Atg9a is a critical factor in the immune response of grouper (Epinephelus coioides) to viral infection

Autophagosome generation, development and maturation are largely dependent on Atg9a, an important member of the autophagy-related protein family. However, the potential role of fish Atg9a in viral infections is poorly understood. This research involved cloning and characterizing an Atg9a homologue from the orange-spotted grouper ( Epinephelus coioides ), known as EcAtg9a . The ORF (open reading frame) of EcAtg9a is composed of 2583 nucleotides and encodes 860 amino acids. EcAtg9a was detected in every tissue that was analyzed, and was particularly abundant in liver, kidney, head and blood. EcAtg9a expression levels in grouper spleen (GS) cells increased following infection with SGIV and RGNNV. EcAtg9a was uniformly present in the cytoplasm, while found to co-exist with the endoplasmic reticulum (ER), Golgi apparatus, mitochondria and lysosomes. EcAtg9a suppressed the expression of interferon-related genes and factors linked to inflammation while promoting the replication of SGIV as well as RGNNV in GS cells. In addition, EcAtg9a could interact with the key molecules of the cGAS-STING pathway including EccGAS, EcTBK1, EcSTING, and even EcIRF3. EcAtg9a facilitated the upregulation of Beclin1 and LC3-II synthesis within cells, resulting in an augmented formation of LC3 fluorescent aggregates. The level of eIF2α phosphorylation (S51) was increased in EcAtg9a-overexpressing cells, and the p53 protein moved from the cytoplasm into the nucleus. Findings may offer insight into how grouper's innate immunity works against viral infections. • EcAtg9a was uniformly distributed in the cytoplasm. • EcAtg9a promotes the replication of SGIV and RGNNV in grouper spleen (GS) cells. • EcAtg9a interacts with EcSTING, EccGAS, EcIRF3, and EcTBK1. • EcAtg9a promotes the synthesis of LC3-II and Beclin1 in GS cells.