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CRISPR Screens Identify Potential Host Targets to Reverse HIV-1 Latency

NEW YORK — Using genome-wide CRISPR screens, researchers have identified host genes that can be targeted to help induce expression of latent HIV-1, potentially enabling the otherwise unreachable virus to be targeted by drugs.

HIV-1 can persist among affected individuals within their CD4+ T cells, even in face of antiretroviral therapy. This latency in which the virus is present, though not reproducing, presents a barrier to curing HIV-1, and some scientists have suggested that reversing latency could provide an opportunity to then kill infected cells.

Researchers from Johns Hopkins University School of Medicine used a CRISPR-based approach to screen for host genes that, when knocked out, boost viral gene expression in combination with known latency reversing agents like second mitochondrial-derived activator of caspases (SMAC) mimetics. Combinations, they noted, are preferred, since no single latency reversing agent has been able to reduce latency in people with HIV-1 on its own.

As they reported in Science Translational Medicine on Wednesday, the researchers identified a number of such genes, such as histone deacetylase 2 (HDAC2) and bromodomain-containing protein 2 (BRD2).

"Our approach was based on the concept that host genes whose knockout enhances latency reversal in these screens are potential targets for synergistic drugs," senior author Robert Siliciano, a professor of medicine at Hopkins, and colleagues wrote in their paper.

For their study, the researchers first adapted an in vitro model of HIV-1 latency, so that the cells carried Cas9 and a green fluorescent protein reporter gene. The researchers then screened those cells against a range of sgRNAs to identify which genes are needed to enable viral reactivation upon treatment with AZD5582, a SMAC mimetic. SMAC mimetics activate the noncanonical NFκB (ncNF-κB) pathway and induce apoptosis among CD4+ T cells infected with HIV-1.

The researchers similarly conducted other CRISPR screens using tumor necrosis factor-α, PMA and ionomycin, and T-cell receptor cross-linking, which are also used for T-cell activation and to induce HIV transcription.

Together, these screens identified 578 genes required in at least one of the conditions for viral reactivation, including HDAC2 and BRD2, suggesting that targeting them with inhibitors like SAHA or JQ1, respectively, could have a synergistic effect with AZD5582.

Using CD4+ T cells isolated from people living with HIV, the researchers confirmed that there was such synergy between AZD5582 and HDAC inhibitors or between AZD5582 and JQ1. There was no synergy, however, between SAHA and JQ1 in the CD4+ T cells from people living with HIV.

In a single-cell RNA sequencing analysis of CD4+ T cells isolated from someone living with HIV, they found that genes enriched in the ncNF-κB pathway were upregulated by AZD5582 and JQ1, while cell-cycle genes were not.

Additionally, though reciprocal screens using an HDAC inhibitor as the latency reversing agent, the researchers uncovered other genes that could be candidate targets for synergistic effect with HDAC inhibitors themselves. These screens suggested synergistic effects with BRD2 and ncNF-κB regulators like BIRC2.

"Looking forward, a combination of screens in in vitro models with in vivo and clinical validations will be crucial to better understand the mechanism of latent HIV reactivation and to find [latency reversing agent] drug combinations to eliminate latent HIV virus," Siliciano and colleagues wrote.