Positive selection detection in 40,000 human immunodeficiency virus (HIV) type 1 sequences automatically identifies drug resistance and positive fitness mutations in HIV protease and reverse transcriptase

Abstract

Drug resistance is a major problem in the treatment of AIDS, due to the very high mutation rate of human immunodeficiency virus (HIV) and subsequent rapid development of resistance to new drugs. Identification of mutations associated with drug resistance is critical for both individualized treatment selection and new drug design. We have performed an automated mutation analysis of HIV Type 1 (HIV-1) protease and reverse transcriptase (RT) from approximately 40,000 AIDS patient plasma samples sequenced by Specialty Laboratories Inc. from 1999 to mid-2002. This data set provides a nearly complete mutagenesis of HIV protease and enables the calculation of statistically significant K(a)/K(s) values for each individual amino acid mutation in protease and RT. Positive selection (i.e., a K(a)/K(s) ratio of >1, indicating increased reproductive fitness) detected 19 of 23 known drug-resistant mutation positions in protease and 20 of 34 such positions in RT. We also discovered 163 new amino acid mutations in HIV protease and RT that are strong candidates for drug resistance or fitness. Our results match available independent data on protease mutations associated with specific drug treatments and mutations with positive reproductive fitness, with high statistical significance (the P values for the observed matches to occur by random chance are 10(-5.2) and 10(-16.6), respectively). Our mutation analysis provides a valuable resource for AIDS research and will be available to academic researchers upon publication at http://www.bioinformatics.ucla.edu/HIV. Our data indicate that positive selection mapping is an analysis that can yield powerful insights from high-throughput sequencing of rapidly mutating pathogens.

FIG. 1.

RT-PCR and sequencing of the HIV-1 protease and RT regions. HIV-1 RNA was isolated from AIDS patient plasma samples. Reverse transcription was performed to obtain the cDNA from single-stranded viral RNA. The HIV protease and RT region around 1.4 kb was amplified by PCR using two (forward and backward) unique primers. This was followed by a nested PCR, which split the target sequence into three shorter fragments with the use of six unique primers. These fragments were then cycle sequenced in forward and reverse directions.

FIG. 2.

Chromatogram evidence for an HIV-1 protease mutation. The program snp_assess identified an A→G mutation with an LOD score of 11.6 (top). Chromatograms for the forward (Seq214224) and reverse (Seq214232) strand sequencing are shown in the lower panels. Seq214232 is shown in reverse complement for the purposes of comparison.

FIG. 3.

Positive selection mapping of HIV-1 protease from 40,000 patient samples. The K_a/K_s value represents the greatest selection pressure among all the individual amino acid mutations at each codon. The dotted line indicates the K_a/K_s value of 1.

FIG. 4.

Positive selection mapping of HIV-1 RT from 40,000 patient samples. The K_a/K_s value represents the greatest selection pressure among all the individual amino acid mutations at each codon. The dotted line indicates the K_a/K_s value of 1.

FIG. 5.

Positive selection identifies drug resistance and positive fitness mutations. (a) Identification of codons with positive selection, either from the set of all positions in HIV protease (All codons), positions reported in the literature as sites of drug-resistant mutations (Known drug resistance associated codons), or positions reported as sites of mutations specifically associated with adaptation to drug treatment (Treatment associated codons). (b) Identification of specific amino acid mutations with positive selection, either from the set of all HIV protease mutations found in our data set (All mutations), or mutations reported in the literature as causing drug resistance (Known drug resistance associated mutations). (c) Phenotypic fitness, as measured by a protease activity assay by Loeb et al., for a random sample of HIV protease mutants (All mutations tested), or the subset of those mutations found to have positive selection in our study (Positive selected mutations). active, protease mutants with normal or greater-than-wild-type proteolytic activity; intermediate, partial cleavage was observed in the assay; inactive, no proteolytic cleavage was observed.