The HBV RNA pre-genome encodes specific motifs that mediate interactions with the viral core protein that promote nucleocapsid assembly

Formation of the Hepatitis B (HBV) nucleocapsid (NC) is an essential step in the viral lifecycle but its assembly is not fully understood. We report the discovery of sequence-specific interactions between the viral pre-genome and HBV core protein (Cp) that play roles in defining the NC assembly pathway. Using RNA SELEX and bioinformatics we identified multiple regions in the pre-genomic RNA with high-affinity for Cp dimers. These RNAs form stem-loops with a conserved loop motif that trigger sequence-specific assembly of virus-like particles (VLPs) at much higher fidelity and yield than in the absence of RNA. The RNA oligos do not interact with preformed RNA-free VLPs, so their effects must occur during particle assembly. Asymmetric cryo-EM reconstruction of the T=4 VLPs assembled in the presence of one of the RNAs reveals a unique internal feature connected to the main Cp shell via lobes of density. Biophysical assays suggest that this is a complex involving several RNA oligos interacting with the C-terminal arginine-rich domains of Cp. These Cp-RNA contacts may play a role(s) in regulating the organization of the pre-genome during nucleocapsid assembly, facilitating subsequent reverse transcription and acting as a nucleation complex for NC assembly.