Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure

Abstract

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.

Author summary

The liver-pathogenic hepatitis B virus (HBV) is a small enveloped DNA virus that replicates through reverse transcription of a pregenomic (pg)RNA. This requires specific encapsidation of pgRNA and viral polymerase into a shell of 240 core protein (HBc) subunits. Capsid-internal formation of relaxed circular (RC) DNA enables the particle to leave the cell as stable virion; yet, when infecting a new cell it must release the RC-DNA for conversion into another, plasmid-like DNA that templates new viral RNAs. This up and down in nucleic acid interactions is presumably regulated by transient phosphorylation of HBc, mainly in its arginine-rich C terminal domain (CTD) which displays strong non-sequence-specific RNA binding. However, neither the phosphorylation sites nor the relevant enzymes are well defined. We developed a recombinant system to produce kinase-specific phospho-HBc species, and adapted a feasible gel assay for their separation. By mutagenesis and mass spectrometry we identified seven target sites for a major candidate kinase, SRPK1, in the CTD. As full SRPK1 phosphorylation thwarted non-specific RNA binding the comparably high phosphorylation of HBc in human cells suggests how specific pgRNA encapsidation might be achieved. Our new tool set will facilitate disentangling the role of HBc phosphorylation in HBV infection and exploiting it as potential therapeutic target.