Frequent polymorphism at drug resistance sites in HIV-1 protease and reverse transcriptase

Abstract

Background-Failure of antiretroviral therapy may result from the selection of pre-existing, drug-resistant HIV-1 variants, but the frequency and type of such variants have not been defined. Objective-We used single genome sequencing (SGS) to characterize the frequency of polymorphism at drug resistance sites in protease (PR) and reverse transcriptase (RT) in plasma samples from antiretroviral naive individuals. Methods-A total of 2229 pro-pol sequences in 79 plasma samples from 30 patients were analyzed by SGS. A mean of 28 single genome sequences was obtained from each sample. The frequency of mutations at all PR and RT sites was compared to those associated with drug resistance. Results-We detected polymorphism at one or more drug resistance sites in 27 of 30 (90%) patients. Polymorphism at positions 179 and 215 of RT was most common, both occurring in 23% of patients. Most (68%) of other drug resistance sites were polymorphic with an average of 3.2% of genomes per sample containing at least one variant from wild type. Seven drug resistance sites were polymorphic in more than 1% of genomes: PR position 33; RT positions 69, 98, 118, 179, 210, and 215. Although frequencies of synonymous polymorphism were similar at resistance and nonresistance sites, nonsynonymous polymorphism were significantly less common at drug resistance sites, implying stronger purifying selection at these positions. Conclusions-HIV-1 variants that are polymorphic at drug resistance sites pre-exist frequently as minor species in antiretroviral naive individuals. Standard genotype techniques have grossly underestimated their frequency.

Ultrasensitive Allele-Specific PCR Reveals Rare Preexisting Drug-Resistant Variants and a Large Replicating Virus Population in Macaques Infected with a Simian Immunodeficiency Virus Containing Human Immunodeficiency Virus Reverse Transcriptase

“…It is, therefore, important to understand the frequency of such mutations, as well as their variability and kinetics of appearance in drug-naïve infected individuals. Although we (14,19, We have used UsASP to investigate the evolution of drug resistance before and after EFV monotherapy in 2 RT-SHIV mne infected macaques. We were able to reproducibly detect the K65R and M184I drug resistance mutations before and after drug exposure and observe how they were maintained in the same proportion relative to the total virus population, consistent with the predicted balance between mutation and selection at these sites.…”

HIV-1 Virion Production from Single Inducible Proviruses following T-Cell Activation Ex Vivo

“…According to Poisson's distribution, ϳ80% of wells with detectable HIV-1 RNA were expected to contain a single expressing HIV-1 provirus. Single-genome sequencing (SGS) (11,12) revealed that three of four wells had monotypic sequences with infrequent single-base-pair differences within the known error rate of SGS (ϳ1.1 ϫ 10 Ϫ4 errors/nucleotide) ( Fig. 3) (11), confirming that most wells with detectable HIV-1 RNA contained a single expressing provirus.…”

The HIV-1 reservoir in eight patients on long-term suppressive antiretroviral therapy is stable with few genetic changes over time

“…To compare the intracellular populations identified using the single-proviral sequencing assay to HIV-1 RNA populations found in plasma collected before initiation of antiretroviral therapy, we performed singlegenome sequencing (SGS) on the plasma samples from each of the eight patients as described earlier (48)(49)(50). To extract viral RNA from plasma collected during suppressive therapy, we used a modified version of the SGS method.…”