Grass Carp Autophagy-Related Gene 5 Shows Inflammatory Regulation Function During Grass Carp Reovirus Infection

Abstract

Grass carp (Ctenopharyngodon idella) aquaculture faces significant challenges due to grass carp hemorrhagic disease, which is instigated by the grass carp reovirus (GCRV). A major contributor to the high mortality rates associated with this disease is an excessive inflammatory response. Autophagy contributes to host disease resistance partly through modulating inflammation, in which autophagy-related gene 5 (ATG5) serves as a core component. In this study, we generated an ATG5-edited cell model with biallelic frameshift mutations in C. idella kidney (CIK) cells using the CRISPR/Cas9 system. Transcriptome and qPCR analyses indicated that ATG5 gene editing significantly influenced multiple biological pathways, including oxidative phosphorylation, phagosome formation, and arachidonic acid metabolism, and altered the expression of inflammation-related genes such as IL-1β, IL-8, TNF-α, IL-10, and TGF-β in CIK cells during GCRV infection. Further investigations through Western blot and ubiquitination assays demonstrated that ATG5 gene editing led to a notable decrease in the LC3-II/LC3-I ratio in CIK cells, along with a buildup of pro-IL-1β protein. The overexpression of grass carp ATG5 and IL-1β in HEK-293T cells reduced the ubiquitination of grass carp pro-IL-1β, facilitating its accumulation. Collectively, our results indicate that grass carp ATG5 modulates the expression of multiple inflammatory mediators and may shape pro-IL-1β protein abundance in a ubiquitination-associated manner. This study provides insights into autophagy-linked inflammatory regulation underlying grass carp hemorrhagic disease.