Background
Genetic analysis of HIV-1 during acute infection can reveal early drivers of diversity and evolution. Here, we used ultrasensitive single-genome sequencing (uSGS) on plasma virus collected from participants in the early weeks after transmission to determine the relative roles of mutation and selection in shaping the pol and env genes.
Methods
HIV-1 RNA was extracted from plasma of 10 participants with acute infection (Fiebig II-III) enrolled in the RV254/SEARCH010 Trial. Participant HLA types were previously determined. Our uSGS method used primer IDs and Illumina sequencing (234bp) to characterize the genetics of >10,000 pol (RT) and env (C2-V3) genomes per sample. We measured average pairwise distances (APD), substitution rates, transition/transversion ratios (Ti/Tv), non-synonymous/synonymous ratios (dN/dS), frequencies of amino acid reversions back to consensus CRF01AE (conAE), CTL mutations away from conAE, and drug resistance mutations.
Results
Sequences from an average of 11,733 pol and 12,096 env single genomes were obtained from each participant. In these 10 single transmitted founder (TF) infections, an average 93% of the genomes were identical. APD was a median of 0.07% in pol and 0.05% in env (p=0.10), or ~6-fold lower than expected from the HIV-1 mutation rate and time since transmission. A preference toward transitions was observed with a median Ti/Tv of 5.1 in pol and 3.8 in env (p=0.21). In pol, 52% of transitions were G>A and in env, 42% were G>A, with the split divided over the other 3 mutations. The average dN/dS was 0.38 in pol and 1.0 in env (p=0.02). Reversions to conAE were observed at 57% of sites that varied from the TF, but these mutations only comprised 0.3% of all amino acid changes in pol and 2.8% in env (p<0.0003). The remainder were mutations away from conAE, with 42% in pol and 51% in env falling within CTL epitopes that matched the participants’ HLA. Drug resistance mutations were detected in all participants (0.2-4.0% of genomes), but were not linked to one another.
Conclusions
Genetic diversity in Fiebig II-III was ~6-fold lower than expected from the HIV-1 mutation rate over the first 21.5 days of infection, implying purifying selection during acute infection. While many transmitted amino acid mutations reverted to conAE; these mutations were a small contribution to the overall genetic diversity. The main drivers of early HIV-1 diversity were synonymous mutations and amino acid changes in CTL epitopes, suggesting the possibility of early CTL escape.
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