Background:  The K70E HIV-1 reverse transcriptase (RT) adefovir (ADV)-associated mutation has become more prevalent in clinical samples since the introduction of tenofovir use. Specifically, it was recently reported that K70E was selected in 10% of ART-naïve subjects receiving tenofovir (TDF), abacavir (ABC), and lamivudine (3TC) triple nucleoside reverse transcriptase inhibitor (NRTI) therapy. Since the mechanism by which K70E confers resistance to nucleoside analogs is unknown, we conducted biochemical analyses to define the role of this residue in resistance to TDF, ABC, and 3TC.

Methods:  Pre-steady state kinetic parameters for the single nucleotide incorporation of the natural substrates dATP, dCTP, and dGTP, as well as the nucleoside analogs TDF-diphosphate (TDF-DP), 3TC-triphosphate (3TC-TP), and carbovir-triphosphate (CBV-TP, the active metabolite of abacavir) were determined for purified recombinant wild type, K65R, and K70E HIV-1 RT. The zidovudine-monophosphate (AZT-MP) phosphorolytic excision activity of the recombinant enzymes was also analyzed.   

Results:  Compared to the wild type enzyme, K70E RT showed 2.1-, 2.3-, and 3.5-fold discrimination against TDF-DP, CBV-TP, and 3TC-TP, respectively. In comparison, K65R RT showed 12.4-, 12.0-, and 13.1-fold discrimination against the same substrates. The discrimination imparted by both the K65R and K70E mutations in HIV-1 RT was due to a decreased rate of nucleotide analog incorporation (k_pol) and not to changes in nucleotide analog binding affinity (K_d). Furthermore, RT containing either K65R or K70E exhibited significantly decreased rates of ATP-mediated excision of AZT-MP from a chain-terminated template/primer.

Conclusions:  The K70E mutation in HIV-1 RT confers resistance to NRTI via a nucleotide discrimination phenotype. The K70E mutation also significantly impairs the enzyme’s ability to excise AZT-MP, suggesting that this mutation may be antagonistic toward thymidine analog mutations. Therefore, the recent emergence of K70E in TDF- containing triple NRTI regimens might be counteracted by the addition of AZT.