A nuanced role of the small loop of hepatitis B virus small envelope protein in virion morphogenesis and secretion

Background The virion secretion mechanism of human hepatitis B virus (HBV) remains to be investigated. In our current study, we characterized a reverse transcriptase mutant, which changed from the YMDD motif to YMHA. We noted that this mutant YMHA secreted no virions in the medium. Because of the overlapping open reading frame (ORF) between the polymerase and the envelope genes, the lack of virion secretion is likely due to corresponding concurrent mutations in a small loop of the envelope protein (HBsAg, HBV surface antigen). In literature, small loop mutations are thought to affect virion secretion of hepatitis delta virus (HDV), but not HBV. Methods Here, we revisited the relationship between the small loop and virion secretion by site-directed mutagenesis and native agarose gel electrophoresis. Results A proline substitution at residue 196 or 198 in the small loop blocked both HBV genome-containing and genome-free virion secretion, but not the secretion of 22-nm HBsAg subviral particles. Surprisingly, a leucine substitution at residue 196 enhanced genome-containing virion secretion. It is also intriguing that a proline-197, sandwiched by residue 196 and 198, exhibited no apparent defect in secreted virions, with or without containing an HBV genome. By complementation assay, we demonstrated that the wild type small envelope protein alone is sufficient to rescue the virion secretion defect of a small loop mutant M198P. Conclusions The effect of the small loop mutation of HBV small envelope protein on virion secretion is position-dependent. It warrants further investigation how the small loop of HBsAg plays a subtle role in HBV morphogenesis and secretion of virions with or without containing an HBV genome.

Inflammation Pharmacological Reaction and YMDD Mutational Patterns in Lamivudine Therapeutics Hepatitis B Virus

Background/Aims: Emergence of tyrosine-methionine-aspartate-aspartate (YMDD) motif in reverse transcriptase is a serious problem in chronic hepatitis B(CHB) patients after Lamivudine (LAM) therapy. However, the relationship between inflammation pharmacological reaction and YMDD mutational patterns of CHB has not been well-characterized. The aim of this study was to investigate the inflammation pharmacological reaction and different YMDD mutants patterns of CHB patients.Methods: We investigated the inflammation pharmacological reaction and YMDD mutational patterns through biochemical, serological and virological detection among 83 CHB patients, including 25 YMDD mutants, 25 under detection, and 33 control patients without YMDD mutants.Results: Prevalence of YMDD mutation patterns is different. Among 25 YMDD mutants patients, YIDD was the dominant mutation (72%), followed YVDD (16%) and the hybrid YIDD + YVDD (12%). The time course during the YMDD mutations was also different. 52.4% patients developed the mutation less than 12 months after the LAM therapy. Serum hepatitis B virus (HBV) DNA level in patients with YMDD mutants were significantly higher than that in control and negative groups. Serum HbsAg and HbeAg in patients with YMDD mutants were also higher than those in control and negative groups, despite no significant difference was found forserum HbeAb. ALT and AST levels were also significantly higher in mutants group.Conclusions: Illuminating inflammation pharmacological reaction and YMDD mutational patterns of CHB during pathological process may have implications for future therapy in YMDD mutation patients. This may have impact on the choice of treatment strategies for lamivudine-resistant HBV.

THE EXISTENCE OF GENE DNA POLYMERASE MUTATION FROM POSITIVE HEPATITIS B SAMPLES IN BANDUNG, INDONESIA

Objective: To complete mutation existence in reverse transcriptase domain of the viral Polymerase.Methods: The study was done by amplification step of viral polymerase gene fragment, agarose gel electrophoresis, PCR product purification with GFX column kit, sequencing and sequencing result analysis. The samples were derived from Clinical Laboratory in Bandung Indonesia.Results: The result showed mutations in DNA fragment encoding for RT domain viral polymerase in sample 6, 7 and 8. There were mutations leading to amino acid substitution L526S in sample 6, D551E in sample 7 and D552E in sample 8.Conclusion: D551E and D552E substitution occurred in YMDD motif RT DNA polymerase that produced YMDE mutant. L526S, D551E and D552E were estimated as antivirus-resistance mutants that have never been reported before.