Abstract
Abstract
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.
Importance
Retroviruses such as human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), and human T-lymphotropic virus type 1 (HTLV-1) cause lifelong infections that can lead to AIDS, leukaemia, and other severe diseases, respectively. After infection, these viruses convert their RNA into DNA and integrate it into the host genome, creating proviruses that can persist for years. Most proviruses are defective and often lack large parts of their genome, particularly in people with HIV receiving antiretroviral therapy, but the viral features that drive this instability have remained incompletely understood. Here we show that the long terminal repeats, which are specialized DNA sequences at the ends of the viral genome, influence the formation of defective proviruses. These insights are expected to improve our understanding of defective provirus landscapes in infected individuals and can inform the design and interpretation of retrovirus-based vectors in research and therapy.