Grass carp reovirus (GCRV) fibrin VP56 interacts with grass carp (Ctenopharyngodon idella) GRP78 for inducting endoplasmic reticulum stress

Grass carp (Ctenopharyngodon idella) is one of the most important aquaculture fish species in China. C. idella hemorrhagic disease caused by grass carp reovirus (GCRV) results in severe outbreaks annually. It causes great losses to aquaculture industry and a threat to food safety. GCRV is the most virulent in aquareovirus. VP56 is a fibrin which is specific to type Ⅱ/Ⅲ. It attaches the surface of host cells during GCRV infection. In order to clarify the infection mechanism of GCRV, this study delved into the interaction between VP56 and host cell proteins. First, co-immunoprecipitation (co-IP) was performed. The specific antibodies to different tags respectively fused on the two molecules were used. Empty vectors and negative immunoglobulin G were used as control. It was found that VP56 interacts with the molecular chaperone protein glucose regulated protein 78 ku (GRP78) located in the endoplasmic reticulum. Then, the interaction between VP56 and GRP78 was verified by the bimolecular fluorescence complementation (BiFC) based on the far-red fluorescent protein mNeptune. According to the BiFC results, red fluorescence of mNeptune was observed in CIK cells transfected with both VP56 and GRP78 respectively linked with each terminal of the mNeptune protein, showing that these two fusion proteins presented closely and sent out red fluorescence signal. In VP56 stably expressed C. idella kidney (CIK) cells, the endoplasmic reticulum morphology underwent tremendous changes, compared with empty vector stably expressed CIK cells resulting in swelling, expansion, and degranulation, indicating that VP56 activates endoplasmic reticulum stress. mRNA levels of transcription factors downstream of endoplasmic reticulum stress were detected, which revealed that VP56 activates the signal transduction pathway regulated by activating transcription factor 6 (ATF6) and triggers the unfolded protein response. The above results indicate that during GCRV-Ⅱ infection, cell homeostasis is destroyed, and endoplasmic reticulum stress is activated. This study provides new ideas for the study of GCRV infection mechanism and anti-GCRV research, reveals a new virus escape strategy, provides in-depth antiviral research in C. idella, and contributes to the prevention and control of C. idella hemorrhagic disease in the freshwater aquaculture industry.