Switching between raltegravir resistance pathways analyzed by deep sequencing

Abstract

Our objective was to analyze the pathways leading to resistance of HIV to the integrase (IN) inhibitor raltegravir (RAL). Three HIV-infected individuals exhibiting RAL resistance pathway switching were characterized using longitudinal analysis of viral samples from plasma. 454/Roche pyrosequencing was used to generate approximately 74,000 sequence reads from the integrase coding region. Effects of error were controlled by denoising with Pyronoise, and by comparison to approximately 142,000 control reads from HIV(NL4-3). Viral lineages were modeled quantitatively using viral serial pathway analysis (vSPA). All three patients showed transitions from the N155H pathway to the Q148H/G140S pathway. Analysis with vSPA revealed complex pathways to the final genotype, probably involving both de-novo mutation and recombination. No reads contained both the N155H and Q148H drug resistance mutations (DRMs), indicating that the double mutant is not a prominent intermediate, consistent with low fitness. To characterize possible drug-resistant variants circulating prior to therapy, we sequenced approximately 70,000 reads from samples collected prior to initiating treatment. Although some preexisting drug-resistant variants were detected, N155H, the first major DRM present after initiating RAL therapy, was not detected. The main DRMs are present at very low levels if at all prior to initiating therapy. We also outline general methods for deep sequence analysis of DRMs in longitudinal HIV samples.