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

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.