Relative replication fitness of a high-level 3′-azido-3′-deoxythymidine-resistant variant of human immunodeficiency virus type 1 possessing an amino acid deletion at codon 67 and a novel substitution (Thr→Gly) at codon 69

The combination of an amino acid deletion at codon 67 (Delta 67) and Thr-to-Gly change at codon 69 (T69G) in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is associated with high-level resistance to multiple RT inhibitors. To determine the relative contributions of the Delta 67 and T69G mutations on viral fitness, we performed a series of studies of HIV replication using recombinant variants. A high-level 3 ' -azido-3 ' -deoxythymidine (AZT)-resistant variant containing Delta 67 plus T69G/K70R/L741/KI03N/T215F/ K219Q in RT replicated as efficiently as wild-type virus (Wt). In contrast, the construct without Delta 67 exhibited impaired replication (23% of growth of Wt). A competitive fitness study failed to reveal any differences in replication rates between the Delta 67+T69G/K70R/L741/KI03N/T215F/K219Q mutant and Wt. Evaluation of proviral DNA sequences over a 3-year period in a patient harboring the multiresistant HIV revealed that the T69G mutation emerged in the context of a D67NiK70R/T215F/K219Q mutant backbone prior to appearance of the Delta 67 deletion. To assess the impact of this stepwise accumulation of mutations on viral replication, a series of recombinant variants was constructed and analyzed for replication competence. The T69G mutation was found to confer 2 ' ,3 ' -dideoxyinosine resistance at the expense of fitness. Subsequently, the development of the Delta 67 deletion led to a virus with improved replication and high-level AZT resistance.