Molecular characterization of ATG5 in Larimichthys crocea and its role in promoting virus proliferation

Autophagy is an important process that maintains homeostasis in eukaryotic cells and is involved in cell differentiation, development, and immunity. However, little is known about the function of autophagy-related genes (ATGs) in the immune response of fish. To explore the function of ATG5 in virus infection of the Larimichthys croaker, we cloned a ATG5 gene from large yellow croaker (Larimichthys crocea) and its open reading frame (ORF) was 828 nucleotides long, encoding a protein of 275 amino acids. The predicted molecular weight of LcATG5 was 32.3 ku and its theoretical isoelectric point was 5.7. Homology comparison showed that LcATG5 had a highly conserved APG5 domain as found in other vertebrate ATG5 proteins. Phylogenetic analysis showed that LcATG5 was closely related to Collichthys lucidus ATG5. LcATG5 was detected in all eleven tissues of L. crocea, with highest expression in blood and lowest in spleen. LcATG5 transcripts were also detected in primary head kidney granulocytes, lymphocytes and macrophages, and L. crocea head kidney cell line (LYCK), with highest expression in primary head kidney granulocytes. After induction by poly(I:C), LcATG5 transcripts were significantly increased in these cells, and were more responsive in LYCK cells, with 3.93-fold increase at 12 h. In the LcATG5-overexpressed epithelioma papulosum cyprini (EPC) cells, the cytopathic effects (CPE) caused by spring viremia of carp virus (SVCV) infection were increased at 48 h post-infection. The viral titer of SVCV in the culture supernatant of the LcATG5-overexpressed EPC cells was 10^13.82 TCID₅₀/mL, which was higher than that of control group (10^9.27 TCID₅₀/mL). Besides, the expression levels of three viral genes (SVCV-G, SVCV-M, and SVCV-P) were significantly up-regulated in the LcATG5-overexpressed cells with 13.77-, 15.72-, and 11.39-fold increases, respectively. These results indicate that LcATG5 plays a role in promoting virus replication, which may provide a basis for in-depth study of the function and mechanism of autophagy and ATGs during virus infection in fish.