Mechanisms Of Autophagy Induced By Swine Acute Diarrhea Syndrome Coronavirus

Coronavirus is regarded as a worldwide pathogen,is susceptible for most types of domestic poultry and mammals as well as human.Coronavirus has caused panic of human beings since the epidemic of SARS-CoV(Severe Acute Respiratory Syndrome Coronavirus,SARS-CoV)in 2003,MERS-CoV(Middle East Respiratory Syndrome Coronavirus,MERS-CoV)in 2012 and COVID-19 in 2020.The Porcine epidemic diarrhea virus(PEDV)from 2010 to 2013 and the newly discovered Swine Acute Diarrhea Syndrome Coronavirus(SADS-CoV)in 2017 have caused a major economic losses of livestock industry.More and more investigations were conducted in Coronavirus concerning its pathogenic severity and ability of rapidly spread.A novel coronavirus,SADS-CoV,is an enveloped,positive and single-stranded sense RNA virus with a genome size of approximately 27 kb.SADS-CoV belongs to the family Coronaviridae and the genus Alphacoronavirus.It was first discovered in Guangdong province in 2017 and caused a large scale outbreak of fatal diarrheal disease in piglets.As obligate intracellular parasites,viruses are extremely reliant on host cells and must interact with host response.Autophagy,as a highly conserved cellular adaptive response,plays important roles in keeping homeostasis and promoting metabolism.Autophagy is a dynamic process in eukaryotes that involves the formation of double-membrane autophagosomes,with damaged organelles or microbes wrapped.Then these substrates would be delivered for digestion and recycling via a lysosomal degradative pathway.Autophagy has intracellular antimicrobial properties.However,accumulating lines of evidence suggest that many viruses have evolved to evade,escape or exploit autophagy for their own benefit,such as influenza virus,hepatitis C virus(HCV),dengue virus and rotavirus.Regarding SADS-CoV,the relationship between SADS-CoV infection and autophagy has not been thoroughly examined and deserves to be explored.To begin with,three classical methods were adopted to examine whether SADS-CoV infection would induce autophagy in Vero E6 and ST cells,including the observation of autophagosomes formation by transmission electron microscopy(TEM),the analysis of LC3 conversion by Western Blot and the detection of GFP-LC3 puncta accumulation by confocal microscopy.Results showed that,compared with the negative control,SADS-CoV infection induced numerous autophagosome-like double-membrane vesicles in the cytoplasm and more GFP-LC3 dots accumulation.Besides,the LC3 conversion was significant after SADS-CoV infection.We also demonstrated that SADS-CoV induced autophagy was dependent on virus replication.In sum,based on these results,we concluded that notable autophagy could be triggered by SADS-CoV infection.Next,given that autophagy is induced by SADS-CoV infection,we then determined whether cellular autophagy regulates.The results showed that treating Vero E6 and ST cells with rapamycin,an inducer of autophagy,promoted SADS-CoV replication.In contrast,treatment Vero E6 and ST cells with 3-MA,an inhibiter of autophagy,suppressed SADS-CoV replication.At the same time,viral protein N levels and the infectious progeny viruses in extracellular exhibited a significantly decrease in ATG5 or LC3 knocked-down cells.These findings conclude that the effective replication of SADS-CoV is the critical factor for autophagy activation,and in turn autophagy is utilized for the virus replicationBesides,the autophagic flux in Vero E6 and ST cells based on the analysis of the autophagic degradation was measured.Autophagic flux is a continuous and complete process of autophagy and can be comprehensively assessed by p62 degradation,LC3-?turnover and colocalization analysis of autophagosomes and lysosomes.Immunoblotting results revealed significant progressive degradation of p62 in SADS-CoV-infected cells.The 3D live cell imaging system analysis revealed that SADS-CoV infection promoted the fusion of autophagosomes with lysosomes.Moreover,the colocalization of GFP-LC3-tagged autophagosomes and LysoTracker-stained lysosomes was detected in SADS-CoV-infected Vero E6 cells.And the accumulation of LC3-? and p62 was observed along with the employment of CQ in SADS-CoV-infected cells.Collectively,these results indicated that SADS-CoV induce autophagy initiation and autophagic degradation,proving that SADS-CoV infection induces the occurrence of enhanced autophagic flux.Finally,with a view to understand the underlying mechanisms of autophagy initiation by SADS-CoV infection,the relationship between ER stress and autophagy was systematically analyzed.The results show that SADS-CoV infection can induce ER stress and activated all three UPR pathways(PERK-EIF2S1,ATF6 and IRE1)in vitro and in vivo.Further use of specific inhibitors or activators,RNA interference and overexpression of key molecules,we found that the IRE1 pathway is involved in autophagy induction and enhanced SADS-CoV replication.In conclusion,this study demonstrated that(1)SADS-CoV infection induced autophagy in Vero E6 and ST cells;(2)Autophagy could facilitate virus replication;(3)SADS-CoV induce autophagy via UPR IRE1 signaling pathway in host cells.Our achieved findings here identify a crucial step in driving the progress of interplay between SADS-CoV and autophagy.It will provide new insights into the pathogenesis of SADS-CoV and contribute to antiviral drugs development.