title: Lamivudine (3TC) resistance in HIV-1 reverse transcriptase involves steric hindrance with beta-branched amino acids.
abstract: An important component of triple-drug anti-AIDS therapy is 2', 3'-dideoxy-3'-thiacytidine (3TC, lamivudine). Single mutations at residue 184 of the reverse transcriptase (RT) in HIV cause high-level resistance to 3TC and contribute to the failure of anti-AIDS combination therapy. We have determined crystal structures of the 3TC-resistant mutant HIV-1 RT (M184I) in both the presence and absence of a DNA/DNA template-primer. In the absence of a DNA substrate, the wild-type and mutant structures are very similar. However, comparison of crystal structures of M184I mutant and wild-type HIV-1 RT with and without DNA reveals repositioning of the template-primer in the M184I/DNA binary complex and other smaller changes in residues in the dNTP-binding site. On the basis of these structural results, we developed a model that explains the ability of the 3TC-resistant mutant M184I to incorporate dNTPs but not the nucleotide analog 3TCTP. In this model, steric hindrance is expected for NRTIs with beta- or L- ring configurations, as with the enantiomer of 3TC that is used in therapy. Steric conflict between the oxathiolane ring of 3TCTP and the side chain of beta-branched amino acids (Val, Ile, Thr) at position 184 perturbs inhibitor binding, leading to a reduction in incorporation of the analog. The model can also explain the 3TC resistance of analogous hepatitis B polymerase mutants. Repositioning of the template-primer as observed in the binary complex (M184I/DNA) may also occur in the catalytic ternary complex (M184I/DNA/3TCTP) and contribute to 3TC resistance by interfering with the formation of a catalytically competent closed complex.
authors.affiliation: Center for Advanced Biotechnology and Medicine (CABM) and Rutgers University Chemistry Department, 679 Hoes Lane, Piscataway, NJ 08854-5638, USA.
authors.forename: S G
authors.initials: SG
authors.lastname: Sarafianos
authors.forename: K
authors.initials: K
authors.lastname: Das
authors.forename: A D
authors.initials: AD
authors.lastname: Clark
authors.forename: J
authors.initials: J
authors.lastname: Ding
authors.forename: P L
authors.initials: PL
authors.lastname: Boyer
authors.forename: S H
authors.initials: SH
authors.lastname: Hughes
authors.forename: E
authors.initials: E
authors.lastname: Arnold
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017124:Conserved Sequence; D019281:Dimerization; D004352:Drug Resistance, Microbial; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D006801:Humans; D019259:Lamivudine / Q000737:chemistry* / Q000494:pharmacology*; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D011487:Protein Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D013698:Templates, Genetic
chemical_list: D046911:Macromolecular Substances; D012367:RNA, Viral; D019259:Lamivudine; D054303:HIV Reverse Transcriptase