Figure 6

Role of NNV capsid protein in ER stress. (A) Co-immunoprecipitation of BiP protein and NNV capsid protein. GF-1 cells were infected with NNV at MOI of 5. The cell lysate was incubated with Protein A agarose and anti-BiP IgG. The immunocomplexes were separated on a 12% SDS-PAGE gel, and were further analyzed by western blotting with mouse anti-BiP and horseradish peroxidase-conjugated anti-mouse IgG. Chemiluminescence signal was captured after incubation with ECL substrate. For NNV capsid protein, the membrane was stripped, and re-probed with rabbit anti-NNV capsid antiserum, followed by horseradish peroxidase-conjugated anti-rabbit IgG. The result shows that BiP protein could interact with NNV capsid protein after NNV infection. (B) Induction of XBP1 mRNA splicing by NNV capsid protein in vivo. Zebrafish embryos at one-cell stage were mock-injected with 9.2 nl of sterile water or injected with 9.2 nl of pIR8 at the concentration of 150 ng/μl. The larvae were sacrificed at 20 hr post-fertilization and subjected to RNA extraction. RT- PCR was carried out to analyze the transcript of xbp-1 gene. Splicing of XBP1 mRNA, an indicator of ER stress, was detected in the embryos expressing exogenous NNV capsid protein.