Abstract
Autophagy is an essential and highly conserved process in mammals, which is critical to maintaining physiological homeostasis, including cell growth, development, repair, and survival. However, the understanding of autophagy in fish virus replication is limited. In this study, we found that grass carp reovirus (GCRV) infection stimulated autophagy in the spleen of grass carp (Ctenopharyngodon idella). Moreover, both Western blot (WB) analysis and fluorescent tracer tests showed that GCRV infection induced the enhancement of autophagy activation in Ctenopharyngodon idella kidney (CIK) cells. Autophagy inducer rapamycin and autophagy inhibitor 3-MA pretreatment can inhibit and promote the proliferation of GCRV, respectively. In addition, grass carp autophagy-related gene 5 (CiATG5)-induced autophagy, as well as rapamycin, showed effects on GCRV replication in CIK cells. Transcriptome analysis revealed that the total number of differentially expressed genes (DEGs) in CiATG5 overexpression groups was less than that of the control during GCRV infection. Enrichment analysis showed that CiATG5 overexpression induced the enhancement of autophagy, lysosome, phagosome, and apoptosis in the early stage of GCRV infection, which led to the clearance of viruses. In the late stage, steroid biosynthesis, DNA replication, terpenoid backbone biosynthesis, and carbon metabolism were upregulated, which contributed to cell survival. Moreover, signaling pathways involved in the immune response and cell death were downregulated in CiATG5 overexpression groups. Further study showed that CiATG5 repressed the expression of inflammatory response genes, including cytokines and type I interferons. Taken together, the results demonstrate that autophagy represses virus replication and attenuates acute inflammatory responses to protect cells.
Highlights
Autophagy is a highly conserved process in eukaryotes by which intracellular material is degraded and recycled in response to nutrient starvation or other hostile environments [1,2,3]
To determine whether autophagy was activated by grass carp reovirus (GCRV) infection, grass carp were infected with GCRV
Over the past few years, the studies on autophagy have become a top subject of particular scientific interest because of accumulating evidence that indicates that autophagy is closely related to viral infection and replication [13,14,15,16,17,18,19,20,21,22,23]
Summary
Autophagy is a highly conserved process in eukaryotes by which intracellular material is degraded and recycled in response to nutrient starvation or other hostile environments [1,2,3]. More than 40 autophagy-related genes (ATGs) are involved in the regulation of the autophagy process [4,5]. Nutrient starvation is a classic pathway to activate autophagy. Mammalian target of rapamycin complex 1 (mTORC1) activity is switched off when cells sense insufficient nutrition, and, subsequently, the cells release unc-51-like autophagy activating kinase 1 (ULK1), initiating autophagy signaling [6,7]. ULKs are thought to be the upstream components of the autophagy pathway that initiate the downstream ATG conjugate cascades [8]. Phosphatidylethanolamine (PE)-conjugated LC3 (LC3-II) is located in the isolation membrane, where the ATG16L1 complex accumulates, while unconjugated LC3 (LC3-I) is evenly distributed in cells [10]. LC3-II and LC3-I can be detected separately by immunoblot; LC3 is used as a preferred marker for microscopic detection of isolation membranes and autophagosomes [5,11,12]
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