title: Exposure of RNA templates and encapsidation of spliced viral RNA are influenced by the arginine-rich domain of human hepatitis B virus core antigen (HBcAg 165-173).
abstract: Previously, human hepatitis B virus (HBV) mutant 164, which has a truncation at the C terminus of the HBV core antigen (HBcAg), was speculated to secrete immature genomes. For this study, we further characterized mutant 164 by different approaches. In addition to the 3.5-kb pregenomic RNA (pgRNA), the mutant preferentially encapsidated the 2.2-kb or shorter species of spliced RNA, which can be reverse transcribed into double-stranded DNA before virion secretion. We observed that mutant 164 produced less 2.2-kb spliced RNA than the wild type. Furthermore, it appeared to produce at least two different populations of capsids: one encapsidated a nuclease-sensitive 3.5-kb pgRNA while the other encapsidated a nuclease-resistant 2.2-kb spliced RNA. In contrast, the wild-type core-associated RNA appeared to be resistant to nuclease. When arginines and serines were systematically restored at the truncated C terminus, the core-associated DNA and nuclease-resistant RNA gradually increased in both size and signal intensity. Full protection of encapsidated pgRNA from nuclease was observed for HBcAg 1-171. A full-length positive-strand DNA phenotype requires positive charges at amino acids 172 and 173. Phosphorylation at serine 170 is required for optimal RNA encapsidation and a full-length positive-strand DNA phenotype. RNAs encapsidated in Escherichia coli by capsids of HBcAg 154, 164, and 167, but not HBcAg 183, exhibited nuclease sensitivity; however, capsid instability after nuclease treatment was observed only for HBcAg 164 and 167. A new hypothesis is proposed here to highlight the importance of a balanced charge density for capsid stability and intracapsid anchoring of RNA templates.
authors.affiliation: Department of Pathology, WHO Collaborating Center for Tropical Diseases, and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX 77555-0609, USA.
authors.forename: Sophie
authors.initials: S
authors.lastname: Le Pogam
authors.forename: Pong Kian
authors.initials: PK
authors.lastname: Chua
authors.forename: Margaret
authors.initials: M
authors.lastname: Newman
authors.forename: Chiaho
authors.initials: C
authors.lastname: Shih
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D002213:Capsid / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D006512:Hepatitis B Core Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008836:Micrococcal Nuclease / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D012326:RNA Splicing; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D013698:Templates, Genetic
chemical_list: D004279:DNA, Viral; D006512:Hepatitis B Core Antigens; D012367:RNA, Viral; D001120:Arginine; D008836:Micrococcal Nuclease