3′-Azido-3′-Deoxythymidine (AZT) and AZT-Resistant Reverse Transcriptase Can Increase the In Vivo Mutation Rate of Human Immunodeficiency Virus Type 1

How antiretroviral drug resistance influences human immunodeficiency virus type 1 (HIV-1) evolution is not clear. This study tested the hypothesis that antiretroviral drugs such as 3*-azido-3*-deoxythymidine (AZT) can influence the in vivo mutation rate of HIV-1. It was observed that AZT can increase the rate of HIV-1 mutation by a factor of 7 in a single round of replication. In addition, (2)2*,3*-dideoxy-3*-thiacytidine (3TC) was also found to increase the mutation rate of HIV-1 by a factor of 3. It was also found that HIV-1 drug-resistant reverse transcriptase (RT) variants can influence the in vivo mutation rate. Replication of HIV-1 with AZTresistant RTs increased the mutation rate by as much as a factor of 3, while replication of HIV-1 with a 3TCresistant RT (M184V) had no significant effect on the mutation rate. It was observed that only high-level, AZT-resistant RT variants could influence the in vivo mutation rate (i.e., M41L/T215Y and M41L/D67N/K70R/ T215Y). In total, these observations indicate that both antiretroviral drugs and drug resistance mutations can influence the in vivo mutation rate of HIV-1. The emergence of drug-resistant variants of human immunodeficiency virus type 1 (HIV-1) has frustrated efforts to effectively block virus replication at a clinically meaningful level (4, 16). These variants are not only resistant to antiretroviral drugs such as 39-azido-39-deoxythymidine (AZT) and (2)29,39dideoxy-39-thiacytidine (3TC) but can also have altered tropism and virulence properties (29). Reverse transcriptase (RT) is thought to play a role in the generation of retrovirus diversity (38). Antiretroviral drugs have been previously shown to influence the in vivo mutation rate of spleen necrosis virus (SNV) and murine leukemia virus (MLV). First, 5-azacytidine, which is a nucleoside analog that is incorporated into RNA and inhibits protein synthesis, was found to increase the in vivo SNV mutation rate by a factor of 13 (30). Second, AZT was found to increase the SNV mutant frequency by a factor of 10, while it increased the MLV mutant frequency by a factor of 3 (10). It has been shown that deoxynucleoside triphosphate (dNTP) pool imbalances can influence the SNV and MLV mutation rates but that AZT influences the SNV and MLV rates by a mechanism not involving alterations in dNTP pools (11). A tractable genetic system has been developed to measure the forward mutation rate of HIV-1 with a vector containing the lacZa peptide gene as a reporter for mutations (21, 23, 24). This system allows for the study of mutations that occur during a single round of HIV-1 replication. The mutation rate of HIV-1 in this system was determined to be 3 3 10 25 mutations per target base pair per cycle in HeLa cells (24) and 4 3 10 25 mutations per target base pair per cycle in a T-lymphoid cell line (20). Three issues were addressed in the present study. First, we tested the hypothesis that the antiretroviral drugs AZT and 3TC could influence the HIV-1 mutation rate. Second, we tested whether specific mutations in HIV-1 RT that conferred resistance to either AZT or 3TC could influence the rate of HIV-1 mutation. Third, we tested whether there was a correlation between increased AZT drug resistance and the HIV-1 mutation rate. We found that replication of HIV-1 in the presence of either AZT or 3TC can increase the HIV-1 mutation rate. In addition, we found that two AZT-resistant variants, but not a 3TC variant, increased the mutation rate. Finally, we observed that high-level AZT resistance in HIV-1 correlated with significant increases in the in vivo mutation rate.