Hepatitis B viral core proteins with an N-terminal extension can assemble into core-like particles but cannot be enveloped

The structure of hepatitis B virus (HBV) nucleocapsids has been revealed in great detail by cryoelectron microscopy. How nucleocapsids interact with s urface antigens to form enveloped virions remains unknown. In this study, c ore mutants with N-terminal additions were created to address two questions : (1) can these mutant core proteins still form nucleocapsids and (2) if so , can the mutant nucleocapsids interact with surface antigens to form virio n-like particles. One plasmid encoding an extra stretch of 23 aa, including six histidine residues, fused to the N terminus of the core protein (desig nated HisC 183) was expressed in Escherichia coli and detected by Western b lot. CsCl gradient and electron microscopy analyses indicated that HisC183 could self-assemble into nucleocapsids. When HisC183 or another similar N-t erminal fusion core protein (designated FlagC183) was co-expressed with a c ore-negative plasmid in human hepatoma cells, both mutant core proteins sel f-assembled into nucleocapsids, These particles also retained kinase activi ty. Using an endogenous polymerase assay, a fill-in HBV DNA labelled with i sotope was obtained from intracellular nucleocapsids formed by mutant cores . In contrast, no such signal was detected from the transfection medium, wh ich was consistent with PCR and Southern blot analyses. Results indicate th at core mutants with N-terminal extensions can form nucleocapsids, but are blocked during the envelopment process and cannot form secreted virions. Th e mutant nucleocapsids generated from this work should facilitate further s tudy on how nucleocapsids interact with surface antigens.