A RAPID METHOD FOR SIMULTANEOUS DETECTION OF PHENOTYPIC RESISTANCE TOINHIBITORS OF PROTEASE AND REVERSE-TRANSCRIPTASE IN RECOMBINANT HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 ISOLATES FROM PATIENTS TREATED WITH ANTIRETROVIRAL DRUGS

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 h igh-throughput analysis of clinical samples that permits the simultane ous detection of HN type 1 (HIV-1) phenotypic resistance to both RT an d 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 rev erse transcription-PCR. The pool of PR-RT-coding sequences is then cot ransfected into CD4+ T lymphocytes (MT4) with the pGEMT3 Delta PRT pla smid from which most of the PR (codons 10 to 99) and RT (codons 1 to 4 82) sequences are deleted. Homologous recombination leads to the gener ation of chimeric viruses containing PR- and RT-coding sequences deriv ed from HIV-1 RNA in plasma. The susceptibilities of the chimeric viru ses to all currently available RT and/or PR inhibitors is determined b y an MT4 (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-ba sed cell viability assay in an automated system that allows high sampl e throughput. The profile of resistance to all RT and PR inhibitors is displayed graphically in a single PR-RT-Antivirogram. This assay syst em facilitates the rapid large-scale phenotypic resistance determinati ons for all RT and PR inhibitors in one standardized assay.