Proliferation of cells with HIV integrated into cancer genes contributes to persistent infection

To especially examine whether or not prolonged Integrase inhibitor HIV-1 suppression resulted in changes in amino acid sequences, we investigated nonsynonymous Integrase inhibitor changes alone in clients from groups 1 and 2 for which there had been much more than 7 sequences at time details with <50 copies/ml (N = 8) (Table S2). We found that amino acid frequencies were remarkably stable during cART. As all enrolled patients underwent HLA testing, we were able to investigate, using in silico techniques, the predicted positions of all the CTL epitopes in the HIV-1 sequence as well as the estimated binding affinity of all the HIV-1 peptides at each epitope site [39]. As shown in Table S2, there was no consistent trend to enrich or deplete CTL escape mutations after prolonged cART suppression, including in those patients who underwent a significant population shift (e.g., PID 1). Taken together, these data suggest that the population of virus-producing cells present after prolonged suppression is not shaped in a substantial way by new CTL selection following introduction of cART. This finding is in stark contrast to the strong selection at CTL epitopes in elite controllers ranging from 11–66% of epitopes carrying escape mutations [28].
Effect of cART on HIV-1 population structure and divergence

Divergence of HIV-1 populations during cART could result either from on-going cycles of replication leading to the emergence of new variants or as a consequence of shifts in the viral variants present in the plasma during suppression, indicating a dynamic reservoir. To investigate the possibility of population shift (divergence) during cART, we used a test for panmixia to detect changes in the population structure during therapy compared to pretherapy virus. The panmixia test compares populations of single-genome sequences obtained from longitudinal samples and provides a p-value for the probability that the populations are the same [34]. Probabilities of <10−3 were considered to indicate significantly different populations, taking into account the large numbers of comparisons. Figure 3 and Table 3 show the panmixia results for single-genome sequences from group 1 (Figure 3a, Table 3), group 2, (Figure 3b, Table 3), and group 3 (Figure 3c, Table 3) compared to pretherapy sequences. Panmixia probabilities of virus populations in samples collected from patients on cART compared to pre-therapy populations did not achieve significance (Figure 3a) in 8/10 patients from group 1. These results indicate that there is typically no significant shift in the plasma virus population during the first and second phases of decay after initiating cART despite up to 10,000-fold declines in levels of viremia. Two patients in group 1 (PID 6, 7), however, did show a significant change in population structure after 173 and 193 days on therapy. Additional analyses describing the nature of these changes are presented below. Three of 5 patients in group 2 (long-term cART) showed a significant change in population structure during cART for 4–12 years with no treatment interruptions, suggesting either that new variants emerged during therapy or that the reservoir for persistent viremia is dynamic. Four of 5 patients in group 3 (long-term cART but with brief treatment interruptions) showed a significant shift in population structure using the panmixia test.