Targeted Chromatinization and Repression of HIV-1 Provirus Transcription with Repurposed CRISPR/Cas9

The major barrier to HIV-1 cure is the persistence of latent provirus, which is not eradicated by antiretroviral therapy. The “shock and kill” approach entails stimulating viral production with latency-reversing agents followed by the killing of cells actively producing the virus by immune clearance. However, this approach does not induce all intact proviruses, leaving a residual reservoir. CRISPR/Cas9 has been utilized to excise integrated Human Immunodeficiency Virus (HIV) DNA from infected cells in an RNA-guided, sequence-specific manner. Here, we seek to epigenetically silence the proviral DNA by introducing nuclease-deficient disabled Cas9 (dCas9) coupled with a transcriptional repressor domain derived from Kruppel-associated box (KRAB). We show that specific guide RNAs (gRNAs) and dCas9-KRAB repress HIV-1 transcription and reactivation of latent HIV-1 provirus. This repression is correlated with chromatin changes, including decreased H3 histone acetylation and increased histone H3 lysine 9 trimethylation, histone marks that are associated with transcriptional repression. dCas9-KRAB-mediated inhibition of HIV-1 transcription suggests that CRISPR can be engineered as a tool for block-and-lock strategies.

Posttranscriptional Regulation of HIV-1 Gene Expression during Replication and Reactivation from Latency by Nuclear Matrix Protein MATR3

ABSTRACTPosttranscriptional regulation of HIV-1 replication is finely controlled by viral and host factors. Among the former, Rev controls the export of partially spliced and unspliced viral RNAs from the nucleus and their translation in the cytoplasm or incorporation into new virions as genomic viral RNA. To investigate the functional role of the Rev cofactor MATR3 in the context of HIV infection, we modulated its expression in Jurkat cells and primary peripheral blood lymphocytes (PBLs). We confirmed that MATR3 is a positive regulator of HIV-1 acting at a posttranscriptional level. By applying the same approach to J-lat cells, a well-established model for the study of HIV-1 latency, we observed that MATR3 depletion did not affect transcriptional reactivation of the integrated provirus, but caused a reduction of Gag production. Following these observations, we hypothesized that MATR3 could be involved in the establishment of HIV-1 posttranscriptional latency. Indeed, mechanisms acting at the posttranscriptional level have been greatly overlooked in favor of transcriptional pathways. MATR3 was almost undetectable in resting PBLs, but could be promptly upregulated upon cellular stimulation with PHA. However, HIV latency-reversing agents were poor inducers of MATR3 levels, providing a rationale for their inability to fully reactivate the virus. These data have been confirmedex vivoin cells derived from patients under suppressive ART. Finally, in the context of MATR3-depleted J-lat cells, impaired reactivation by SAHA could be fully rescued by MATR3 reconstitution, demonstrating a direct role of MATR3 in the posttranscriptional regulation of HIV-1 latency.IMPORTANCEThe life cycle of HIV-1 requires integration of a DNA copy into the genome of the host cell. Transcription of the viral genes generates RNAs that are exported to the cytoplasm with the contribution of viral and cellular factors to get translated or incorporated in the newly synthesized virions. It has been observed that highly effective antiretroviral therapy, which is able to reduce circulating virus to undetectable levels, cannot fully eradicate the virus from cellular reservoirs that harbor a transcriptionally latent provirus. Thus, persistence of latently infected cells is the major barrier to a cure for HIV-1 infection. In order to purge these reservoirs of latently infected cells, it has been proposed to activate transcription to stimulate the virus to complete its life cycle. This strategy is believed to unmask these reservoirs, making them vulnerable to the immune system. However, limited successes of this approach may indicate additional posttranscriptional restrictions that need to be overcome for full virus reactivation. In this work we identify the cellular protein MATR3 as an essential cofactor of viral RNA processing. Reactivation of HIV-1 transcriptionper seis not sufficient to allow completion of a full life cycle of the virus if MATR3 is depleted. Furthermore, MATR3 is poorly expressed in quiescent CD4+T lymphocytes that are the major reservoir of latent HIV-1. Cells derived from aviremic HIV-1 patients under antiretroviral therapy didn’t express MATR3, and most importantly, latency-reversing agents proposed for the rescue of latent provirus were ineffective for MATR3 upregulation. To conclude, our work identifies a cellular factor required for full HIV-1 reactivation and points to the revision of the current strategies for purging viral reservoirs that focus only on transcription.

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

ABSTRACTUnderstanding the mechanisms of HIV proviral latency is essential for development of a means to eradicate infection and achieve a cure. We have previously described anin vitrolatency 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.IMPORTANCEThere 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.