Hepatitis B virus DNA replication is coordinated by core protein serine phosphorylation and HBx expression

The hepatitis B virus (HBV) core protein forms the capsid of viral particles and is essential for viral genome DNA replication and maturation. The C terminus of core protein contains three serines at positions 155, 162, and 170, phosphorylation of which is important for viral DNA replication. We demonstrate that the phosphorylation of these serines is stimulated by the viral HBx protein, a regulatory protein that activates signal transduction pathways and viral replication. HBx is therefore shown to stimulate HBV replication by increasing core serine phosphorylation. Mutational, biochemical, and mixing studies of C-terminal core serine mutants demonstrate that multiple serine phosphorylations occur on the same core protein. Mutation of individual core protein serines is shown to inhibit HBV replication at distinct stages corresponding to encapsidation of viral pregenomic RNA, reverse transcription, and restriction to synthesis of specific DNA replicative intermediates. We therefore demonstrate that a primary target of HBV replication that is regulated by HBx protein corresponds to increased phosphorylation of the viral core protein. We also demonstrate that core phosphorylation mediated by HBx promotes sequential progression of viral replication through the assembly of capsids primed for different stages of DNA synthesis.