Background:  After almost 20 years of HIV treatment with the reverse transcriptase inhibitor zidovudine (AZT), the complete pattern of resistance against AZT is still not completely clear. Classic AZT-resistance-associated amino acid substitutions have been mapped at positions 41, 67, 70, 210, 215, and 219 in RT. We have identified several patient-derived viruses containing the E40F change in the background of the classical AZT-mutations. In this study we have investigated the effects of this change on resistance and replication capacity (RC) and its frequency in the treated population.

Methods:  Patient-derived HIV RT variants harboring the E40F change in the background of classical AZT-mutations were investigated. We have cloned the N-terminal part of the RT gene (amino acid 25 through 314) in a reference HIV strain and the E40F change was reverted to wild type by site-directed mutagenesis. The impact on RC and drug susceptibility for AZT and stavudine (d4T) were analyzed. 

Results:  The patient-derived virus clones contained the E40F change in the background of the classical AZT-mutations M41L, L210W, T215Y ± D67N. These viral clones demonstrated high-level resistance against both AZT and d4T but had a reduced RC compared to wild type. Changing the E40F substitution back to wild type resulted in a 5-fold decrease in resistance for AZT and a slight increase in RC. Analysis of a large database revealed the presence of this change in 0.45% of the treated population, while it was not present in the naïve population.

Conclusions:  In this study we found a novel amino acid substitution in HIV-1 RT that contributes to AZT resistance. Selection of the E40F change results in a 5-fold increase in resistance to AZT at the price of a reduction in RC. Further research is warranted to determine why this amino acid change is relatively seldom observed in treated patients.