Characterization of the nonstructural protein NS80 of grass carp reovirus

Abstract

Nonstructural proteins of members of the family Reoviridae are believed to play significant roles in the virus replication cycle. Phylogenetic analyses indicate that the nonstructural protein NS80 of grass carp reovirus, encoded by a gene of Segment 4 (S4), is a primary determinant that is related to the formation of viroplasmic inclusion bodies (VIB), where viral replication and assembly are thought to occur. To understand the role of the NS80 protein in viral replication, an initial investigation of NS80 gene expression in both infected and transfected cells was conducted. Transmission electron microscopy results indicate that replication and assembly of GCRV occur within VIB-like structures in the perinuclear region of the cell cytoplasm. Furthermore, expression of the S4 gene in infected cells was detected with an NS80-specific antibody by western blot and immunofluorescence. Moreover, globular VIB-like structures were observed when expressing GFP-derived full-length NS80 (pEGFP-C1/NS80) and recombinants containing the C-terminal conserved region (pEGFP-C1/NS80₃₃₅₋₇₂₄) in transfected Vero. No such structures were detected in cells transfected with an N-terminal recombinant (pEGFP-C1/NS80₁₋₃₃₄), suggesting that the NS80 C-terminal conserved region may be involved in the formation of inclusion structures. These data provide a foundation for further functional studies of NS80 related to viral inclusion formation in viral replication.

Identification and characterization of two cleavage fragments from the Aquareovirus nonstructural protein NS80

“…Similarly, a shorter fragment with NS80 expression has also been found in GCRV-infected cells (Fan et al, 2009b;Fan et al, 2010;Shao et al, 2013;Yan et al, 2015). These results suggest that NS80 presents different forms or structural conformations in infected cells to regulate viral replication and assembly at different life cycle stages.…”

“…VP5 and VP7 make up the outer capsid required for viral entry into host cells during infection . In addition, the nonstructural proteins are involved in the viral replication cycle (Fan et al, 2009a;Fan et al, 2010;Cai et al, 2011;Shao et al, 2013;Yan et al, 2015). Phylogenetic and three-dimensional structural analyses have revealed that there is a close evolutionary relationship between aquareoviruses and orthoreoviruses, including mammalian orthoreoviruses (MRV) and avian reoviruses (ARV) (Shaw et al, 1996;Attoui et al, 2002;Fang et al, 2005;Cheng et al, 2008;Cheng et al, 2010).…”

“…A single μNS of MRV can form VIBs in transfected cells and can recruit five core structural proteins (λ1, λ2, λ3, μ2, and σ2), the nonstructural protein σNS, and cellular clathrin (Sharpe et al, 1982;Parker et al, 2002;Becker et al, 2003;Miller et al, 2003;Broering et al, 2004;Touris-Otero et al, 2004;Miller et al, 2010;Ivanovic et al, 2011). The nonstructural protein NS80 of GCRV, the analogue of the μNS protein of MRV and ARV, consists of 742 amino acids with a molecular weight of approximately 80 kDa (Fan et al, 2009b;Fan et al, 2010). Previous studies in our lab have demonstrated that NS80 can form VIBs in singly expressed or infected cells.…”

Proteomic analysis of cellular protein expression profiles in response to grass carp reovirus infection

“…The grass carp is the most economically important fish species cultivated in China, with an annual production of more than 3.6 million metric tons. Frequent outbreaks of hemorrhagic disease resulting from GCRV infection have resulted in significant economic losses in the grass carp cultivation industry [2,3]. GCRV is a dsRNA virus with a double-layered protein capsid, while the GCRV genome comprises 11 dsRNA genomic fragments encoding 12 viral proteins [4].…”

Identification of a functional motif in the AqRV NS26 protein required for enhancing the fusogenic activity of FAST protein NS16

“…Based on analyses of the AqRV-C genome and of 3D structure, the properties of the viral structural proteins have been well characterized (Cheng et al, 2008(Cheng et al, , 2010Zhang et al, 2010). The major nonstructural protein NS80, the analogue of the mNS protein in mammalian orthoreoviruses (MRVs) and avian reoviruses (ARVs), which is encoded by the S4 genome segment, has been demonstrated to be involved in viral factory or viral inclusion formation during infection (Cai et al, 2011;Fan et al, 2010). Recently, we demonstrated that NS80 can recruit the minor core protein VP4 and nonstructural protein NS38 to its inclusions, and the C-terminal domain of NS80 is the primary driving force for inclusion formation .…”