A Novel Polymorphism at Codon 333 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Can Facilitate Dual Resistance to Zidovudine and l-2′,3′-Dideoxy-3′-Thiacytidine

Zidovudine (3′-azido-3′-doexythymidine, AZT, or Retrovir) is commonly used in combination with other antiretroviral agents for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. AZT therapy delays the development of AIDS and increases the survival of patients with AIDS (6, 12, 39). Long-term treatment with AZT monotherapy results in the eventual development of resistance to AZT (22, 26, 33), which ultimately leads to treatment failure (4, 32). Site-directed mutagenesis experiments have demonstrated that at least five amino acid changes in reverse transcriptase (RT) of HIV-1 (at codons 41, 67, 70, 215, and 219) are responsible for AZT resistance (16, 20; for reviews, see references 19 and 31). The first mutation to arise after several months of AZT monotherapy is typically at codon 70, which results in an approximate eightfold increase in the 50% inhibitory concentration (IC50). More resistant viruses, usually having combinations of mutations that include changes at codons 41 and 215, subsequently become dominant in the resistant virus population (3, 17). Highly AZT-resistant variants (with IC50s increased more than 100-fold) require the accumulation of four to six mutations in RT, frequently including a recently recognized mutation at codon 210 of RT (10, 11). In contrast to AZT resistance, high-level resistance to the nucleoside analog l-2′,3′-dideoxy-3′-thiacytidine (3TC or lamivudine) is conferred by a single mutation in HIV-1 RT at codon 184 (Met-184 to Val or occasionally Ile) (2, 7, 34, 38). The appearance of this mutation during 3TC therapy is associated with an increase in plasma HIV-1 levels and treatment failure (35). Of note is that the 184 Val mutation causes a concomitant increase in AZT sensitivity in genotypically AZT-resistant backgrounds (2, 24, 38). Furthermore, AZT-3TC combination therapy in drug-naive patients leads to a delay in the appearance of AZT resistance mutations even though 3TC resistance occurs rapidly (18, 24). These observations have prompted speculation that dual resistance to both drugs may not develop easily, as phenotypic AZT resistance in the presence of the 184 Val mutation may be rare. The results of several clinical trials examining the safety and efficacy of AZT-3TC combination therapy have recently been published (1, 5, 14, 37). Collectively, these studies showed substantial effects on virological markers and significant clinical benefit in either therapy-naive or AZT-experienced patients. One plausible explanation for the duration of this benefit in therapy-naive patients is the observed delay in the development of AZT resistance, as discussed above. In AZT-experienced patients, more complex patterns of virological response and resistance have been observed, ranging from restoration of AZT susceptibility to the development of AZT-3TC dual resistance (13, 27, 28). An HIV-1 variant that was selected in cell culture and that became simultaneously highly resistant to AZT and 3TC was recently described (8). This mutant was obtained by cell culture passage in both AZT and 3TC of a preexisting highly AZT-resistant clinical isolate (1373). To increase our understanding of how HIV-1 can become resistant to both AZT and 3TC, we describe a mapping and site-directed mutagenesis study designed to define the genetic basis of dual resistance of this cell culture-selected variant. A novel polymorphism at RT codon 333 was shown to be responsible for facilitating dual resistance in the context of AZT and 3TC resistance mutations. To determine the relevance of this codon 333 polymorphism in AZT- and 3TC-treated patients, we studied HIV-1 clinical isolates by genetic mapping of RT molecular clones. In some cases, polymorphism at codon 333 was responsible for facilitating dual resistance.