In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus

The roles of RNA sequence/structure motifs, Packaging Signals (PSs), for regulating assembly of an HBV genome transcript have been investigated in an efficient in vitro assay containing only core protein (Cp) and RNA. Variants of three conserved PSs, within the genome of a strain not used previously, preventing correct presentation of a Cp-recognition loop motif are differentially deleterious for assembly of nucleocapsid-like particles (NCPs). Cryo-electron microscopy reconstruction of the T = 4 NCPs formed with the wild-type gRNA transcript, reveal that the interior of the Cp shell is in contact with lower resolution density, potentially encompassing the arginine-rich protein domains and gRNA. Symmetry relaxation followed by asymmetric reconstruction reveal that such contacts are made at every symmetry axis. We infer from their regulation of assembly that some of these contacts would involve gRNA PSs, and confirmed this by X-ray RNA footprinting. Mutation of the epsilon stem-loop in the gRNA, where polymerase binds in vivo, produces a poor RNA assembly substrate with Cp alone, largely due to alterations in its conformation. The results show that RNA PSs regulate assembly of HBV genomic transcripts in vitro, and therefore may play similar roles in vivo, in concert with other molecular factors. Patel et al. investigate the role of RNA sequence/structure motif 'Packaging Signals' (PSs) in regulating the assembly of an HBV genome transcript using an in vitro assay containing only core protein (Cp) and RNA. They show that RNA PSs regulate assembly of HBV genomic transcripts in vitro, and therefore may play similar roles in vivo, in concert with other molecular factors.

Automated summary

The study investigated the roles of RNA sequence/structure motifs and Packaging Signals (PSs) in regulating assembly of an HBV genome transcript, revealing their crucial importance for correct assembly of core protein and RNA, and suggesting potential similar roles in vivo in coordination with other molecular factors.