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

Characterization of cleavage sites and protease activity in the polyprotein precursor of Japanese... 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 ↓ Ser 509 and Ala 734 ↓ 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

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

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Publisher
Springer Journals
Copyright
Copyright © 2007 by Springer-Verlag
Subject
Biomedicine; Virology; Medical Microbiology; Infectious Diseases
ISSN
0304-8608
eISSN
1432-8798
D.O.I.
10.1007/s00705-007-0935-z
Publisher site
See Article on Publisher Site

Abstract

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 ↓ Ser 509 and Ala 734 ↓ 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.

Journal

Archives of VirologySpringer Journals

Published: Jun 1, 2007

References

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