The Roles Of Autophagy In Singapore Grouper Iridovirus And Red-spotted Grouper Nervous Necrosis Virus Infections In Host Cells

Grouper,Epinephelus spp.,are important marine cultured fish in the coastal areas of south China and southeast Asian countries.However,the frequent occurrence of viral diseases has seriously restricted the healthy and sustainable development of grouper aquaculture.Singapore grouper iridovirus(SGIV)and Red-spotted grouper nervous necrosis virus(RGNNV)are two representative viral pathogens.Further studies on viral pathogenesis and virus-host interactions are significant for the prevention of grouper viral diseases.Autophagy,a conservative physiological activity to maintain intracellular homeostasis,is also a defense mechanism for cells to cope with adverse conditions.For virus infection,autophagy may transport it to lysosomes to degrade or activate the host immune response against the virus replication.However,many viruses have evolved some mechanisms that prevent autophagy occurring or use membrane bubbles formed during autophagy as replication factory.The game between autophagy and virus affects the fate of host cells and viruses to some extent.This paper focuses on the effects of SGIV and RGNNV on autophagy in grouper spleen cells(GS)and its mechanism.From the perspective of the host,we also demonstrated the role of autophagy in viral replication.Moreover,the immune regulation effects of autophagy related genes on virus replication were analyzed.The main innovative research results are as follows.1.SGIV inhibited autophagy for efficient viral replication.In this study,we confirmed SGIV entry triggered GS cells autophagy activity by three classic method,including detecting autophagy related protein levels by Western blot,observing autophagy related structures under a transmission electron microscope and GFP-LC3 gathered under a fluorescence microscope.As the infection progresses,SGIV obviously inhibited autophagy.Next,we explored the inhibition mechanism of SGIV from the regulation of p53 on autophagy and the interaction between viral protein and autophagy protein.The strategies that SGIV inhibited autophagy include at least two aspects:(1)SGIV infection altered p53 localization,so that p53 can transfer to the cytoplasm from the nucleus to inhibit autophagy occur;(2)SGIV encode some viral proteins(VP48,VP122 and VP132)that interacted with autophagy related proteins 5,blocking the conversion of LC3-I to LC3-II so that the formation of autophagosome was impeded.In order to investigate the effect of autophagy on SGIV replication,an autophagy inducer(Rapamycin,Rap)and an inhibitor(Wortmannin,WM)were used to regulated autophagy.We found that autophagy inhibited SGIV replication in GS cells.Combining Rap with the lysosomal acid inhibitors Bafilomycin A1(Baf A1)and Chloroquine(CQ),we found that autophagosome inhibited SGIV replication.In addition,LC3,a key autophagy protein,promotes SGIV entry but plays an antiviral role in SGIV replication.2.RGNNV infection caused obvious autophagy in GS cells.In order to explore whether autophagy induced by RGNNV was dependent on virus infectivity,RGNNV was inactivated by UV irradiation.Then we found that RGNNV induced autophagy was independent of virus infectivity.In addition,overexpression of capsid protein CP also induced autophagy.In order to explore the effect of autophagy on RGNNV replication,we used the autophagy regulator(Rap,WM and Baf A1)to interfere the autophagy pathway,then we found that RGNNV utilized autophagosomes and autolysosomes for its replication.In addition,by detecting proteins and their phosphorylation levels in autophagy pathway,we preliminarily revealed that RGNNV induced autophagy by activating e IF2α phosphorylation and inhibiting m TOR phosphorylation.3.Autophagy related genes(Atg)from grouper(Epinephelus coioides)promoted virus replication by negatively regulating immune response.Three autophagy related genes(Atg)from grouper were cloned and identified,namely EcAtg5,EcAtg12 and EcAtg16L1.EcAtg5 and EcAtg16L1 were distributed in cytoplasm,mainly in the form of point-like aggregation.EcAtg12 was evenly distributed in the cytoplasm and nucleus.Compared with non-infected cells,the expression levels of EcAtg5,EcAtg12 and EcAtg16L1 were decreased in SGIVinfected cells.However,the expression levels of EcAtg5,EcAtg12 and EcAtg16L1 were increased in RGNNV-infected cells.Using transfecting recombinant plasmids or si RNA,we demonstrated that EcAtg5,EcAtg12 or EcAtg16L1 promoted virus replication through down-regulating interferon-related molecules and pro-inflammatory factors.In addition,overexpressing EcAtg5 promoted the level of LC3-II protein and affected cell cycle progression from the G1 to the S phase and arrested cells in the G1 phase.Overexpression of EcAtg16L1 promoted the recruitment of LC3-II to the autophagosome membrane.These results revealed the functions of Atg5,Atg12 and Atg16L1 in autophagy,host immunity and virus replication.In conclusion,we revealed the effects and mechanisms of SGIV and RGNNV infection on autophagy,respectively.Then,we investigated the effects of autophagy on SGIV and RGNNV replication.Moreover,we illuminated the roles of Atgs on viral replication by regulating host immune and.These results lay the foundation for searching antiviral strategies from the perspective of autophagy.