Screening of potential host proteins interacting with VP56 of type Ⅱ grass carp reovirus by yeast two-hybrid system

Among the three genotypes of grass carp reovirus (GCRV), type II GCRV accounts for the major epidemic of grass carp hemorrhagic disease at present in China. Bioinformatic analysis predicted that VP56 of type II GCRV might function as the fiber-like protein of reoviruses. This study aimed to screen potential host proteins that interact with VP56, which might present clues on the biological role of VP56. VP56 gene was amplified by RT-PCR and was used to generate the bait plasmid, pGBKT7-VP56. Subsequently, cDNA library plasmid of Ctenopharyngodon idella kidney cells was screened to test potential interaction partners in yeast AH109 transformed with pGBKT7-VP56. The positive clones were analyzed by plasmid sequencing and nucleotide sequence blasting. To test whether JAM-A served as a potential interaction partner for reoviral fiber protein in grass carp like in mammalian cells, pGADT7-GcJAM-A vector was constructed for the sake of further validation of its interaction with VP56 protein. Results in this study showed that the bait plasmid pGBKT7-VP56 has no selfactivation in yeast, 9 positive clones were obtained, and 7 cellular proteins and an extracellular matrix protein were suggested to bind VP56, and no interaction between VP56 protein and GcJAM-A protein was detected in yeast cell. These results may pave the way for further functional analysis of VP56 protein.