Cys residues of the hepatitis B virus capsid protein are not essential for the assembly of viral core particles but can influence their stability.
In the spherical capsid of hepatitis B virus (HBV), intermolecular disulfide bonds cross-link the approximately 180 p21.5 capsid protein subunits into a stable lattice. In this study, we used mutant capsid proteins to investigate the role that disulfide bonds and the four p21.5 Cys residues (positions 48, 61, 107, and 185) play in capsid assembly and/or stabilization. p21.5 Cys residues were either replaced by Ala or removed (Cys-185) by carboxyl-terminal truncation, creating Cys-minus mutants which were expressed in Xenopus oocytes via microinjected synthetic mRNAs. Fractionation of radiolabeled oocyte extracts on 10 to 60% sucrose gradients revealed that Cys-minus core proteins resolved into the nonparticulate and capsid forms seen for wild-type p21.5. On 5 to 30% sucrose gradients, nonparticulate Cys-minus core proteins sedimented as dimers of approximately 40 kDa. We conclude that Cys residues and disulfides are not required for the assembly of either HBV capsids or the dimers that provide the precursors for capsid assembly. Since assembly presumably demands an appropriate p21.5 tertiary structure, it is unlikely that Cys residues are required for proper p21.5 folding. However, Cys residues stabilize isolated p21.5 structures, as evidenced by the marked reduction in stability of Cys-minus dimers and capsids (i) in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and (ii) upon protease digestion. We discuss these results in the context of the HBV life cycle and the role of Cys residues in other proteins.
Zhou
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Hormone Research Institute, University of California, San Francisco 94143-0534.
Standring
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D000409:Alanine / Q000378:metabolism; D001483:Base Sequence; D002213:Capsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D006515:Hepatitis B virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014779:Virus Replication
D046911:Macromolecular Substances; D010450:Endopeptidases; D003545:Cysteine; D000409:Alanine