Mechanistic characterization and molecular modeling of hepatitis B virus polymerase resistance to entecavir

• Published in: PloS one Vol. 5; no. 2; p. e9195
• Main Authors: Walsh, Ann W, Langley, David R, Colonno, Richard J, Tenney, Daniel J
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 12.02.2010; Public Library of Science (PLoS)
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Analysis; Antiviral Agents - pharmacology; Binding sites; Binding Sites - genetics; Cell culture; Chemical bonds; Cloning; Computational chemistry; Deoxyguanosine; Deoxyribonucleic acid; Disease resistance; DNA; DNA biosynthesis; DNA synthesis; Drug resistance; Drug Resistance, Viral - genetics; Energy; Entecavir; Enzymatic activity; Enzyme activity; Enzymes; Guanine - analogs & derivatives; Guanine - pharmacology; Guanosine Triphosphate - chemistry; Guanosine Triphosphate - metabolism; Hep G2 Cells; Hepatitis; Hepatitis B; Hepatitis B virus; Hepatitis B virus - enzymology; Hepatitis B virus - genetics; Humans; Hydrogen; Hydrogen Bonding; Infectious Diseases/Antimicrobials and Drug Resistance; Kinetics; Lamivudine; Lamivudine - pharmacology; Modelling; Models, Molecular; Molecular modelling; Protein Binding; Protein Structure, Tertiary; Proteins; R&D; Research & development; Ribonucleic acid; RNA; RNA-Directed DNA Polymerase - chemistry; RNA-Directed DNA Polymerase - genetics; RNA-Directed DNA Polymerase - metabolism; Studies; Substrate Specificity; Transcription; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism; Virology/Antivirals, including Modes of Action and Resistance; Virology/Viral and Gene Regulation; Virus Replication - drug effects; Virus Replication - genetics; Viruses; United States > US; Connecticut
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0009195

Entecavir (ETV) is a deoxyguanosine analog competitive inhibitor of hepatitis B virus (HBV) polymerase that exhibits delayed chain termination of HBV DNA. A high barrier to entecavir-resistance (ETVr) is observed clinically, likely due to its potency and a requirement for multiple resistance changes to overcome suppression. Changes in the HBV polymerase reverse-transcriptase (RT) domain involve lamivudine-resistance (LVDr) substitutions in the conserved YMDD motif (M204V/I +/- L180M), plus an additional ETV-specific change at residues T184, S202 or M250. These substitutions surround the putative dNTP binding site or primer grip regions of the HBV RT. To determine the mechanistic basis for ETVr, wildtype, lamivudine-resistant (M204V, L180M) and ETVr HBVs were studied using in vitro RT enzyme and cell culture assays, as well as molecular modeling. Resistance substitutions significantly reduced ETV incorporation and chain termination in HBV DNA and increased the ETV-TP inhibition constant (K(i)) for HBV RT. Resistant HBVs exhibited impaired replication in culture and reduced enzyme activity (k(cat)) in vitro. Molecular modeling of the HBV RT suggested that ETVr residue T184 was adjacent to and stabilized S202 within the LVDr YMDD loop. ETVr arose through steric changes at T184 or S202 or by disruption of hydrogen-bonding between the two, both of which repositioned the loop and reduced the ETV-triphosphate (ETV-TP) binding pocket. In contrast to T184 and S202 changes, ETVr at primer grip residue M250 was observed during RNA-directed DNA synthesis only. Experimentally, M250 changes also impacted the dNTP-binding site. Modeling suggested a novel mechanism for M250 resistance, whereby repositioning of the primer-template component of the dNTP-binding site shifted the ETV-TP binding pocket. No structural data are available to confirm the HBV RT modeling, however, results were consistent with phenotypic analysis of comprehensive substitutions of each ETVr position. Altogether, ETVr occurred through exclusion of ETV-TP from the dNTP-binding site, through different, novel mechanisms that involved lamivudine-resistance, ETV-specific substitutions, and the primer-template.