Interaction And Functional Studies Of Major Structural Proteins In Infectious Spleen And Kidney Necrosis Virus (ISKNV)

The predatory freshwater mandarin fish, Siniperca chuatsi (Basilewsky), is animportant cultured freshwaterborne fish species in China. In recent years, the spread ofInfectious Spleen and Kidney Neurosis Virus (ISKNV) has been causing high mortalityand great economic loss in mandarin fish industry. Analysis of purified ISKNV virus,we found the fourth high molecular weight protein as virus-inducible stress protein, anddesignated as mVISP. Western-blot and immunogold experiment showed that mVISPprotein was the structure of the virus and located in the viral envelope. Real timequantitative PCR results showed mVISP expression increased significantly in ISKNVinfected mandarin fish tissue and MFF-1cell. The immunofluorescence assay displayedmVISP antibody could effectively identify the virus, but could not neutralize the virus.RNA interference silencing of mVISP had no significant effect on ISKNV replication.A variety of important physiological activities and the response to external andinternal environment of host cells, are all based on protein interaction as a link and forma signal transduction network system. There are interactions between viral proteins tomaintain the hierarchical structure of the viral protein and the stability of the virus form.In this paper, we used the yeast two-hybrid technology to explore the interactionbetween viral proteins under the core of the major capsid protein. Using MCP as target protein, MCP, VP007, VP036, VP037, VP041, VP056, VP063, VP071, VP101, VP117as bait protein, co-transfected yeast cells and by reporter gene screening, we found thereis interaction between MCP and VP071. In reverse yeast two-hybrid experiment, usingVP071as target protein and other proteins as bait protein co-transfected yeast cells, weverified the interaction between the MCP and VP071and found the interaction betweenVP071and VP071.We further verity the interaction between MCP and VP071by immuneco-precipitation technology. Real time quantitative PCR result showed ORF071wasviral late gene. Western blot result showed that VP071maybe was viral capsid proteinand in the same structural level of the virus as MCP. Cells transfected with071-EGFPplasmid found VP071could induce apoptosis and we confirmed it using annexin V Kit.By microinjected a recombinant plasmid containing the DNA sequence of ORF071intozebrafish embryos, we observed ORF071over-expression resulted in apoptotic signalby TdT-mediated dUTP nick end labeling (TUNEL).ORF071induced apoptosis wasclearly associated with significant caspase8up-regulation and activation. Byhomologous recombination method, we constructed a recombinant virus strain△m-071and the recombination rate of ORF071was50%-65%. Comparision of wild and△m-071viruses in MFF-1cells showed that no obvious differentiation were observedin viral titers infection to MFF-1cell, which indicated ISKNV-ORF071is not a essentialgene in megalocytivirus production in vitro infection.