Rapid, phenotypic HIV-1 drug sensitivity assay for protease and reverse transcriptase inhibitors.
BACKGROUND
Development of drug resistance is one of the major reasons for the failure of antiretroviral therapy of HIV-1 infection. Knowing the drug sensitivity-resistance profile of viruses present in a patient prior to treatment or change in treatment could help to optimize therapy.


OBJECTIVE
Development of a rapid standardized phenotypic HIV-1 drug sensitivity assay for protease (PR) and reverse transcriptase (RT) inhibitors.


METHODS
The PR gene (codons 1-99) and the 5' part of the RT gene (codons 1-300) of HIV-1 is amplified from the plasma of infected individuals by RT-PCR and ligated into a proviral clone of HIV-1 containing a deletion of the PR gene and the 5' part of the RT gene. Bacteria are transformed with the ligation product and plasmid DNA is prepared from a library of transformed bacteria. The plasmid DNA is transfected into 293 T cells and recombinant virus is harvested from the supernatant of the transfected cells 2 days after transfection. The sensitivity of the recombinant virus is determined with the help of a sensitive indicator cell line.


RESULTS
Recombinant viruses were generated with high efficiency. Determination of the drug sensitivity of the recombinant viruses with an indicator cell line was highly reproducible. The recombinant viruses accurately reflected the sensitivity-resistance profile of the parental viruses. The phenotypic drug sensitivity determined by this assay correlated well with the treatment history of patients.


CONCLUSIONS
This assay system should allow rapid, high-throughput analyses of phenotypic HIV-1 drug sensitivity for PR and RT inhibitors. Due to the efficient generation of recombinant viruses, propagation of the recombinant viruses in cell culture is not required prior to the determination of the sensitivity of the recombinant viruses. The risk of selecting fitter non-resistant viruses due to culture conditions is minimized.
Walter
H
H
Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Germany.
Schmidt
B
B
Korn
K
K
Vandamme
A M
AM
Harrer
T
T
Uberla
K
K
D019380:Anti-HIV Agents / Q000494:pharmacology*; D002460:Cell Line; D015658:HIV Infections / Q000821:virology; D016333:HIV Protease / Q000235:genetics; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D054303:HIV Reverse Transcriptase / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D010641:Phenotype; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D013997:Time Factors
D019380:Anti-HIV Agents; D017320:HIV Protease Inhibitors; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase; D016333:HIV Protease