multiple different regions within the host genome.…”

Diversity of small molecule HIV‐1 latency reversing agents identified in low‐ and high‐throughput small molecule screens

“…This limitation can likely be attributed to inadequate anti‐HIV‐1 cellular immune response in these patients, but also because of limitations of the HDACIs, which are only capable of inducing a subset of provirus integrations. Several studies have confirmed this limitation using cell lines with HIV‐1 reporter virus where it was shown that the site of chromosomal integration influences responsiveness to signaling agonists and chromatin‐modifying agents . Importantly, therefore, a critical property of LRAs is the capacity to induce a broad range of provirus integrated at diverse chromosomal locations.…”

BI-2536 and BI-6727, dual Polo-like kinase/bromodomain inhibitors, effectively reactivate latent HIV-1

“…Although the causes of the differences in responsiveness are not clear, the following possible mechanisms may be involved. Cell types or subtypes contributing to HIV-1 latency or ratios of each of them to the total number of latently infected cells may be different among HIV-1 infected patients, resulting in a divergence in drug sensitivity among the samples; the difference in drug responsiveness may be due to different epigenetic modification of the integrated provirus among infected cells, as it has been reported that the epigenetic environment at the site of chromosomal integration significantly affects responsiveness to LRAs 21 . If this is the case, different bromodomain proteins may be involved in HIV-1 latency in each patient.…”

“…Despite clinical trials on the therapeutic ‘shock and kill” potential of several LRAs 7 , none have effectively reduced the size of HIV-1 reservoirs possibly because the responsiveness of the latent provirus to each LRA may depend on the reservoir cell type. In addition, the chromosomal location of the integrated provirus may influence the responsiveness of the infected cells to LRAs 21 . Therefore, development of new LRAs or combination therapy using multiple LRAs to efficiently reactivate latent HIV-1 provirus is a priority…”