Characterizing the proviral landscape and APOBEC3 innate immune responses within immune cells targeted by HIV-1.

Abstract

Introduction: HIV-1 persistence despite combination antiretroviral therapy (cART) is a key barrier to curing HIV, as latent reservoirs of integrated proviral DNA persist, primarily in resting CD4+ T-cells and possibly other immune cells including myeloid cells. A cure for HIV-1 can be achieved either through total eradication of the virus or a functional cure, defined as controlling HIV without ongoing ART, but both approaches remain scientifically challenging. Antiretroviral-free control of HIV by the immune system is promising but requires a better understanding of the underlying mechanisms for widespread efficacy and application. The apolipoprotein B mRNA editing catalytic polypeptide-like 3 (A3) family of cytidine deaminase proteins, particularly A3G and A3F, potently inhibit HIV-1 by inducing mutations in viral DNA, potentially rendering the virus defective. This study aimed to provide a comprehensive longitudinal analysis of the HIV-1 subtype C reservoir with immune cells from the blood of PLWH and establish the impact of A3 proteins on the viral reservoir in a cohort of young women from Durban, South Africa. Methods: This study analysed ten women from Durban, South Africa, diagnosed with HIV during hyperacute HIV infection to evaluate the effects of ART initiation timing on HIV reservoirs. Five participants initiated ART early, within one day of plasma viremiadetection (median: 1 day post-positive viral load [DPOPV]), while the other five started ART late, during chronic infection (median: 774 DPOPV, range: 399–1202 days). Peripheral blood mononuclear cells (PBMCs) were sorted into CD4+ T-cell subsets, including central memory (CM), effector memory (EM), transitional memory (TM), naïve (N), and myeloid cells (M), for analysis at multiple time points: pre-infection, and at 1, 6, and 12 months post-infection and post-ART initiation. Total HIV DNA was quantified using ddPCR. Intact and defective HIV-1 genomes were measured using the intact proviral DNA assay (IPDA). The inducible reservoir was assessed in study participants at 3 years post-treatment using the Specific Quantification of inducible HIV-1 reservoir by LAMP (SQuHIVLa) assay. Additionally, A3G and A3F mRNA levels and proviral hypermutation were evaluated to explore the impact of host restriction factors on viral persistence. Results: CM and EM CD4+ T cells harboured the highest total HIV DNA levels, averaging 7,695 and 5,680 DNA copies/million cells, respectively. Naïve CD4+ T cells contained detectable levels (744 copies/million cells), while myeloid cells also exhibited detectable, albeit lower than CM and EM levels of viral DNA (1,050 copies/million cells). At 12 months post-ART, proviral DNA persisted in the myeloid compartment of 4/5 late-treated participants but only 1/5 early treated participants. Late-treated participants showed no significant decline in proviral DNA levels over 12 months, while early treated participants demonstrated significant reductions across CM (p = 0.008), EM (p = 0.007), and transitional memory, TM (p = 0.007) CD4+ T-cell subsets. Early treated participants had significantly lower proviral DNA across all subsets compared to late-treated participants at 12 months (CM; p= 0.0079, EM; p= 0.0079, TM; p= 0.0070, N; p= 0.0476, M; p= 0.0400). Intact proviral DNA was more abundant in EM cells than CM cells in late-treated participants, while ~80% of earlytreated participants had undetectable intact HIV DNA in the EM subset after one year. Inducible reservoir levels were higher in late-treated participants (p = 0.0038). A3G expression exceeded A3F by ~1.5-fold pre-infection. Untreated HIV-1 infections led to significant downregulation of both, with greater reductions in A3G. ART partially restored A3 expression, with robust recovery in early treated participants. Defective proviral DNA levels were higher in late-treated participants across most subsets, except naïve CD4+ T cells. A3G expression correlated with larger defective reservoirs in TM CD4+ T cells (p = 0.003). Proviral DNA sequencing revealed frequent A3Ginduced hypermutation (p = 0.0006), clustered at HIV genome regions prone to single stranded states during reverse transcription. A3G levels positively correlated with hypermutation percentages pre-infection (p = 0.027, r = 0.91) and post-ART (p = 0.032, r = 0.90). Early ART initiation preserves A3 activity, reducing intact proviruses and enhancing immune restoration. Conclusion: This study highlights the persistence of HIV proviral DNA across immune cell subsets, including CD4+ T cells and myeloid cells. In late-treated individuals, no significant change in reservoir size was observed, whereas early ART initiation led to substantial reductions, particularly in CD4+ T cells, underscoring the critical role of early treatment in minimizing HIV reservoirs. CM and EM CD4+ T cells were identified as key contributors to reservoir maintenance, with EM cells harbouring higher levels of intact proviral DNA, making them essential targets for intervention. Early ART initiation not only reduces reservoir size but also preserves A3G mRNA expression, which plays a pivotal role in inducing hypermutation of proviral DNA. This preservation supports immune function and creates an environment favourable for targeting and inactivating the HIV reservoir. Enhancing A3G-mediated hypermutation may be a promising therapeutic strategy to disrupt HIV reservoirs. Overall, these findings advance the goal of a functional HIV cure by providing insights into reservoir seeding and decay in diverse immune cells following ART in acute or chronic infection and through studies of the expression and impact of a key innate immune mechanism. The results have implications for achieving an HIV-1 cure through combination mechanisms. Future research should focus on cell-specific regulation of A3 activity through transcriptional profiling and the development of interventions to augment A3 function.