White Kirsten L, Margot Nicolas A, Ly John K, Chen James M, Ray Adrian S, Pavelko Martina, Wang Ruth, McDermott Martin, Swaminathan S, Miller Michael D
Gilead Sciences Inc. Foster City, California 94404, USA.
AIDS. 2005 Nov 4;19(16):1751-60. doi: 10.1097/01.aids.0000189851.21441.f1.
To determine the mechanisms of resistance of K65R mutant reverse transcriptase (RT) to the currently approved nucleoside and nucleotide RT inhibitors (NRTI).
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.
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.
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.
确定K65R突变型逆转录酶（RT）对目前已获批的核苷类和核苷酸类逆转录酶抑制剂（NRTI）产生耐药性的机制。
在细胞培养中测定K65R突变型HIV-1对NRTI的敏感性。测量Ki/Km值以确定NRTI的相对结合或掺入情况，并测量ATP介导的已掺入NRTI的切除情况以确定NRTI作为链终止剂的稳定性。
K65R HIV-1对大多数NRTI的敏感性降低，但对齐多夫定（ZDV）的敏感性增加。与野生型RT相比，K65R对所有NRTI的Ki/Km值增加了2至13倍，表明结合或掺入减少。然而，与野生型相比，K65R对所有NRTI的切除也减少，表明掺入后稳定性更高。在生理核苷酸浓度下，ZDV、卡博韦（阿巴卡韦的活性代谢物；ABC）、司他夫定（d4T）和替诺福韦的切除进一步减少，而地丹诺辛（ddI）、扎西他滨（ddC）、拉米夫定（3TC）和恩曲他滨（FTC）的切除未改变。K65R对ZDV的结合或掺入减少似乎被切除减少所抵消，导致细胞培养中对ZDV的总体敏感性增加。对于ABC、替诺福韦和d4T，尽管切除减少，但结合或掺入减少导致对K65R的敏感性降低。对于ddI、ddC、3TC和FTC，K65R结合或掺入减少似乎是细胞培养中敏感性降低的原因。
细胞中的NRTI耐药性可能包括NRTI结合或掺入的改变以及切除的改变。对于K65R，这些相反机制的组合导致对大多数NRTI的敏感性降低，但对ZDV的敏感性增加。
