Characterization of cleavage sites and protease activity in the polyprotein precursor of Japanese marine aquabirnavirus and expression analysis of generated proteins by a VP4 protease activity in four distinct cell lines.
A polyprotein precursor NH(2)-pVP2-VP4-VP3-COOH is encoded in genomic segment A of members of the family Birnaviridae. By N-terminal sequencing analysis, primary cleavage sites of a marine birnavirus (MABV) polyprotein were identified as Ala(508) downward arrow Ser(509) and Ala(734) downward arrow Ser(735), where the cleavage motif was the same as that of infectious pancreatic necrosis virus (IPNV). However, further VP4 and VP3 cleavages occurred at novel sites. Ser(633) and Lys(674) mutations affected the cleavage activity by site-directed mutagenesis. Additional catalytic residues including Ile(543) and Val(686) were MABV-specific. As shown by electron microscopy, pVP2 and further cleaved VP3s (fcVP3s) could not form virus-like particles (VLPs). This suggests that VP3 is necessary for VLP formation. By Western blot analysis of the VP3 expression, fcVP3s were found in RSBK-2 cells and FHM cells, while VP3 was cleaved less in EPC cells, suggesting that fcVP3s might merely be a degraded form. Alternatively, if fcVP3s play functional roles other than in viral assembly, the further VP3 cleavage is, at least, not restricted in FHM cells. Strangely, VP3 was not completely further cleaved in CHSE-214 cells despite the fact that this cell line has a potential proteolytic factor, implying that complicated factors are associated with the further VP3 cleavage.
Imajoh
M
M
Laboratory of Cell Structure and Function, Division of Marine Bioresource Science, Graduate School of Kuroshio Science, Kochi University, Kochi, Japan.
Goto
T
T
Oshima
S
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019201:Aquabirnavirus / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D005399:Fishes / Q000821:virology; D018061:Infectious pancreatic necrosis virus / Q000235:genetics / Q000378:metabolism; D007564:Japan; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism
D004279:DNA, Viral; D011498:Protein Precursors; D015678:Viral Structural Proteins; D012697:Serine Endopeptidases; C414992:VP4 protease, birnavirus