A rapid method for simultaneous detection of phenotypic resistance to inhibitors of protease and reverse transcriptase in recombinant human immunodeficiency virus type 1 isolates from patients treated with antiretroviral drugs

Abstract

Combination therapy with protease (PR) and reverse transcriptase (RT) inhibitors can efficiently suppress human immunodeficiency virus (HIV) replication, but the emergence of drug-resistant variants correlates strongly with therapeutic failure. Here we describe a new method for high-throughput analysis of clinical samples that permits the simultaneous detection of HIV type 1 (HIV-1) phenotypic resistance to both RT and PR inhibitors by means of recombinant virus assay technology. HIV-1 RNA is extracted from plasma samples, and a 2.2-kb fragment containing the entire HIV-1 PR- and RT-coding sequence is amplified by nested reverse transcription-PCR. The pool of PR-RT-coding sequences is then cotransfected into CD4+ T lymphocytes (MT4) with the pGEMT3deltaPRT plasmid from which most of the PR (codons 10 to 99) and RT (codons 1 to 482) sequences are deleted. Homologous recombination leads to the generation of chimeric viruses containing PR- and RT-coding sequences derived from HIV-1 RNA in plasma. The susceptibilities of the chimeric viruses to all currently available RT and/or PR inhibitors is determined by an MT4 cell-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based cell viability assay in an automated system that allows high sample throughput. The profile of resistance to all RT and PR inhibitors is displayed graphically in a single PR-RT-Antivirogram. This assay system facilitates the rapid large-scale phenotypic resistance determinations for all RT and PR inhibitors in one standardized assay.

FIG. 1

(A) Proviral clone pGEMT3ΔPRT from which the sequences encoding PR and RT were deleted was constructed by removing a 750-bp ApaI-BstEII fragment upstream of the RT deletion of the proviral clone pHIVΔRTBstEII and replacing it with a 270-bp ApaI-XmaI fragment recovered from the intermediate construct pGEM7Apa/Sma (see Materials and Methods). This resulted in a clone from which coding sequences starting from amino acid position 10 in the HIV-1 PR gene to amino acid position 484 in the HIV-1 RT gene have been removed. (B) Nested PCR is used to amplify either RT-only coding sequences (1.7 kb) (primer pairs OUT3-OUT5 and IN3-IN5) or combined PR-RT-coding sequences (2.2 kb) (primer pairs OUT3-PRTO5 and IN3-PRTI5). RT-only coding sequences encompass all resistance-associated amino acid changes between residues 41 and 483 of the HIV-1 RT gene. Combined PR- and RT-coding sequences encode for all resistance-associated mutations between amino acid residue 9 of the PR gene and residue 483 of the RT gene.

FIG. 2

Phenotypic AZT resistance was determined either by the HeLa CD4 plaque reduction assay (y axis) or the MT4-MTT cell viability assay (x axis). The correlation between AZT resistance data obtained by both methods and presented as log fold AZT resistance values (fold increase in the mean IC₅₀ relative to the mean wild-type IC₅₀ for the wild type; the mean IC₅₀ for both patient and wild-type viruses is derived from two to four separate susceptibility determinations) was statistically determined (n = 43; r = 0.8; P = < 0.00001).