Background:  Two clinical isolates with no known non-nucleoside reverse transcriptase inhibitor (NNRTI) -resistance mutations exhibited a surprising high-level resistance phenotype to NVP. Both isolates emerged during a non-NNRTI treatment, and disappeared immediately after interruption of the treatment.

Methods:  To identify mechanism(s) of nevirapine (NVP) resistance in the clinical isolates, we generated molecular clones of HIV-1 by replacing the N-terminal (amino acids 15 to 267) or C-terminal region (amino acids 268 to 560) of reverse transcriptase (RT) with the corresponding regions from the RT derived from the clinical isolates. The mutations were introduced by site-directed mutagenesis. The effect of N348I on replication capacity and drug susceptibility to NRTI and NNRTI was analyzed using standard protocols.

Results:  NVP resistance (30-fold) was observed only in recombinant viruses that contained RT with C-terminal substitutions from the corresponding regions derived from clinical isolates. Phenotypic assays for a panel of single amino acid substituted recombinant viruses revealed that the N348I mutation was responsible for the NVP resistance (22-fold). Interestingly, N348I also conferred zidovudine (AZT) and didanosine (ddI) resistance (23- and 5.4-fold, respectively). In the absence of NVP, HIV-1_WT and HIV-1_N348I exhibited comparable replication capacities. However, HIV-1_WT was outgrown by HIV-1_N348I in the presence of NVP. Acquisition of N348I observed in 5 patients (n = 39, 12%) who received zidovudine (AZT) or didanosine (ddI) -containing regimens, was significantly higher than that in the Los Alamos database (n = 328, 0.9%) (Fisher’s exact test, p <0.0001).

Conclusions:  Our current hypothesis is that the novel N348I mutation, which is located at the connection domain, confers resistance to NVP by contributing to structural changes that may affect the positioning of the nucleic acid and abrogate the inhibitory effects of NVP. Cross-resistance to AZT and ddI may be explained by an enhanced efficiency of the resistance-causing excision reaction that may be the result of possible changes in nucleic acid binding. Such changes would have to be relatively small and selective, so that they would not affect the replication capacity of the mutant. We are currently testing this hypothesis. The N348I mutation appears to be frequently acquired during AZT- or ddI-based regimens. However, accurate evaluation of the detection frequency is hampered by the fact that residue 348 is outside the region that is typically tested for drug-resistance mutations.