Pyrosequencing reveals restricted patterns of CD8 ⁺ T cell escape-associated compensatory mutations in simian immunodeficiency virus

CD8 ⁺ T cells play a major role in the containment of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. CD8 ⁺ T cell-driven variations in conserved regions under functional constraints result in diminished viral replicative capacity. While compensatory mutations outside an epitope can restore replicative capacity, the kinetics with which they arise remains unknown. Additionally, certain patterns of linked mutations associated with CD8 ⁺ T cell epitope escape in these highly conserved regions may lead to variable levels of viral fitness. Here, we used pyrosequencing to investigate the kinetics and patterns of mutations surrounding the Mamu-A1*00101-bound Gag _181-189CM9 CD8 ⁺ T cell epitope. We obtained more than 400 reads for each sequencing time point, allowing us to confidently detect the emergence of viral variants bearing escape mutations with frequencies as low as 1% of the circulating virus. With this level of detail, we demonstrate that compensatory mutations generally arise concomitantly with Gag _181-189CM9 escape mutations. We observed distinct patterns of linked flanking mutations, most of which were found downstream of Gag _181-189CM9. Our data indicate that, whereas Gag _181-189CM9 escape is much more complex that previously appreciated, it occurs in a coordinated fashion, with very specific patterns of flanking mutations required for immune evasion. This is the first detailed report of the ontogeny of compensatory mutations that allow CD8 ⁺ T cell epitope escape in infected individuals.