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 surface antigens to form enveloped virions remains unknown. In this study, core 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 virion-like particles. One plasmid encoding an extra stretch of 23 aa, including six histidine residues, fused to the N terminus of the core protein (designated HisC183) was expressed in Escherichia coli and detected by Western blot. CsCl gradient and electron microscopy analyses indicated that HisC183 could self-assemble into nucleocapsids. When HisC183 or another similar N-terminal fusion core protein (designated FlagC183) was co-expressed with a core-negative plasmid in human hepatoma cells, both mutant core proteins self-assembled into nucleocapsids. These particles also retained kinase activity. Using an endogenous polymerase assay, a fill-in HBV DNA labelled with isotope was obtained from intracellular nucleocapsids formed by mutant cores. In contrast, no such signal was detected from the transfection medium, which was consistent with PCR and Southern blot analyses. Results indicate that core mutants with N-terminal extensions can form nucleocapsids, but are blocked during the envelopment process and cannot form secreted virions. The mutant nucleocapsids generated from this work should facilitate further study on how nucleocapsids interact with surface antigens.
Hui
Eric Ka-Wai
EK
Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei, Taiwan 112, Republic of China 1.
Yi
Yong Shyang
YS
Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei, Taiwan 112, Republic of China 1.
Lo
Szecheng J
SJ
Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei, Taiwan 112, Republic of China 1.
D000595:Amino Acid Sequence; D006528:Carcinoma, Hepatocellular; D004260:DNA Repair; D019281:Dimerization; D004926:Escherichia coli; D015870:Gene Expression; D006512:Hepatitis B Core Antigens / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006515:Hepatitis B virus / Q000235:genetics / Q000502:physiology*; D006639:Histidine; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019251:Nucleocapsid / Q000378:metabolism; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014771:Virion; D019065:Virus Assembly
D006512:Hepatitis B Core Antigens; D011993:Recombinant Fusion Proteins; D006639:Histidine