Determination of HIV-1 susceptibility to reverse transcriptase (RT) inhibitors by a quantitative cell-free RT assay.
BACKGROUND
Mutations in the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) gene confer resistance to antiviral drugs acting on RT. Current methods employed to detect such resistance require time-consuming culture techniques during which selective pressures may affect the outcome of the test.


OBJECTIVE
We sought to determine whether drug-susceptible and drug-resistant HIV-1 derived from clinical specimens could be distinguished by the effects of the active form of the drug on the enzyme activity in a quantitative, cell-free RT assay.


METHODS
Polyethylene glycol (PEG)-precipitated virus was obtained from 7-day culture supernatants. RT activity in the lysed viral extracts was measured in the presence of increasing concentrations of the active form of the drug being tested. IC50 (50% inhibitory concentration) values were determined by application of the median effect equation.


RESULTS
Assays from nine post-nevirapine therapy isolates gave IC50 values at least 2 logs greater than pre-nevirapine isolates. The method also correctly distinguished between isolates sensitive and resistant to 2',3'-dideoxyinosine (ddI), but not between the ZDV-sensitive and ZDV-resistant isolates tested. The results agreed with data obtained by sequencing and by culture-based susceptibility assays.
Greene
R A
RA
NEN Life Science Products, Boston, MA 02118, USA.
Japour
A J
AJ
Brewster
F
F
Joseph
R A
RA
Chung
P H
PH
Kasila
P A
PA
Chatis
P A
PA
D002474:Cell-Free System; D016049:Didanosine / Q000494:pharmacology*; D004351:Drug Resistance; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D015215:Zidovudine / Q000494:pharmacology
D018894:Reverse Transcriptase Inhibitors; D015215:Zidovudine; D054303:HIV Reverse Transcriptase; D016049:Didanosine