IDENTIFICATION AND CHARACTERIZATION OF MUTATIONS IN HEPATITIS-B VIRUSRESISTANT TO LAMIVUDINE

Cirrhosis and hepatocellular carcinoma occur as longterm complications of chronic hepatitis B virus (HBV) infection. Antiviral therapy is po tentially a successful approach for the treatment of patients with HBV infection, which includes the nucleoside analog, lamivudine [(-)2'-de oxy-3'-thiacytidine, 3TC], Although resistance to lamivudine therapy h as been reported in several HBV-infected patients, the pattern of resi stance-associated mutations in HBV has not been fully characterized. W e report a DNA sequence database that includes a 500-base pair region of the HBV polymerase gene from 20 patients with clinical manifestatio ns of lamivudine resistance. Analysis of the database reveals two patt erns of amino acid substitutions in the tyrosine, methionine, aspartat e, aspartate (YMDD) nucleotide-binding locus of the HBV polymerase. HB V DN4 from the sera of patients in Group I exhibits a substitution of valine for methionine at residue 552, accompanied by a substitution of methionine for leucine at residue 528. Patients in Group II had only an isoleucine-for-methionine substitution at position 552, Reconstruct ion of these mutations in an HBV replication-competent plasmid was per formed in a transient transfection cell assay to determine the functio n/relevance of these mutations to lamivudine resistance. Both Group I and Group II mutations resulted in a substantial decrease in sensitivi ty to lamivudine treatment (>10,000-fold shift in IC50 over wild-type [wt] IC50), strongly indicating that these mutations were involved in resistance to lamivudine. A hypothetical model of the HBV reverse tran scriptase has been generated for further study of the role of these mu tations in lamivudine resistance.