Hepatitis B virus (HBV) replicates its 3 kb DNA genome through capsid-internal reverse transcription, initiated by assembly of 120 core protein (HBc) dimers around a complex of viral pregenomic (pg) RNA and polymerase. Following synthesis of relaxed circular (RC) DNA capsids can be enveloped and secreted as stable virions. Upon infection of a new cell, however, the capsid disintegrates to release the RC-DNA into the nucleus for conversion into covalently closed circular (ccc) DNA. HBc´s interactions with nucleic acids are mediated by an arginine-rich C terminal domain (CTD) with intrinsically strong non-specific RNA binding activity. Adaptation to the changing demands for nucleic acid binding during the viral life cycle is thought to involve dynamic phosphorylation / dephosphorylation events. However, neither the relevant enzymes nor their target sites in HBc are firmly established. Here we developed a bacterial coexpression system enabling access to definably phosphorylated HBc. Combining Phos-tag gel electrophoresis, mass spectrometry and mutagenesis we identified seven of the eight hydroxy amino acids in the CTD as target sites for serine-arginine rich protein kinase 1 (SRPK1); fewer sites were phosphorylated by PKA and PKC. Phosphorylation of all seven sites reduced nonspecific RNA encapsidation as drastically as deletion of the entire CTD and altered CTD surface accessibility, without major structure changes in the capsid shell. The bulk of capsids from human hepatoma cells was similarly highly, yet non-identically, phosphorylated as by SRPK1. While not proving SRPK1 as the infection-relevant HBc kinase the data suggest a mechanism whereby high-level HBc phosphorylation principally suppresses RNA binding whereas one or few strategic dephosphorylation events enable selective packaging of the pgRNA/polymerase complex. The tools developed in this study should greatly facilitate the further deciphering of the role of HBc phosphorylation in HBV infection and its evaluation as a potential new therapeutic target.

The highly immunogenic icosahedral capsid of hepatitis B virus (HBV) can be exploited as a nanoparticulate display platform for heterologous molecules. Its constituent core protein (HBc) of only ~180 amino acids spontaneously forms capsid-like particles (CLPs) even in E. coli. The immunodominant c/e1 epitope in the center of the HBc primary sequence comprises a solvent-exposed loop that tolerates insertions of flexible peptide sequences yet also of selected whole proteins as long as their 3D structures fit into the two acceptor sites. This constraint is largely overcome in the SplitCore system, where the sequences flanking the loop are expressed as two separate but self-complementing entities, with the foreign sequence fixed to the carrier at one end only. Both the contiguous and the split type of CLP strongly enhance immunogenicity of the displayed sequence but also nonvaccine applications can easily be envisaged. After a brief survey of the basic features of the two HBc carrier forms, we provide conceptual guidelines concerning which foreign proteins are likely to be presentable, or not, on either carrier type. We describe generally applicable protocols for CLP expression in E. coli, cell lysis and CLP enrichment by sucrose gradient velocity sedimentation, plus a simple but meaningful gel electrophoretic assay to assess proper particle formation.