K70Q adds high-level tenofovir resistance to "Q151M complex" HIV reverse transcriptase through the enhanced discrimination mechanism.
HIV-1 carrying the "Q151M complex" reverse transcriptase (RT) mutations (A62V/V75I/F77L/F116Y/Q151M, or Q151Mc) is resistant to many FDA-approved nucleoside RT inhibitors (NRTIs), but has been considered susceptible to tenofovir disoproxil fumarate (TFV-DF or TDF). We have isolated from a TFV-DF-treated HIV patient a Q151Mc-containing clinical isolate with high phenotypic resistance to TFV-DF. Analysis of the genotypic and phenotypic testing over the course of this patient's therapy lead us to hypothesize that TFV-DF resistance emerged upon appearance of the previously unreported K70Q mutation in the Q151Mc background. Virological analysis showed that HIV with only K70Q was not significantly resistant to TFV-DF. However, addition of K70Q to the Q151Mc background significantly enhanced resistance to several approved NRTIs, and also resulted in high-level (10-fold) resistance to TFV-DF. Biochemical experiments established that the increased resistance to tenofovir is not the result of enhanced excision, as K70Q/Q151Mc RT exhibited diminished, rather than enhanced ATP-based primer unblocking activity. Pre-steady state kinetic analysis of the recombinant enzymes demonstrated that addition of the K70Q mutation selectively decreases the binding of tenofovir-diphosphate (TFV-DP), resulting in reduced incorporation of TFV into the nascent DNA chain. Molecular dynamics simulations suggest that changes in the hydrogen bonding pattern in the polymerase active site of K70Q/Q151Mc RT may contribute to the observed changes in binding and incorporation of TFV-DP. The novel pattern of TFV-resistance may help adjust therapeutic strategies for NRTI-experienced patients with multi-drug resistant (MDR) mutations.
Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, Missouri, United States of America.
Atsuko
A
Hachiya
Eiichi N
EN
Kodama
Matthew M
MM
Schuckmann
Karen A
KA
Kirby
Eleftherios
E
Michailidis
Yasuko
Y
Sakagami
Shinichi
S
Oka
Kamalendra
K
Singh
Stefan G
SG
Sarafianos
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D020134:Catalytic Domain; D003198:Computer Simulation; D024882:Drug Resistance, Viral / Q000235:genetics*; D006678:HIV / Q000235:genetics / Q000302:isolation & purification; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D020125:Mutation, Missense; D063065:Organophosphonates / Q000494:pharmacology*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D000068698:Tenofovir
D063065:Organophosphonates; D018894:Reverse Transcriptase Inhibitors; D000068698:Tenofovir; D054303:HIV Reverse Transcriptase; D000225:Adenine
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