Background:  The HIV-1 reverse transcriptase (RT) mutation K65R is selected by the nucleoside and nucleotide RT inhibitors (NRTI) ddI, abacavir, tenofovir, and d4T. Viruses containing K65R display decreased susceptibility to tenofovir, ddI, 3TC, ddC, abacavir, and d4T, but slight hypersusceptibility to AZT in vitro.  This study characterizes the molecular mechanisms of resistance of K65R RT to NRTI.

Methods:  Virus susceptibility was determined using the PhenoSense assay (ViroLogic).  Steady-state enzymatic analyses measured the relative inhibitory capacities (K_i/K_m) for the active metabolites of tenofovir, ddI, abacavir, d4T, and AZT for wild-type (WT) and K65R mutant RT. ATP-mediated excision of NRTI was measured for WT and K65R RT. Computer modeling and molecular dynamics were utilized to examine the structure and mobility of the WT and K65R RT active site bound to DNA and tenofovir-diphosphate.

Results: The K_i/K_m of tenofovir, ddI, abacavir, d4T, and AZT were increased 5- to 17-fold for K65R compared to WT RT resulting in decreased incorporation of these inhibitors.  ATP-mediated excision by WT RT was efficient for AZT and abacavir, intermediate for d4T and tenofovir, and low for ddA.  However, for all the NRTI, ATP-mediated excision was decreased approximately 3-fold for K65R compared to WT RT.  In the presence of relevant concentrations of the next complementary nucleotide, excision by WT and K65R RT became minimal for ddA, tenofovir, and d4T (<8%). Abacavir was excised at intermediate levels (26% and 11% excised by WT and K65R RT, respectively), and consistent with previous reports, excision remained high for AZT (43% and 19%, respectively). Molecular models suggest that the K65R-containing fingers subdomain of RT has decreased mobility compared to WT RT that may interfere with polymerization and excision. 

Conclusions:  For K65R RT, decreased binding/incorporation and decreased excision of NRTI must be considered together when explaining the resistance profile for this mutant. For AZT, even though there is decreased binding/incorporation by K65R, the decreased excision yields greater chain-termination stability, thus increasing overall susceptibility to AZT in cell culture. For abacavir, despite decreased excision by K65R, more strongly decreased binding/incorporation contributed to its overall reduced susceptibility to K65R. For the other NRTI, decreased binding/incorporation appeared solely responsible for the decreased susceptibilities.

Keywords: K65R; reverse transcriptase; tenofovir