Homogeneous hepatitis A virus particles. Proteolytic release of the assembly signal 2A from procapsids by factor Xa.
Among the picornaviridae, hepatitis A virus (HAV) is unique in that its assembly is driven by domain 2A of P1-2A, the precursor of the structural proteins (Probst, C., Jecht, M., and Gauss-Müller, V. (1999) J. Biol. Chem. 274, 4527-4531). Whereas infected individuals excrete in stool mature HAV capsids with VP1 as the major structural protein, its C-terminal extended form VP1-2A is the main component of immature procapsids produced in HAV-infected cells in culture. Obviously, a postassembly proteolytic step is required to remove the primary assembly signal 2A from VP1-2A of procapsids. Mutants of VP1-2A were expressed in COS7 cells to determine the cleavage site in VP1-2A and to test for the cleavage potential of viral and host proteinases (factor Xa and thrombin). Site-specific in vitro cleavage by factor Xa and thrombin occurred in procapsids that contained VP1-2A with engineered cognate cleavage sites for these proteinases. Interestingly, factor Xa but not thrombin liberated mature VP1 also from native procapsids in an assembly-dependent manner. The data show that domain 2A, which is required for pentamerization of its precursor polypeptides and thus for the primary step of HAV assembly, is removed from the surface of immature procapsid by a host proteinase. Moreover, our data open a novel avenue to produce homogeneous HAV particles from recombinant intermediates by in vitro treatment with exogenously added proteases such as factor Xa or thrombin.
Rachow
Andrea
A
Institute of Medical Molecular Biology, University of Luebeck, 23538 Luebeck, Germany.
Gauss-Müller
Verena
V
Probst
Christian
C
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002213:Capsid / Q000737:chemistry; D036022:Capsid Proteins / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry; D015951:Factor Xa / Q000737:chemistry* / Q000378:metabolism; D030041:Hepatitis A virus / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013917:Thrombin / Q000737:chemistry; D015678:Viral Structural Proteins / Q000737:chemistry*
D036022:Capsid Proteins; D018076:DNA, Complementary; D011994:Recombinant Proteins; C096539:VP1 protein, hepatitis A virus; D015678:Viral Structural Proteins; D010450:Endopeptidases; D013917:Thrombin; D015951:Factor Xa