Proteomic analysis of Singapore grouper iridovirus envelope proteins and characterization of a novel envelope protein VP088

Abstract

Singapore grouper iridovirus (SGIV) is an enveloped virus causing heavy economic losses to marine fish culture. The envelope fractions of SGIV were separated from the purified virions by Triton X-100 treatment, and subjected to 1-DE-MALDI-TOF/TOF-MS/MS and LC-MALDI-TOF/TOF-MS/MS analysis. A total of 19 virus-encoded envelope proteins were identified in this study and 73.7% (13/17) of them were predicted to be membrane proteins. Three viral envelope proteins were uniquely identified by 1-DE-MALDI, whereas another ten proteins were identified only by LC-MALDI, with six proteins identified by both workflows. VP088 was chosen as a representative of proteomic identification and characterized further. VP088 was predicted to be a viral transmembrane envelope protein which contains two RGD (Arg-Gly-Asp) motifs, three transmembrane domains, and five N-glycosylation sites. VP088 gene transcript was first detected at 12 h p.i. and reached the peak at 48 h p.i. Combined with the drug inhibition assay, VP088 gene was identified as a late (L) gene. Recombinant VP088 (rVP088) was expressed in Escherichia coli, and the specific antiserum against rVP088 was raised. VP088 was proved to be a viral envelope protein by Western blot and immunoelectron microscopy (IEM). Furthermore, rVP088 can bind to a 94 kDa host cell membrane protein, suggesting that VP088 might function as an attaching protein. Neutralization assay also suggested that VP088 is involved in SGIV infection. This study will lead to a better understanding of molecular mechanisms of the iridoviral pathogenesis and virus-host interactions.

Subcellular redistribution and sequential recruitment of macromolecular components during SGIV assembly

“…This protein is expressed late during SGIV infection in grouper cells and represents a putative SGIV envelope protein (Zhou et al, 2011), which is conserved in several iridoviruses (Fig. S1).…”

“…This study was aimed to identify a molecule that is suitable for visualizing dynamic processes of SGIV assembly and release in a living cell. The SGIV late gene encoded protein VP088 was identified as a putative myristylated envelope protein (Zhou et al, 2011). We overexpressed this protein in host cells and show that the transgene expression of VP88GFP, a fusion between VP088 and green fluorescent protein (GFP), does not compromise the ES cell properties and susceptibility to SGIV.…”

PROTEOMICS in aquaculture: Applications and trends

“…A few research papers have been published recently with promising results. A recent study on three of the major fish species of mariculture in southeast Asia describes the isolation of a pathogenic iridovirus and proteomics analysis of its envelope proteins [37]. This virus is responsible from 30% (adult fish) to 100% (fry) mortality in cultured grouper and has the ability of inducing an advanced cytopathic effect in a grouper cell line (GP).…”

“…The effect of probiotics administration in diets has also been reported in rainbow trout and cod [35,36] with interesting results at the level of immune-stimulation. In vitro [37] and in vivo infections proteomic studies on cultured fish like gilthead seabream [38], rainbow trout [39] and Atlantic salmon [40,41] are also commonly addressed since infection and concomitant death is one of the main causes of heavy economic losses in marine fish cultures. These works lead not only to a better understanding of the molecular mechanisms of pathogenesis as well as provide novel protein candidates for vaccines development.…”

Ranavirus Replication: Molecular, Cellular, and Immunological Events

“…Two late genes, SGIV ORF088 and ORF019, encode viral envelope proteins. Furthermore, rVP88 was shown to bind a 94 kDa host cell membrane protein, suggesting that VP88 might function as an attachment protein and play a role in viral entry (Huang et al 2013a ;Zhou et al 2011 ). Finally, similar approaches have been applied to ISKNV (genus Megalocytivirus ) and used to identify putative functions among a viral TRAF protein (He et al 2012a ), a viral protein that mediates formation of a mock basement membrane and provides attachment sites for lymphatic endothelial cells (Xu et al 2010 ), a viral ankyrin repeat protein that may inhibit TNFα-induced NF-κB signaling (Guo et al 2011a ), and a viral-encoded vascular endothelial growth factor (Wang et al 2008c ).…”