Abstract
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 ε 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.
Highlights
The roles of RNA sequence/structure motifs, Packaging Signals (PSs), for regulating assembly of an Hepatitis B Virus (HBV) genome transcript have been investigated in an efficient in vitro assay containing only core protein (Cp) and RNA
Mfold[33] suggests that each of the matched sites is potentially able to fold into a stem-loop with an over-represented sequence, 5′RGAG-3′, in the loop[12]
The assembly of infectious HBV is a complex process that many people have studied in vivo in suitable cell culture systems
Summary
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. We show that homologous PSs occur in similar genome locations in a commercially-available strain variant (JQ707375.1), which was not included in the previous analysis These sites regulate in vitro assembly of T = 3 and T = 4 nucleocapsid-like particles (NCPs) in the context of a long genomic gRNA fragment lacking a 5′ cap and a poly-A tail. Mutation to prevent ε interacting with the distal φ site creates a genomic fragment that is considerably larger than both wild-type and PS mutant RNAs, as well as being a poor in vitro assembly substrate These data highlight that PS-mediated assembly via the formation of multiple PS-Cp contacts promotes NCP-like formation, in the absence of other mechanisms regulating assembly
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