E3 Ligase Parkin Mediates The Immune Response Against White Spot Syndrome Virus By Ubiquitin-Proteasome And Xenophage Pathways

White spot syndrome virus (White spot syndrome virus, WSSV) is one of the main diseases of shellfish, and it can infect a variety of crustaceans like shrimp, crab. The high fatality rate for aquaculture brings great financial losses. And yet, the study of host innate immune mechanism is of great significance for the control of outbreak of viral disease. Previous studies have found that ubiquitination and sumoylation play an important role in shrimp antiviral immunity. As an E3 ubiquitin ligase and a mitochondrial quality control molecule, Parkin plays a vital role in the mitophagy. Recent studies reported that Parkin can clear intracellular bacterial pathogens with similar means to mitochondrial autophagy. But the detailed mechanism is indefinite. We identified an E3 ubiquitin ligase Parkin in Marsupenaeus japonicus named shrimp Parkin (sParkin). The total cDNA length of sParkin is 1656 bp. It contains a complete open reading frame which encodes 552 amino acids. It consists of a ubiquitin-like (UBQ) domain at the N-terminus followed by RINGO, RING1, RING2 in the C terminus. In this study, we found sParkin was expressed in the heart, stomach, hepatopancreas, gills, and intestine five organs and hemocytes. After white spot syndrome virus (WSSV) stimulation, its expression level was up-regulated at both mRNA and protein level. Injection of recombinant Parkin protein (rsParkin) reduced the mortality of shrimp, whereas knocking down the expression of sParkin increased WSSV replication in vivo. The experimental results show that sParkin may participate in the antiviral immunity in shrimp. Further study found that sParkin could bind to WSSV envelope proteins VP19, VP24, VP26, and VP28 by pulldown assay. Electron microscopy (SEM) observation showed that rsParkin was located on the surface of the WSSV. Immunocytochemical analysis found that sParkin was colocalized with WSSV in hemocytes. Furthermore, rsParkin could ubiquitinate the four envelope proteins of WSSV in vitro. Inhibitor of proteasome or autophagy could accelerate the replication of WSSV. To found whether the antiviral function of sParkin was related with autophage, We firstly analyzed the expression patterns of ATG5 and LC3 and the results showed that they were upregulated by WSSV infection. The expression of Atg5 and Lc3 was declined after interference of sParkin. On the other hand, after interference of Atg5, the expression of VP28 was increased. After injection of sParkin into Atg5-RNAi shrimp, the WSSV inhibition ability was recovered.In conclusion, our study demonstrates that sParkin can inhibit WSSV replication by Ubiquitin-Proteasome and xenophage pathway. This is the first report on the antiviral function of sParkin.