DNA Damage Response Proteins Are Involved in the Formation of Defective HIV-1 Proviruses

Abstract Latent HIV-1 proviruses remain the major barrier to curing HIV infection. Although many of these proviruses are defective, with large internal deletions and hypermutations, the mechanisms underlying their formation are still poorly understood. In this study, we applied CRISPR/Cas9 knockout screens to identify DNA damage response (DDR) proteins that contribute to the formation of defective HIV-1 proviruses carrying large internal deletions. Using an HIV-1-based dual-fluorophore vector as a model, we distinguished cells harbouring intact proviruses from those carrying large internal deletions by flow cytometry and cell sorting. We then validated top candidates using CRISPR-mediated gene activation and small interfering RNA-mediated knockdown, and we measured gene and protein expression by quantitative PCR and Western blotting. Across these approaches, the helicase-like transcription factor HLTF emerged as a consistent modulator of large internal deletions: increased HLTF expression raised the proportion of cells carrying defective proviruses, whereas reduced HLTF expression had the opposite effect. Additional repair factors, including RAD1, RAD18, TREX2, and ZRANB3, also influenced the balance between intact and defective proviruses, suggesting that multiple DNA repair pathways cooperate in this process. Deep sequencing of reporter proviruses confirmed the presence of large internal deletions in the populations identified as defective. Our data indicate that several DNA damage response proteins, including HLTF, are involved in the generation of defective proviruses and may constitute a previously undescribed host defense mechanism against HIV-1. Authors Summary When HIV-1 infects a cell, it copies its genetic material (RNA) into DNA and inserts this DNA into the cell’s genome, giving rise to proviruses that can persist for long periods and become part of the host DNA. Many of these viral DNA copies are defective, often missing large parts of their genome, but we still do not fully understand how these large deletions arise. In this study, we used a genetic screening approach to switch off many human DNA repair genes and asked how this affected the balance between intact and defective HIV proviral DNA. We used an HIV-1-based dual-colour reporter vector allowing us to distinguish intact from deleted viral DNA by simple fluorescence read-outs. We found that several human DNA repair factors, in particular a protein called HLTF, change how often large deletions appear. Our results suggest that normal DNA repair processes in infected cells can sometimes turn incoming HIV-1 DNA into defective forms that cannot support productive infection. This work points to host DNA repair as a contributor to the large pool of defective HIV-1 DNA seen in people with HIV (PWH) and raises the possibility that these pathways could one day be harnessed to make infections less harmful. Competing Interest Statement Within the last 5 years, Prof. Dr. Karin J. Metzner has received advisory board honoraria from ViiV; and the University of Zurich received research grants from Gilead Sciences and Novartis for studies that Dr Metzner serves as principal investigator. Abbreviations - AIDS - Acquired immunodeficiency syndrome - ART - Antiretroviral therapy - CEG - Core-essential genes - CRISPRa - CRISPR activation - CRISPRko - CRISPR knockout - DDR - DNA damage response - gRNA - guide RNA - HIV-1 - Human immunodeficiency virus type 1 - LID - Large internal deletion - LTatC[M]L - 5’ LTR Tat-Cerulean-insulator-mCherry-insulator-3’ LTR - LTR - Long terminal repeat - MAGeCK - Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout - MOI - Multiplicity of infection - NEG - Non-essential genes - NGS - Next-generation sequencing - NHEJ - Non-homologous end joining - PCA - Principal component analysis - PFGE - Pulsed-field gel electrophoresis - PWH - People with HIV - TNFα - Tumor necrosis factor - SAHA - Suberoylanilide hydroxamic acid - siRNA - Small interfering RNA