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 (Δ67) 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 Δ67 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 Δ67 plus T69G/K70R/L74I/K103N/T215F/K219Q in RT replicated as efficiently as wild-type virus (Wt). In contrast, the construct without Δ67 exhibited impaired replication (23% of growth of Wt). A competitive fitness study failed to reveal any differences in replication rates between the Δ67+T69G/K70R/L74I/K103N/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 D67N/K70R/T215F/K219Q mutant backbone prior to appearance of the Δ67 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 Δ67 deletion led to a virus with improved replication and high-level AZT resistance.