Screening and characterization of host proteins interacting with rhabdovirus glycoprotein

Spring viraemia of carp virus (SVCV), a member of the Rhabdoviridae family, is responsible for triggering epidemic diseases in cyprinid fish. Viral envelope glycoprotein plays a crucial role in mediating viral invasion, assembly, and release by interacting with host proteins. Using the yeast membrane two-hybrid screening in zebrafish embryo libraries, several proteins that interact with SVCV glycoprotein were identified. Annotation analysis showed that host proteins were mainly implicated in binding, molecular function regulator, and transcription regulator activity, as well as involvement in translation, folding, and transport. Through one-on-one Y2H verification experiment, 9 positive clones associated with viral infection process were obtained, including CCTα, GFAP, ACTB2, RPL24, and RPS3. Molecular docking analysis indicated that the interaction between G and CCTα occurs primarily through hydrogen bonding and hydrophobic interactions, with binding site located at ARG89∼HIS98 of glycoprotein. Site-directed mutagenesis showed that TYR91, GLN94, and TYR95 were critical binding sites for the interaction between viral G protein and host CCTα protein. Moreover, overexpression of CCTα protein in SVCV-infected ZF4 cells promoted viral G protein gene expression. These results demonstrated that G-CCTα could act as a promising new target for anti-SVCV drug development.