Myristylation of the hepatitis B virus large surface protein is essential for viral infectivity

The hepatitis B virus (HBV) envelope contains equimolar amounts of three viral proteins: the major (S), middle, and large (L) polypeptides. Their roles in the adsorption and penetration of the virus have not yet been elucidated. We have used a highly efficient in vitro model that permits reproducible HBV infection to investigate whether N-myristylation, a posttranslational modification of the L protein, was essential for viral infectivity. A point mutation abolishing myristylation was introduced into the HBV genome. Mutant virions were produced by transfecting viral DNA into hepatoma cells and their infectivity was evaluated on primary human hepatocyte cultures. No difference between mutant and wild-type viral RNA production could be observed. Furthermore, intermediate DNA replicative forms were observed in transfected cells demonstrating replication competence of mutant viral genomes. In addition, complete virions were produced in the cell supernatant. However, we found that mutant viral particles contained viral DNA with a reduced mean size, probably corresponding to a larger single-stranded region in the relaxed circular DNA form. We have evidenced the presence of pre-S1, pre-S2, and S epitopes at the outer surface of these virions by using immunoprecipitation with specific monoclonal antibodies. This result confirmed that mutant viruses were normally assembled. By contrast, myristylation-defective mutants completely lost their infectivity for human hepatocytes in primary cultures as shown by the absence of HBs antigen production and viral intermediate replicative forms in hepatocytes. In conclusion, the myristylation of the L protein is not required for the production of Dane-like particles but it is absolutely necessary for HBV infectivity.