A combination of decreased NRTI incorporation and decreased excision determines the resistance profile of HIV-1 K65R RT.
OBJECTIVE
To determine the mechanisms of resistance of K65R mutant reverse transcriptase (RT) to the currently approved nucleoside and nucleotide RT inhibitors (NRTI).


METHODS
Susceptibilities of K65R mutant HIV-1 to NRTI were determined in cell culture. The Ki/Km values were measured to determine the relative binding or incorporation of the NRTI, and ATP-mediated excision of incorporated NRTI was measured to determine NRTI stability as chain terminators.


RESULTS
K65R HIV-1 had decreased susceptibility to most NRTI, but increased susceptibility to zidovudine (ZDV). Ki/Km values were increased 2- to 13-fold for K65R compared to wild-type RT for all NRTI, indicating decreased binding or incorporation. However, K65R also showed decreased excision of all NRTI compared to wild-type, indicating greater stability once incorporated. At physiological nucleotide concentrations, excision of ZDV, carbovir (the active metabolite of abacavir; ABC), stavudine (d4T), and tenofovir was further decreased, while excision of didanosine (ddI), zalcitabine (ddC), lamivudine (3TC), and emtricitabine (FTC) was unchanged. The decreased binding or incorporation of ZDV by K65R appeared counteracted by decreased excision resulting in overall increased susceptibility to ZDV in cell culture. For ABC, tenofovir, and d4T, despite having decreased excision, decreased binding or incorporation resulted in reduced susceptibilities to K65R. For ddI, ddC, 3TC, and FTC, decreased binding or incorporation by K65R appeared responsible for the decreased susceptibilities in cell culture.


CONCLUSIONS
NRTI resistance in cells can consist of both altered binding or incorporation and altered excision of the NRTI. For K65R, the combination of these opposing mechanisms results in decreased susceptibility to most NRTI but increased susceptibility to ZDV.
White
Kirsten L
KL
Gilead Sciences Inc. Foster City, California 94404, USA. kwhite@gilead.com
Margot
Nicolas A
NA
Ly
John K
JK
Chen
James M
JM
Ray
Adrian S
AS
Pavelko
Martina
M
Wang
Ruth
R
McDermott
Martin
M
Swaminathan
S
S
Miller
Michael D
MD
D019380:Anti-HIV Agents / Q000627:therapeutic use*; D002478:Cells, Cultured; D024882:Drug Resistance, Viral / Q000235:genetics*; D015658:HIV Infections / Q000188:drug therapy*; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors / Q000627:therapeutic use*
D019380:Anti-HIV Agents; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase