Coxsackievirus B3 Infection Triggers Autophagy Through Endoplasmic Reticulum Stress

Coxsackieviruses belong to the Enterovirus genus within Picornaviridae family.The virion is a nonenveloped icosahedral particle.The genome is a single-stranded positive strand RNA of approximately 7.5 kb.which has a 5'non-coding region,an open reading frame(ORF)and a 3'non-coding region.The ORF encodes a polypeptide chain,which is subsequently cleaved into 11 proteins by viral-encoded proteases.Depending on antigenicity and pathogenesis,coxsackieviruses are divided into A and B types.Coxsackievirus B type(CVB)has 6 serotypes,and CVB1,CVB2,and CVB3`have significant cardiac tropism.Coxsackievirus B3(CVB3)is a common pathogen of human viral myocarditis,which often leads to dilated cardiomyopathy and heart failure.Endoplasmic reticulum(ER)has important physiological functions involved in protein folding,calcium storage and lipid synthesis.When the environment changes,such as pathogen invasion,glucose starvation or calcium imbalance,a large number of non-folded or misfolded proteins accumulate in the ER,then ER stress and the unfolded protein response(UPR)is activated to regain normal physiology condition When ER stress becomes too intense or lasts long enough,UPR induces apoptosis to clear the damaged cells.Autophagy is a highly conserved catabolic process that delivers cytoplasmic components to lysosomes for degrading and recycling.There exist three types of autophagy:microautophagy,chaperone-mediated autophagy(CMA)and macroautophagy,and macroautophagy(herein referred to as autophagy)is the most studied.More than 30 autophagy-related genes(ATGs)have been identified,and many genes reveal significant conservation among yeast and mammals.ER stress and autophagy are two different biological mechanisms.Recent researches show that ER stress can upregulate autophagy.Autophagy efficiently degrades misfolded proteins to alleviate ER stress.Viral infection may cause endoplasmic reticulum stress and autophagy.In this study,the relationship between ER stress and autophagy in CVB3-infected HeLa cells is investigated.In this study,we first verify whether ER stress and autophagy are activated in CVB3-infected HeLa cells.HeLa cells were infected with CVB3 and collected at 6,8 and 10 h post-infection.The expression level of GRP78 is detected.Western Blot shows that CVB3 infection increased the expression of GRP78,which suggests that CVB3 infection induced ER stress.Western blot and Reverse transcription polymerase chain reaction(RT-PCR)were used to detect the activation level of PERK-eIF2?,IRE1-XBP1 and ATF6 signal pathways in CVB3-infected cells The results show that CVB3 infection respectively up-regulated the phosphorylation levels of PERK,eIF2?,and IRE 1,increased the cleaved XBP1 mRNA,and up-regulated the expression of the cleaved ATF6 protein.These results indicate that CVB3 infection activated PERK-eIF2?,IRE1-XBP1 and ATF6 signaling pathways.To determine whether CVB3 infection induces autophagy,the expression level and localization of LC3 protein and the expression of p62 protein were detected by Western blot and confocal microscopy.The results showed that CVB3 infection increased the expression of LC3-?/LC3-?,dereased expression of p62 protein,and induced the accumulation of green fluorescent protein(GFP)-LC3 punctuation,which indicated that CVB3 infection activated autophagy.Then we detected the activation of ULK1,AMPK and mTOR in HeLa cells infected with CVB3.The results showed that CVB3 infection promoted the phosphorylation of ULK1 and AMPK respectively.while decreased the protein level and phosphorylation of mTOR.The results indicated that CVB3 infection activated ULK1 and AMPK,and inhibited mTOR activity to promote autophagy.In order to fully understand the transcriptional level of autophagy-related genes in CVB3-infected cells,the transcriptional level of 19 autophagy-related genes was detected by dye-based RT-PCR.The results showed that the transcriptional levels of ATG5,ATG10,ATG12,FIP200,GABARAPL1,and GABARAPL2 mRNA were increased in CVB3-infected cells,and the transcription level of these genes was gradually increased with extension of infection time.The above results indicated that CVB3 virus infection differentially regulated the transcription of autophagy-related genes,thereby inducing autophagy.To explore the relationship between autophagy and viral replication in HeLa cells infected with CVB3,autophagy inducer rapamycin,autophagy inhibitor 3-Methyladenine(3MA)or lysosomal inhibitor Aloxistatin(E64D)was respectively exposed to HeLa cells for 2 h,then HeLa cells were infected with CVB3.The expression of CVB3 VP1 was detected at 6 h post infection.The results showed that both rapamycin and E64D treatment increased the expression of VP1 protein in the HeLa cells infected with CVB3,while 3MA treatment decreased the expression of VP1 protein.The results showed that CVB3 infection activated autophagy to facilitate viral replication.Endoplasmic reticulum stress is one of the causes of autophagy.To determine whether CVB3 infection-induced endoplasmic reticulum stress promotes autophagy,PERK inhibitor GSK2656157,ATF6 inhibitor AEBSF,and IRE1 inhibitor STF-083010 were respectively used to inhibit PERK,ATF6,and IRE1 signaling pathways,and the level of autophagy was detected by Western blot.The results showed that,compared with CVB3-infected cells,GSK2656157,AEBSF or STF-083010 decreased the ratio of LC3-II/LC3-I.which indicated that CVB3 infection induced autophagy through ER stress in HeLa cells,and PERK,IRE1,and ATF6a pathways participated in the regulation of autophagy.Subsequently,we investigated whether endoplasmic reticulum stress regulates the transcription of autophagy-associated genes mRNA in CVB3?infected cells,the transcription levels of ATG5,ATG10,ATG12,FIP200,GABARAPL1 and GABARAPL2 mRNA in CVB3-infected cells exprosed with three inhibitores were detected by Real timeRT-PCR.The results showed that,compared with CVB3-infected cells,ATF6 inhibitor down-regulated the transcription levels of ATG5 and ATG10;IRE1 inhibitor down-regulated the transcription of ATG5,ATG12,GABARAPL1,and GABARAPL2;PERK inhibitor had no significant effect on the transcription level of autophagy-related genes,which indicated that ATF6,IRE1 and PERK singnal pathway differentially regulate autophagy in CVB3 virus-infected cells.Summary,this study demonstrated that CVB3 infection induced ER stress,which triggers autophagy to to facilitate viral replication.