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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.
H Walter, and
B Schmidt, and
K Korn, and
A M Vandamme, and
T Harrer, and
K Uberla
January 2007,
AIDS (London, England),
H Walter, and
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K Korn, and
A M Vandamme, and
T Harrer, and
K Uberla
September 1991,
Nihon rinsho. Japanese journal of clinical medicine,
H Walter, and
B Schmidt, and
K Korn, and
A M Vandamme, and
T Harrer, and
K Uberla
June 2007,
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H Walter, and
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Antiviral therapy,
H Walter, and
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January 1993,
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H Walter, and
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H Walter, and
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Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology,
H Walter, and
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K Uberla
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Mini reviews in medicinal chemistry,
H Walter, and
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K Uberla
January 2021,
Bioorganic & medicinal chemistry letters,
H Walter, and
B Schmidt, and
K Korn, and
A M Vandamme, and
T Harrer, and
K Uberla
April 2012,
Nihon rinsho. Japanese journal of clinical medicine,
