Background
Viral diversification complicates immune-based cure strategies. SIV-infected Rhesus macaques (RMs) treated with antiretroviral therapy (ART) provide insights into viral dynamics and reservoir persistence in people with HIV-1. We previously described a shift in the composition of infected cell populations during ART, with recently infected cells harboring mutated proviruses decaying more rapidly, and ancestral variants seeded during early infection becoming more apparent over time (Fray et al, PMID: 36809762). Here we examine env sequences from pre-ART plasma virus to determine the full extent of viral diversification and identify sequence features that may be targeted in cure strategies.
Methods
We used single-genome sequencing (SGS) to collect env sequences from pre-ART plasma from 6 RMs inoculated with SIVmac251 and placed on ART 48 weeks after infection. We analyzed the extent of variation at individual amino acid positions and performed a rarefaction analysis to estimate the number of sequences required to capture the full diversity in a given individual.
Results
800-1400 Env sequences were collected from each animal, providing a comprehensive, longitudinal dataset of viral populations replicating prior to ART within each host. Env mutations became prominent approximately 24 weeks after infection, with accumulation of non-synonymous changes across multiple regions, including the variable loops V1, V2, V4, and V5, and distinct patterns of evolution in individual macaques were observed despite inoculation with a common stock. Even within the V regions, only certain amino acid positions accumulated mutations, whereas others were strikingly conserved, suggesting constraints on viral evolution and adaptation that limit the positions that can tolerate non-synonymous changes. Rarefaction analysis estimated that between >500 sequences are required to adequately sample the env diversity within a single host.
Conclusions
Extensive datasets including pre-ART plasma and on-ART proviral sequences from individual macaques allowed us to estimate the sequencing depth required to accurately estimate the full diversity in a single host. Within each host, we observed both vast diversity and notable conservation in the env gene, likely indicating evolutionary constraints on the env protein structure. These data may be useful to identify potential targets for therapeutic interventions, including personalized vaccine strategies.
Table or Figure
