Virally Suppressed People Living with HIV Who Use Opioids Have Diminished Latency Reversal.

Of the 12 million people who inject drugs worldwide, 13% live with HIV. Whether opioid use impacts HIV pathogenesis and latency is an outstanding question. To gain insight into whether opioid use influences the proviral landscape and latent HIV reservoir, we performed intact proviral DNA assays (IPDA) on peripheral blood mononuclear cells (PBMCs) from antiretroviral therapy (ART)-suppressed people living with HIV (PWH) with or without current opioid use. No differences were observed between PWH with and without opioid use in the frequency of HIV intact and defective proviral genomes. To evaluate the latent reservoir, we activated PBMCs from ART-suppressed PWH with or without opioid use and assessed the induction of HIV RNA. PWH using opioids had diminished responses to ex vivo HIV reactivation, suggesting a smaller reversible reservoir of HIV-1 latently infected cells. However, in vitro studies using primary CD4+ T cells treated with morphine showed no effect of opioids on HIV-1 infection, replication or latency establishment. The discrepancy in our results from in vitro and clinical samples suggests that while opioids may not directly impact HIV replication, latency and reactivation in CD4+ T cells, opioid use may indirectly shape the HIV reservoir in vivo by modulating general immune functions.

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.

Targeting HIV-1 proviral transcription.

Despite the success of antiretroviral therapies, there is no cure for HIV-1 infection due to the establishment of a long-lived latent reservoir that fuels viral rebound upon treatment interruption. 'Shock-and-kill' strategies to diminish the latent reservoir have had modest impact on the reservoir leading to considerations of alternative approaches to target HIV-1 proviruses. This review explores approaches to target HIV-1 transcription as a way to block the provirus expression.