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Elucidating the molecular interactions of the L ‐nucleotide analogs with the Complex of HIV‐1 Reverse Transcriptase and DNA and the mechanism of drug resistance conferred by the M184V mutation
Author(s) -
Suo Zucai
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.02349
Subject(s) - reverse transcriptase , resistance mutation , nucleotide , chemistry , dna , stereochemistry , nucleotidyltransferase , nucleoside , mutant , nucleoside analogue , reverse transcriptase inhibitor , microbiology and biotechnology , biochemistry , biology , rna , gene
Emtricitabine (FTC) and lamivudine (3TC) are widely used nucleoside reverse transcriptase inhibitors (NRTIs) in antiretroviral therapy for HIV. They contain an oxathiolane ring with unnatural (−)‐stereochemistry as a mimic of the ribose ring found in natural deoxynucleotides ( L ‐nucleoside). Treatment with FTC or 3TC primarily selects for the HIV‐1 reverse transcriptase (RT) M184V/I resistance mutants that are characterized by a marked decrease of incorporation of these NRTIs. Pre‐steady state kinetic data and crystal structures of both (−) and (+) oxathiolane analog triphosphates were determined in wild‐type and M184V HIV‐1 RT to elucidate the structural features of RT that recognize these substrates and gain insight into how M184V confers resistance. The pre‐steady state kinetic data reveal that (−)‐FTC‐TP and (−)‐3TC‐TP have higher binding affinities (1/ K d ) for WT RT than dCTP however they have a slower rate of incorporation ( k p ). The crystal structures corroborate these results revealing that the oxathiolane sulfur orients toward the DNA primer terminus and two different triphosphate orientations are observed which would reduce the rate of incorporation. The structure of (+)‐FTC‐TP shows a close match to that of the natural substrate dCTP illustrating how the oxathiolane D ‐isomers adopt a triphosphate orientation conducive for incorporation. The M184V mutation displays significantly greater (>200‐fold) K d for the L isomers and moderately higher (>9‐fold) K d for the D ‐isomers compared to dCTP. The structure of M184V RT illustrates how the mutation repositions the oxathiolane ring, inducing a shift of the triphosphates into a conformation that hinders nucleotide incorporation. This combined study provides a comprehensive kinetic and structural basis for inhibiting WT HIV‐1 RT by the active metabolites of 3TC and FTC and the mechanism of M184V resistance. Support or Funding Information This work was supported by a grant from the National Science Foundation (grant number MCB‐1716168).