Michalek, K. | bioRxiv Science

bioRxiv2026

Long terminal repeats influence deletion propensity and genome instability in proviruses across major retroviruses

A hallmark of retroviral infection is integration of viral genomes into host chromosomes to form proviruses. Most proviruses are defective, particularly in people with HIV receiving antiretroviral therapy, with large internal deletions as a dominant outcome. The contribution of retroviral long terminal repeats (LTRs) in the formation of large internal deletions remains unclear. We used dual-fluorophore reporters based on HIV-1, HIV-2, and HTLV-1 LTRs, integrated at AAVS1 by CRISPR/Cas9, to test whether LTRs influence proviral integrity in the absence of reverse transcriptase or integrase. Cerulean expression was driven by the LTR and mCherry by an independent human promoter. Double-positive (Cerulean/mCherry) cells were a proxy for intact vectors, whereas single mCherry cells indicated latent and/or defective proviruses. Double-positive cells harboured intact vectors for all constructs. Non-inducible single mCherry populations were enriched for extensively deleted proviruses with HIV-1, HIV-2, or HTLV-1 LTRs, whereas the LTR-deficient reporter yielded few single mCherry cells, all with intact proviruses. The presence of LTRs was significantly associated with deleted genomes. HIV-1 and HTLV-1 showed higher observed deletion rates than HIV-2, but this was not statistically significant owing to limited sequence numbers. Single Cerulean populations across all constructs showed recurrent deletions affecting the mCherry cassette and upstream promoter, with short direct repeats at deletion junctions. These likely reflect microhomology-mediated end joining as a contributing repair mechanism during CRISPR/Cas9-mediated integration, in which LTRs may not directly contribute. Together, these data identify retroviral LTRs as contributors to the formation of large internal deletions in proviruses.

microbiology↗

bioRxiv2026

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

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. 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. 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 defence mechanism against HIV-1.

molecular biology↗