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.
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.
Hertogs
K
K
VIRCO, Central Virological Laboratory, Edegem, Belgium. kurt.hertogs@virco.be
de Béthune
M P
MP
Miller
V
V
Ivens
T
T
Schel
P
P
Van Cauwenberge
A
A
Van Den Eynde
C
C
Van Gerwen
V
V
Azijn
H
H
Van Houtte
M
M
Peeters
F
F
Staszewski
S
S
Conant
M
M
Bloor
S
S
Kemp
S
S
Larder
B
B
Pauwels
R
R
D000163:Acquired Immunodeficiency Syndrome / Q000188:drug therapy / Q000821:virology; D004352:Drug Resistance, Microbial / Q000235:genetics; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D010641:Phenotype; D016865:Reassortant Viruses / Q000187:drug effects; D015203:Reproducibility of Results
D017320:HIV Protease Inhibitors; D054303:HIV Reverse Transcriptase