Molecular Mechanism of Tenofovir, Abacavir, and Lamivudine Resistance by the K70E Mutation in HIV-1 Reverse Transcriptase
Nicolas Sluis-Cremer*, P Argoti Torres, J Grzybowski, U Parikh, and J Mellors
Univ of Pittsburgh, PA, US
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 (kpol) and not to changes in nucleotide analog binding
affinity (Kd). 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.