The Hepatitis B Virus (HBV) double-stranded DNA genome is reverse transcribed from its RNA pregenome (pgRNA) within the virus core (or capsid). Phosphorylation of the arginine-rich carboxy-terminal domain (CTD) of the HBV capsid protein (Cp183) is essential for pgRNA encapsidation and reverse transcription. However, the structure of the CTD remains poorly defined. Here we report sub-nanometer resolution cryo-EM structures of in vitro assembled empty and pgRNA-filled Cp183 capsids in unphosphorylated and phosphorylation-mimic states. In empty capsids, we found unexpected evidence of surface accessible CTD density partially occluding pores in the capsid surface. We also observed that CTD organization changed substantively as a function of phosphorylation. In RNA-filled capsids, unphosphorylated CTDs favored thick ropes of RNA, while the phosphorylation-mimic favored a mesh of thin, high-density strands suggestive of single stranded RNA. These results demonstrate that the CTD can regulate nucleic acid structure, supporting the hypothesis that the HBV capsid has a functional role as a nucleic acid chaperone.
Many single stranded RNA virus encapsidate their genome through positively-charged domains of their capsid proteins. Hepatitis B virus (HBV) is a double stranded DNA virus which packages a single-stranded RNA pregenome (pgRNA) that is reverse transcribed within the capsid. RNA packaging requires a phosphorylated form of the HBV capsid protein's RNA-binding carboxy-terminal domain (CTD). Although the capsid has been well studied, the internal structures, the CTDs and the packaged RNA, are poorly characterized. By using in vitro reassembly, we have generated empty and pgRNA-filled capsids using phosphorylation-mimic and unphosphorylated forms of the capsid protein. Using cryo-EM image reconstruction, we have been able to show the structure of encapsidated pgRNA and, independently, the CTD in the absence of RNA to visualize early stages of the HBV assembly. We showed that the structural organization of the CTD changes as a function of the phosphorylation. Changes in CTD structure affect the structure of the encapsidated pgRNA, changing it from thin segments of density in the phosphorylated state, suggestive of single-stranded RNA, to thick rope-like structures consistent with duplex nucleic acid in the unphosphorylated state.