Clearance of the original hepatitis B virus YMDD-motif mutants with emergence of distinct lamivudine-resistant mutants during prolonged lamivudine therapy

Tyrosine-methionine-aspartate-aspartate (YMDD)-motif mutants may emerge and elicit immune clearance during prolonged lamivudine treatment. The aim of this study was to investigate the virological events following development of the original mutants. Twenty-three patients who developed YMDD-motif mut ants during the Asian lamivudine trial were included. Serial serum samples from these patients were subjected to sequence analysis to identify new mut ants. Site-directed mutagenesis experiments were performed to investigate w hether the new mutations were responsible for lamivudine resistance. Of the 23 patients included, 13 harbored either one or a mixture of the two commo n YMDD-motif mutants (methionine 552-to-isoleucine [M552I] and leucine 528- to-methionine/methionine 552-to-valine [L528M/M552V]) throughout the course , whereas in the remaining 10 patients, distinct mutants became dominant ov er the original mutants to cause continuing chronic hepatitis. Of them, 3 d eveloped an alanine 529-to-threonine (A529T) mutant, 6 developed a leucine 528-to-methionine/methionine 552-to-isoleucine (L528M/ M552I) mutant, and 1 developed these two mutants sequentially. Site-directed mutagenesis experi ments confirmed that the aforementioned mutations were responsible for the resistance to lamivudine in vitro. The nucleotide substitution in the A529T mutant concomitantly generated a stop codon at the surface gene, leading t o impaired secretion of HBsAg. Strikingly, the replication of this mutant w as lamivudine dependent. These results suggested that distinct lamivudine-r esistant mutants could emerge and replace the original YMDD-motif mutants a s the cause of continuing chronic hepatitis during prolonged lamivudine the rapy.