Identification of a novel resistance (E40F) and compensatory (K43E) substitution in HIV-1 reverse transcriptase

A large database (Quest Diagnostics database) was analysed to determine the associations of the E40F and K43E changes with known resistance mutations. Both amino acid changes are strongly associated with the well known NRTI-resistance mutations M41L, L210W and T215Y. In addition, a strong positive association between these changes themselves was observed. A panel of recombinant viruses was generated by site-directed mutagenesis and phenotypically analysed. To determine the effect on replication capacity, competition and in vitro evolution experiments were performed. Introduction of E40F results in an increase in Zidovudine resistance ranging from nine to fourteen fold depending on the RT background and at the same time confers a decrease in viral replication capacity. The K43E change does not decrease the susceptibility to Zidovudine but increases viral replication capacity, when combined with E40F, demonstrating a compensatory role for this codon change.In conclusion, we have identified a novel resistance (E40F) and compensatory (K43E) change in HIV-1 RT. Further research is indicated to analyse the clinical importance of these changes.Shortly after the introduction of Zidovudine (AZT) in 1987 it became clear that HIV-1 is able to develop resistance to this drug [1,2]. Now, after twenty years of NRTI usage in the clinic the complete pattern of resistance is still not understood. Multiple studies have identified mutations at (at least) six codons in the reverse transcriptase (RT) enzyme (thymidine analogue associated mutations (TAMs); M41L, D67N, K70R, L210W, T215Y/F and K219Q/E) that can cause a decrease in Zidovudine susceptibility [3-7]. HIV-1 develops these TAMs by two distinct pathways: the TAM-1 pathway consisting of T215Y, M41L, L210W and sometimes D67N or the TAM-2 pathway including T215F, K70R, K219Q/E and D67N [8-10]. These substitutions cluster around the dNTP binding pocket and confer resistance by increasing the excision of the incorporated nucleoside a