title: Molecular mechanism by which the K70E mutation in human immunodeficiency virus type 1 reverse transcriptase confers resistance to nucleoside reverse transcriptase inhibitors.
abstract: The K70E mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) has become more prevalent in clinical samples, particularly in isolates derived from patients for whom triple-nucleoside regimens that include tenofovir (TNV), abacavir, and lamivudine (3TC) failed. 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 the K70E mutation in HIV-1 RT allows the enzyme to discriminate between the natural deoxynucleoside triphosphate substrate and the NRTI triphosphate (NRTI-TP). Compared to the WT enzyme, K70E RT showed 2.1-, 2.3-, and 3.5-fold-higher levels of resistance toward TNV-diphosphate, carbovir-TP, and 3TC-TP, respectively. By comparison, K65R RT demonstrated 12.4-, 12.0-, and 13.1-fold-higher levels of resistance, respectively, toward the same analogs. NRTI-TP discrimination by the K70E (and K65R) mutation 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 (AZT-MP) 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, the K70E mutation inhibited ATP-mediated excision of AZT-MP. Taken together, these findings indicate that the K70E mutation, like the K65R mutation, reduces susceptibility to NRTI by selectively decreasing NRTI-TP incorporation and is antagonistic to TAM-mediated nucleotide excision.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, S817 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, USA. CremerN@dom.pitt.edu
authors.forename: Nicolas
authors.initials: N
authors.lastname: Sluis-Cremer
authors.forename: Chih-Wei
authors.initials: CW
authors.lastname: Sheen
authors.forename: Shannon
authors.initials: S
authors.lastname: Zelina
authors.forename: Pedro S Argoti
authors.initials: PS
authors.lastname: Torres
authors.forename: Urvi M
authors.initials: UM
authors.lastname: Parikh
authors.forename: John W
authors.initials: JW
authors.lastname: Mellors
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D019943:Amino Acid Substitution / Q000235:genetics*; D019380:Anti-HIV Agents / Q000145:classification / Q000494:pharmacology; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D015224:Dideoxynucleosides / Q000494:pharmacology; D054306:Dideoxynucleotides; D024921:Drug Resistance, Multiple, Viral / Q000235:genetics; D005838:Genotype; D018698:Glutamic Acid / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D006801:Humans; D019259:Lamivudine / Q000031:analogs & derivatives / Q000494:pharmacology; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D063065:Organophosphonates / Q000494:pharmacology; D010641:Phenotype; D018894:Reverse Transcriptase Inhibitors / Q000145:classification / Q000494:pharmacology*; D000068698:Tenofovir; D013942:Thymine Nucleotides / Q000494:pharmacology; D015215:Zidovudine / Q000031:analogs & derivatives / Q000494:pharmacology; D000099227:Abacavir
chemical_list: D000225:Adenine; D019380:Anti-HIV Agents; D003570:Cytidine Triphosphate; D015224:Dideoxynucleosides; D054306:Dideoxynucleotides; D018698:Glutamic Acid; D054303:HIV Reverse Transcriptase; D019259:Lamivudine; D008239:Lysine; D063065:Organophosphonates; D018894:Reverse Transcriptase Inhibitors; D000068698:Tenofovir; D013942:Thymine Nucleotides; D015215:Zidovudine; D000099227:Abacavir; C417268:lamivudine triphosphate; C057287:3'-azido-3'-deoxythymidine 5'phosphate