In order to shed light on the nature of the persistent reservoir of human immunodeficiency virus type 1 (HIV-1), we investigated signs of recent evolution in the pool of proviral DNA in patients on successful HAART. Pro-viral DNA, corresponding to the C2-V3-C3 region of the HIV-1 env gene, was collected from PBMCs isolated from 57 patients. Both "consensus" (57 patients) and clonal (7 patients) sequences were obtained from five time points spanning a 24-month period. The main computational strategy was to use maximum likelihood to fit a set of alternative phylogenetic models to the clonal data, and then determine the support for models that imply evolution between time points. Model fit and model-selection uncertainty was assessed using the Akaike information criterion (AIC) and Akaike weights. The consensus sequence data was also analyzed using a range of phylogenetic techniques to determine whether there were temporal trends indicating ongoing replication and evolution. In summary, it was not possible to detect definitive signs of ongoing evolution in either the bulk-sequenced or the clonal data with the methods employed here, but our results could be consistent with localized expression of archival HIV genomes in some patients. Interestingly, stop-codons were present at the same two positions in several independent clones and across patients. Simulation studies indicated that this phenomenon could be explained as the result of parallel evolution and that some sites were inherently more likely to evolve into stop codons.
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