Minority Human Immunodeficiency Virus Type 1 Variants in Antiretroviral-Naive Persons with Reverse Transcriptase Codon 215 Revertant Mutations

Abstract

T215 revertant mutations such as T215C/D/E/S that evolve from the nucleoside reverse transcriptase (RT) inhibitor mutations T215Y/F have been found in about 3% of human immunodeficiency virus type 1 (HIV-1) isolates from newly diagnosed HIV-1-infected persons. We used a newly developed sequencing method-ultradeep pyrosequencing (UDPS; 454 Life Sciences)-to determine the frequency with which T215Y/F or other RT inhibitor resistance mutations could be detected as minority variants in samples from untreated persons that contain T215 revertants ("revertant" samples) compared with samples from untreated persons that lack such revertants ("control" samples). Among the 22 revertant and 29 control samples, UDPS detected a mean of 3.8 and 4.8 additional RT amino acid mutations, respectively. In 6 of 22 (27%) revertant samples and in 4 of 29 control samples (14%; P ‫؍‬ 0.4), UDPS detected one or more RT inhibitor resistance mutations. T215Y or T215F was not detected in any of the revertant or control samples; however, 4 of 22 revertant samples had one or more T215 revertants that were detected by UDPS but not by direct PCR sequencing. The failure to detect viruses with T215Y/F in the 22 revertant samples in this study may result from the overwhelming replacement of transmitted T215Y variants by the more fit T215 revertants or from the primary transmission of a T215 revertant in a subset of persons with T215 revertants.Human immunodeficiency virus type 1 (HIV-1) exists in vivo as a quasispecies consisting of innumerable genomic variants evolving through a process of mutation, recombination, and selection. The quasispecies nature of HIV-1 complicates the detection of drug-resistant HIV-1 variants because standard resistance testing using direct PCR dideoxynucleoside chain terminator sequencing rarely detects mutant variants present in fewer than 20% of circulating plasma viruses (14,20,26). We previously reported that ultradeep pyrosequencing (UDPS) using the 454 Life Sciences sequencing platform reliably detects minor variants that are missed by direct PCR chain terminator sequencing (26). In this study, we used UDPS to characterize the types of minor variants present in previously untreated HIV-1-infected persons who were found by direct PCR sequencing to have a class of mutations, T215 revertants, that occur almost exclusively among persons primarily infected with a virus previously exposed to selective antiretroviral (ARV) drug pressure. MATERIALS AND METHODS Persons and samples.We studied the treatment histories of all persons receiving medical care at the Kaiser Permanente Medical Care Program of Northern California (KPNC) between 1999 and 2007 who had genotypic resistance testing at Stanford University Hospital (SUH) and who were found to have an amino acid other than T, F, or Y at reverse transcriptase (RT) codon 215 ("215 revertant"). Available cryopreserved plasma samples from persons who were ARV naive, who had no evidence of a mixture that included Y or F, and who had plasma HIV-1 RNA levels Ն4.5 log copies/ml w...

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