Functional characterization of BAG3 in orange-spotted grouper (Epinephelus coioides) during viral infection.

Bcl-2-associated athanogene 3 (BAG3) is a cochaperone protein that interacts with Bcl-2 and mediate cell death. However, little is known about the roles of fish BAG3 during viral infection. In this study, we characterized a BAG3 homolog from orange-spotted grouper (Epinephelus coioides) (EcBAG3) and investigated its roles during viral infection. The EcBAG3 protein encoded 579 amino acids with typical WW, PXXP and BAG domains, which shared high identities with reported fish BAG3. Quantitative real-time PCR (qRT-PCR) analysis revealed that EcBAG3 was highly expressed in brain and heart. And the expression of EcBAG3 was significantly up-regulated after red-spotted grouper nervous necrosis virus (RGNNV) stimulation in vitro. EcBAG3 overexpression could promoted the expression of viral genes (coat protein (CP) and RNA-dependent RNA polymerase (RdRp)), which was enhanced by co-transfection with Hsp70 and Hsp22. Also, EcBAG3 overexpression up-regulated the expression of LC3-Ⅱ and down-regulated the expression of Bax and BNIP3, the IFN- (IRF1, IRF3, IRF7, IFP35, Mx1) or inflammation-related (IL-1β and TNFα) factors, as well as decreased the activities of NF-κB, ISRE and IFN-3. While knockdown of EcBAG3 decreased the transcripts of RGNNV CP gene and RdRp gene. Further studies showed that EcBAG3 knockdown impaired the expression level of autophagy factor LC3-Ⅱ, and promoted the expression level of Bax and BNIP3, inflammatory factors and interferon factors. These data indicate that EcBAG3 can affect viral infection through modulating virus-induced cell death, regulating the expression of IFN- and inflammation-related factors, which will be helpful to further explore the immune response of fish during viral infection.