Formation of autophagosomes and redistribution of LC3 upon in vitro infection with infectious salmon anemia virus

Autophagy plays an important role in both cellular homeostasis and cellular stress responses, and is also implicated in viral immunity. Here we show that infectious salmon anemia virus (ISAV) induce autophagy (double membrane autophagosomes observed by EM), in cells from Atlantic salmon. In addition, cells transfected with the standard autophagy marker, microtubule-associated protein light chain kinase 3 (LC3) fused to green fluorescent protein (LC3-GFP), displayed the redistribution of fluorescence into the typical punctate pattern seen in cells undergoing autophagy (a marker for autophagosome formation). This redistribution is caused by transformation of the LC3 protein into a membrane bound form due to lipidation of the C-terminal glycine. RT-QPCR analysis of key autophagy protein transcripts (ATG 3, 5, 6, 7, 8, 10, 12) mRNA revealed no major changes in expression of autophagy genes in the first phase of ISAV infection. Treatment of cells with an inhibitor of autophagosome formation, 3-methyladenine, both reduced LC3-GFP puncta formation and viral production, suggesting a role for autophagosomes in ISAV replication. This is the first report of virus-induced autophagy in cells from a fish species.