CiATG13 induces autophagy to regulate CiHSP70 to promote GCRV replication.
Ctenopharyngodon idella (grass carp) is the dominant species in freshwater aquaculture, but its farming industry is severely threatened by grass carp haemorrhagic disease (GCHD) caused by grass carp reovirus (GCRV). Autophagy plays a crucial role in viral infection, and ATG13 is a core factor for autophagy initiation. However, the functional mechanism of grass carp ATG13 (CiATG13) during GCRV-I/II infection remains unclear. In this study, the CiATG13 gene was cloned and characterized by bioinformatics analysis. The results showed that CiATG13 sequence is highly conserved in evolution, sharing the highest homology and closest evolutionary relationship with Chanodichthys erythropterus. The expression and function of CiATG13 were investigated using RT-qPCR, Western blotting, fluorescence microscopy, and CRISPR-Cas13d knockdown techniques at both cellular and individual levels. The key findings are summarized below: tissue distribution analysis revealed that CiATG13 is widely expressed in various tissues of healthy grass carp, with the highest expression in the liver, brain, and heart, and it responds actively to stimulation by pathogen-associated molecular patterns (PAMPs), such as poly (I:C) and lipopolysaccharide (LPS). GCRV-I/II infection induces the expression of CiATG13. Overexpression of CiATG13 significantly promotes GCRV-I replication, whereas knockdown of CiATG13 inhibits GCRV-I replication. Further mechanistic studies indicated that CiATG13 can induce autophagy and upregulate the expression of heat shock protein 70 (CiHSP70) through this pathway. CiHSP70 promotes GCRV-I replication, and quercetin (Qu) can block its pro-viral effect on GCRV-I/II replication by inhibiting CiHSP70. Moreover, treatment with the autophagy inhibitors chloroquine (CQ) and Spautin-1 suppressed GCRV-I replication and the associated cytopathic effect (CPE), accompanied by reduced CiHSP70 expression, overexpression of CiATG13 partially rescued these inhibitory effects.. This study reveals the molecular mechanism that CiATG13 mediates autophagy to regulate CiHSP70 expression and promote GCRV-I/II replication, enriches the understanding of the interaction between fish viruses and autophagic molecules, and provides the potential strategy targeting CiATG13 for the prevention and control of GCHD.