HEPATITIS-B VIRUS MUTANTS ASSOCIATED WITH 3TC AND FAMCICLOVIR ADMINISTRATION ARE REPLICATION-DEFECTIVE

Hepatitis B virus (HBV) variant strains may develop during therapy for chronic infection with the nucleoside analog 2',3'-dideoxy-3'-thiacyt idine (3TC). HBV mutants result from isoleucine (I) or valine (V) subs titutions in the methionine (M) of the YMDD motif in the viral reverse -transcriptase catalytic domain, In addition, other mutations in the r everse-transcriptase ''B domain'' involving either a phenylalanine (F) -to-leucine (L) at amino acid 501 (F501L) or an L-to-M substitution at amino acid 515 (L515M) have been observed during 3TC and Famciclovir therapy as well, To determine the biologic consequences of these mutat ions on viral replication, variant viral genomes were constructed and transiently transfected into hepatocellular carcinoma (HCC) and HEK 29 3 human embryo kidney-derived cell lines. In transiently transfected H CC cells, the viruses bearing the YI/VDD or F501L mutations had greatl y impaired replication as compared to wild-type virus, whereas the vir us carrying the L515M substitution showed the least defect. Double mut ants with the L515M substitution showed intermediate defect between th e YI/VDD or F501L and the L515M single-mutant strains. In contrast, wh en transfected into HEK 293 cells, the viruses bearing the YI/VDD or L 515M mutation replicated as wild-type. However, under conditions of de oxynucleotide depletion produced by hydroxyurea treatment of HEK 293 c ells, all mutants but not the wild-type virus exhibited a reduced repl ication phenotype similar to that observed in HCC cells. In both HCC a nd HEK 293 cells, the mutant viruses carrying the F501L substitution s howed a decreased pregenomic RNA encapsidation level, suggesting that the defect in HBV DNA synthesis occurs at the RNA packaging level, The se findings show that 3TC and Famciclovir selected mutations alter the properties of the HBV reverse transcriptase, resulting in impaired vi ral replication within the cell.