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

Abstract

Recent clinical trials examining 3'-azido-3'-deoxythymidine (AZT, zidovudine, or Retrovir) combined with L-2', 3'-dideoxy-3'-thiacytidine (3TC or lamivudine) have shown that combination therapy with these nucleoside analogs affords significant virological and clinical benefits. The addition of 3TC to AZT delays AZT resistance in therapy-naive patients and can restore viral AZT susceptibility in patients who previously received AZT alone. In some AZT-experienced patients, the virological response to AZT-3TC therapy is not sustained and virus resistant to both drugs can be identified. To gain insight into the possible mechanism of dual resistance, we studied a recently described variant resistant to both AZT and 3TC and obtained by simultaneous passage of an AZT-resistant clinical isolate in cell culture with AZT and 3TC. Genetic mapping and site-directed mutagenesis experiments demonstrated that a polymorphism at codon 333 (Gly to Glu) of human immunodeficiency virus type 1 reverse transcriptase (RT) was critical in facilitating dual resistance in a complex background of AZT and 3TC resistance mutations. To assess the potential clinical relevance of RT codon 333 changes, we studied dually resistant viruses from patients taking AZT and 3TC. Genetic mapping of RT molecular clones derived from patients' plasma samples demonstrated that in some cases polymorphism at codon 333 was responsible for facilitating dual resistance.

FIG. 1

Mapping AZT and 3TC dual resistance in HIV-1 strain AZT^r3TC^r. In order to map dual resistance in laboratory isolate AZT^r3TC^r, various RT fragments were exchanged between either mpAZT^r3TC^r and the wild-type virus (HIV-1_HXB2-D) or AZT^r3TC^r and RTMQ+184Val (RTMQ+184). RTMQ+184Val contains the following RT mutations in the HIV-1_HXB2-D background: Met41Leu, Asp67Asn, Lys70Arg, Met184Val, and Thr215Tyr. The open bars represent HIV-1_HXB2-D RT, the solid bars represent AZT^r3TC^r RT, and the hatched bars represent RTMQ+184Val RT. The numbers above the bars are the amino acid positions at which RT fragments were exchanged. In recombinant RRcomb2,3, residues 40 to 231 represent the size of the deletion in the deletion clone used to construct the virus; since this virus was constructed by recombination, the maximum possible size of the mutant fragment in the virus is the size of the deletion. Recombinant viruses were assessed for AZT susceptibility with the HeLa-CD4⁺ cell assay as described in Materials and Methods.

FIG. 2

AZT susceptibility of recombinant HIV-1 variants with altered RT codon 333. Recombinant viruses were constructed by site-directed mutagenesis in order to alter RT codon 333. This codon was changed from Glu to Gly in the dually resistant strain AZT^r3TC^r (designated RR) to produce RR/333G. In addition, codon 333 was changed from Gly to Glu in the laboratory isolate RTMQ+184Val (designated RTMQ+184V) to produce Q184V+333E. RTMQ is an AZT-resistant strain based on HIV-1_HXB2-D and containing the following changes in RT; Met41Leu, Asp67Asn, Lys70Arg, and Thr215Tyr. Recombinant viruses were assessed for AZT susceptibility with the HeLa-CD4⁺ cell assay as described in Materials and Methods.