Molecular Mechanism by which K70E in HIV-1 Reverse Transcriptase Confers Resistance to Nucleoside Reverse Transcriptase Inhibitors

Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

^* To whom correspondence should be addressed. Email: CremerN{at}dom.pitt.edu,

	Abstract

The K70E mutation in HIV-1 reverse transcriptase (RT) has become more prevalent in clinical samples, particularly in isolates derived from patients failing triple nucleoside regimens that include tenofovir (TNV), abacavir and lamivudine (3TC). To elucidate the molecular mechanism by which this mutation confers resistance to these nucleoside RT inhibitors (NRTI), we conducted detailed biochemical analyses comparing wild type (WT), K70E, and K65R HIV-1 RT. Pre-steady-state kinetic experiments demonstrate that K70E in HIV-1 RT allows the enzyme to discriminate between the natural dNTP substrate and the NRTI-triphosphate (NRTI-TP). Compared to the WT enzyme, K70E RT showed 2.1-, 2.3- and 3.5-fold resistance toward TNV-diphosphate, carbovir-TP and 3TC-TP, respectively. By comparison, K65R RT demonstrated 12.4-, 12.0- and 13.1-fold resistance, respectively, toward the same analogs. NRTI-TP discrimination by K70E (and K65R) was primarily due to decreased rates of NRTI-TP incorporation and not to changes in analog binding affinity. The K65R and K70E mutations also profoundly impaired the ability of RT to excise 3'-azido-2',3'-dideoxythymidine monophosphate and other NRTI-MP from the 3'-end of a chain-terminated primer. When introduced into an enzyme with the thymidine analog mutations (TAMs) M41L, L210W and T215Y, K70E inhibited ATP-mediated excision of AZT-MP. Taken together, these findings indicate that K70E, like K65R, reduces susceptibility to NRTI by selectively decreasing NRTI-TP incorporation and is antagonistic to TAM-mediated nucleotide excision.