Combination of drugs and drug-resistant reverse transcriptase results in a multiplicative increase of human immunodeficiency virus type 1 mutant frequencies

Abstract

Replication of drug-resistant human immunodeficiency virus type 1 (HIV-1) in the presence of drug can lead to the failure of antiretroviral drug treatment. Drug failure is associated with the accumulation of drug resistance mutations. Previous studies have shown that 3'-azido-3'-deoxythymidine (AZT), (-)2',3'-dideoxy-3'-thiacytidine (3TC), and AZT-resistant HIV-1 reverse transcriptase (RT) can increase the virus in vivo mutation rate. In this study, the combined effects of drug-resistant RT and antiretroviral drugs on the HIV-1 mutant frequency were determined. In most cases, a multiplicative effect was observed with AZT-resistant or AZT/3TC dually resistant RT and several drugs (i.e., AZT, 3TC, hydroxyurea, and thymidine) and led to increases in the odds of recovering virus mutants to over 20 times that of the HIV-1 mutant frequency in the absence of drug or drug-resistance mutations. This observation indicates that HIV-1 can mutate at a significantly higher rate when drug-resistant virus replicates in the presence of drug. These increased mutant frequencies could have important implications for HIV-1 population dynamics and drug therapy regimens.

FIG. 1.

HIV-1 vector used for analysis of virus mutant frequencies. (A) Expression cassette. The HIV-1 vector used has been previously described (23, 27, 28). The vector contains a cassette with the simian virus 40 promoter driving expression of the neo gene, a bacterial origin of replication, and the lacZα peptide gene. (B) Protocol for one cycle of HIV-1 vector virus replication. The steps going from a parental shuttle vector provirus in the step 2 cell to a vector provirus in the step 3 cell constitute a single cycle of replication. The influence of the antiretroviral drugs on HIV-1 mutant frequencies was determined by posttreatment of step 3 cells with drug. Posttreatment refers to maintaining step 3 cells in medium supplemented with drug for 2 h before cocultivation and continuing until 24 h after cocultivation. G418-resistant cells infected with the HIV-1 vector were pooled and the total DNA was purified and digested with restriction enzymes. The vector cassette was selectively purified with the Lac repressor protein, ligated, and introduced into E. coli. The ratio of light blue and white bacterial colonies to total colonies observed was used to determine virus mutant frequencies.

FIG. 2.

HU increases HIV-1 mutant frequencies in a dose-dependent manner. HU was added at the concentrations indicated for 2 h before cocultivation and continuing until 24 h after cocultivation. Analysis of the effects of HU at different concentrations was done in parallel and each concentration was tested in at least two independent experiments. The average mutant frequency for each HU treatment is shown.

FIG. 3.

Thy increases HIV-1 mutant frequencies in a dose-dependent manner. Thy was added at the concentrations indicated for 2 h before cocultivation and continuing until 24 h after cocultivation. Analysis of the effects of Thy at different concentrations was done in parallel and each concentration was tested in at least two independent experiments. The average mutant frequency for each Thy treatment is shown.