Abstract
BackgroundGrass Carp Reovirus (GCRV), a tentative member in the genus Aquareovirus of family Reoviridae, contains eleven segmented (double-stranded RNA) dsRNA genome which encodes 12 proteins. A low-copy core component protein VP4, encoded by the viral genome segment 5(S5), has been suggested to play a key role in viral genome transcription and replication.ResultsTo understand the role of minor core protein VP4 played in molecular pathogenesis during GCRV infection, the recombinant GCRV VP4 gene was constructed and expressed in both prokaryotic and mammalian cells in this investigation. The recombinant His-tag fusion VP4 products expressed in E.coli were identified by Western blotting utilizing His-tag specific monoclonal and GCRV polyclonal antibodies. In addition, the expression of VP4 in GCRV infected cells, appeared in granules structure concentrated mainly in the cytoplasm, can be detected by Immunofluorescence (IF) using prepared anti-VP4 polyclonal antibody. Meanwhile, VP4 protein in GCRV core and infected cell lysate was identified by Immunoblotting (IB) assay. Of particular note, the VP4 protein was exhibited a diffuse distribution in the cytoplasm and nucleus in transfected cells, suggesting that VP4 protein may play a partial role in the nucleus by regulating cell cycle besides its predicted cytoplasmic function in GCRV infection.ConclusionsOur results indicate the VP4 is a core component in GCRV. The cellular localization of VP4 is correlated with its predicted function. The data provide a foundation for further studies aimed at understanding the role of VP4 in viroplasmic inclusion bodies (VIB) formation during GCRV replication and assembly.
Highlights
Grass Carp Reovirus (GCRV), a tentative member in the genus Aquareovirus of family Reoviridae, contains eleven segmented dsRNA genome which encodes 12 proteins
The product of the VP4 fusion protein was concentrated in the pellet rather than supernatant of the cell lysate, indicating that the induced VP4 fusion protein expression was presented in the form of inclusion body
The purified protein and cell lysate could cross immunologically with GCRV polyclonal antibody (Figure 1B and 1B’), demonstrating that the recombinant fusion VP4 protein is GCRV related antigen that belonged to viral structural protein, which could be further used as an immunogen for VP4 antibody production in animal
Summary
Grass Carp Reovirus (GCRV), a tentative member in the genus Aquareovirus of family Reoviridae, contains eleven segmented (double-stranded RNA) dsRNA genome which encodes 12 proteins. The virus particles in the family of Reoviridae appear to be icosahedral in symmetry with an overall diameter of approximately 60–85 nm comprising 9–12 segmented dsRNA genome enclosed within multiple concentric protein capsids. Based on their structure organization, it is possible to divide the members of the family Reoviridae into two subfamilies, Spinareovirinae. Similar to other members of Reoviridae, GCRV is a multilayer spherical particle enclosing a dsRNA genome of 11 segments, which encode 7 structural proteins (VP1-VP7) and 5 nonstructural proteins.
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