The replication and assembly of the Reovirus occurs in distinctive cytoplasmic inclusion bodies in infected cells which are called viral factories. These structures are attribute to viral infection, and are believed to be necessary in virion assembly. While the formation mechanism of this structure is unclear, studies have shown that the reovirus nonstructural protein μNS plays a major role throughout viral replication cycles (2, 24).
μNS, which is present in most species of Reovirus, has a relatively conserved coding sequence. The molecular weight of the protein among different reovirus isolates is around 80kDa. Studies of mammalian orthoreoviruses (MRV) indicate that a single μNS, or even a truncated carboxyl-proximal region (4), is sufficient for forming phase-dense inclusions in transfected cells (3, 7). Interestingly, the two forms of μNS, μNS and μNSC, and a proportion of expression between 1: 1 to 4: 1 can be detected in infected cells (25), indicating that μNSC may be important in for-mation of inclusions and the viron assembly process. In addition, the other viral proteins, λ1, λ2, λ3, μ2, σ2, and the nonstructural protein σNS and viral RNA, are all involved in interacting with μNS during virus assembly (5, 6, 20).
Grass carp reovirus(GCRV), the main pathogen that can cause outbreaks of hemorrhagic disease in aquatic animals, belongs to the genus Aquareovirus in the family Reoviridea (1, 19, 22). It has been recognized that GCRV is the most pathogenic agent among all the isolates of aquareoviruses reported to date (21). Similar to other members of Aquareovirus, GCRV, a multilayered spherically structured particle, contains a genome of 11 segmented dsRNA, which encodes 7 structure proteins (VP1-VP7) and 5 nonstructural proteins (9, 27).
While considerable fundamental and applied research has been carried out on mammalian or human isolates from the genus Orthoreovirus and Rotavirus (8, 10, 15, 17, 23, 26), relatively little progress has been made with members of the genus Aquareovirus, and is mainly focused on the molecular and structural biology of their structural proteins. To understand the assembly of GCRV infection in its host cells, it is necessary to investigate the biological function of non-structural proteins involved in viral replication.
According to the complete genome sequence of GCRV and the analyses of the homology alignment, NS80, the nonstructural protein encoded by GCRV s4, has the highest level sequence similarity with the μNS protein in MRV, which has a function in the formation of the viral factory. As a fusogenic reovirus, GCRV has unique replication characteristics and produces large inclusion matrix like CPE during its infection. To understand the role of the μNS80 protein in virus replication, the expression and immunogenicity identification of the GCRV NS80 protein inclusion forming-related region was investigated in this study.
Expression and Identification of Inclusion Forming-related Domain of NS80 Nonstructural Protein of Grass Carp Reovirus*
- Received Date: 13 January 2009
- Accepted Date: 17 March 2009
Abstract: Grass carp reovirus (GCRV), a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstructural protein NS80, encoded by GCRV segment 4, has a high similarity with μNS in MRV(Mammalian orthoreoviruses), which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region (335-742) was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28oC. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80(335-742) protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody (mouse) and NS80(335-742) specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.