A modular CRISPR screen identifies individual and combination pathways contributing to HIV-1 latency

Area of Science:

• Virology
• Molecular Biology
• Epigenetics

Background:

• Transcriptional silencing of latent HIV-1 proviruses is a significant hurdle for effective HIV-1 eradication.
• Understanding the complex mechanisms maintaining HIV-1 latency is crucial for developing novel therapeutic strategies.

Purpose of the Study:

• To develop and utilize a novel CRISPR screening strategy (Latency HIV-CRISPR) to identify factors involved in HIV-1 latency maintenance.
• To investigate the interplay between epigenetic regulators and latency-reversing agents in controlling HIV-1 transcription.

Main Methods:

• Developed Latency HIV-CRISPR, a screening platform using lentiviral vectors for guide RNA delivery and readout.
• Employed a custom guide RNA library targeting epigenetic genes.
• Paired CRISPR screening with AZD5582, a non-canonical NFκB pathway activator, in J-Lat cells and primary CD4+ T cells.

Main Results:

• Identified ING3, a component of the NuA4 HAT complex, as a critical factor in HIV-1 latency.
• ING3 knockout reduced H4 histone acetylation and BRD4 occupancy at the HIV-1 LTR.
• Combined ING3 knockout and AZD5582 treatment led to a significant increase in RNA Polymerase II initiation and elongation on the HIV-1 provirus.

Conclusions:

• ING3 plays a role in maintaining HIV-1 latency, potentially by influencing histone acetylation and BRD4 binding.
• The combination of targeting ING3 and activating the non-canonical NFκB pathway represents a potent strategy for HIV-1 reactivation from latency.
• This study provides new insights into the epigenetic regulation of HIV-1 latency and offers potential targets for therapeutic intervention.