The Mechanism Study On ER Stress Caused By HRV 16 And Non-structural 2B Through Increase Of Cytoplasmic Calcium

Human rhinovirus(HRV)is one of the most important pathogens which cause viral influenza.HRV can cause about 50%upper respiratory tract infection,and is also the main pathogen of acute lower respiratory tract infection in children.HRV infection can induce acute exacerbation of childhood asthma and is associated with chronic obstructive pulmonary disease(COPD).Endoplasmic reticulum stress(ERS)is an appropriate response on dysfunction of the endoplasmic reticulum(ER).ER is an organelle where proteins are properly folded and modified,and also is a storage place for calcium.ER stress can be induced by many pathogenic stress stimuli,such as hypoxia,misfolded protein,changes in calcium levels,viral infections.To alleviate the adverse effects of ER stress,the cell induces UPR to protect cells.Many evidences have demonstrated ER stress is important in the development and progression of bronchial asthma.Enhanced cytosolic Ca2+ concentration of airway smooth muscle(ASM)cells plays an important role in airway hyperresponsiveness and airway remodelin.To understand the underlying mechanisms of endoplasmic reticulum stress caused by human rhinovirus,we investigated the celluar signaling pathways of ERS caused by HRV 16 infection and its non-structural 2B transfection.The expressions of GRP78 and three signal transduction pathways,including PERK,ATF6 and IRE1,were evaluated after HRV 16 infection and 2B transfection H1-HeLa.After H1-HeLa cells were infected with HRV16 at a MOI of 5,the expression of GRP78 was detected by Western blotting at 3 h,6 h and 9 h post-infection.The result showed that HRV 16 infection induced the expression of GRP78 in a time-dependent manner.Thus,the results indicated that ERS was induced by HRV 16 infection.Western blot showed that p-PERK and p-eIF2a were markedly increased in Hl-HeLa cells infected with HRV 16 in a time-dependent manner,while the expression level of PERK and eIF2a was unchanged.During ER stress,the 90-kDa ATF6 protein is proteolytically cleaved into 50-kDa ATF6 under the activation of activating transcription factor-6(ATF6)pathway.Our results showed that cleaved-ATF6 was markedly increased in H1-HeLa cells infected with HRV 16 in a time-dependent manner.In response to ER stress,IRE1 is activated by its home-oligomerization in the membrane,causing the splicing of a 26-bp intron from an evolutionarily conserved ER stress transcription factor XBP1.The results show that the expression of p-IRE1 was significantly down-regulated in a time-dependent manner in Hl-HeLa cells infected with HRV 16.We could not detect any spliced XBP1 in mRNA level.Taken together,HRV 16 infection induced ER stress through PERK and ATF6 pathways rather than IRE1 pathway.To understand the role of non-structural protein 2B in HRV 16 induction ERS,the eukaryotic expression vector,pcDNA3.1-2B-Flag,was constructed.The analysis software of secondary structure predicted two hydrophobic regions in the HRV 16 2B protein.When H1-HeLa was transfected with pcDNA3.1-2B-Flag,the 2B protein was clearly co-localized with ER by immunofluorescence technique.Then the three typical signaling pathways of ERS were detected in H1-HeLa transfected with pcDNA3.1-2B-Flag at 12 h,24 h,36h post-transfection.Our results showed that GRP78,p-PERK,p-eIF2?,and cleaved-ATF6 were markedly increased in a time-dependent manner.Meanwhile,the luciferase activity from ATF6-luc and ATF4-luc construct was significantly increased.These showed that HRV 16 2B protein activates PERK and ATF6 pathways.Our results showed that HRV 16 2B protein decreased the expression of p-IRE1 suggsting that IRE1 pathway was not activated in Hl-HeLa cells expressing HRV 16 2B protein.Similar with HRV 16 infection,we did not detect XBP1 splicing.To understand why IRE1 was dephosphorylated in cells infected with HRV 16 and transfected with pcDNA3.1-2B-Flag,a specific inhibitor of CaN(Cyclosporin A,CsA)was selected to treat H1-HeLa cells infected with HRV 16 or transfected with pcDNA3.1-2B-Flag.Our results showed that the expression of p-IRE1 was markedly increased in Hl-HeLa cells infected with HRV 16 or transfected with pcDNA3.1-2B-Flag when treated with CsA at each time point.To further understand whether HRV 16 infection and HRV 16 2B protein induced CaN activity through increase of cytoplasmic Ca2+,a Ca2+chelator,BAPTA-AM,was used to treat H1-HeLa cells infected with HRV 16 or transfected with pcDNA3.1-2B-Flag.The results showed that the CaN activity in cells transfected with pcDNA3.1-26-Flag or infected with HRV 16 was significantly increased at each time point post-infection or post-transfection.However,BAPTA significantly inhibited CaN activity in cells transfected with pcDNA3.1-2B-Flag or infected with HRV 16.The results demonstrated that both HRV 16 and HRV 16 2B protein enhance CaN activity through increase of cytoplasm Ca2+leading to dephosphorylate IRE1.To understand whether PERK and ATF6 pathways were induced through increase of cytoplasmic Ca2+by nonstructural 2B protein,BAPTA-AM was used to treat Hl-HeLa cells transfected with pcDNA3.1-2B-Flag.The results showed that both expressing levels of p-PERK and cleaved-ATF6 were clearly inhibited in B APTA-treated cells transfected with pcDNA3.1-2B-Flag compared with cell only transfected with pcDNA3.1-2B-Flag or control vector with increase of post-transfection.Meanwhile BAPTA significantly inhibited both ATF4 land ATF6 luciferase.The results demonstrated that PERK and ATF6 pathways were induced through increase of Ca2+induced by nonstructural 2B protein of HRV 16.To further elucidate the effects of UPR signaling on HRV 16 viral replication,three specific inhibitors were used to rapidly inactivate three UPR signaling pathways.After infected cells were treated with inhibitors of ATF6(AEBSF),PERK(GSK2656157)or IRE1(STF-083010),the level of HRV 16 replication was assessed using plaque forming unit(pfu).AEBSF and GSK2656157 clearly inhibited the viral replication at each concentration.However,STF-083010 did not inhibit viral replication.These observations suggested that the induction of ATF6 and PERK pathways,but not IRE1 pathway,could positively regulate the viral replication.In short,HRV 16 and non-structural protein 2B induce increase of cytoplasmic Ca2+to cause ER Stress through activation of both PERK and ATF6 pathways.However,both HRV 16 and 2B protein enhance CN activity through increase of cytoplasm Ca2+to dephosphorylate IRE 1.The results can provide a theoretical basis for the prevention and treatment of respiratory diseases caused by rhinovirus infection.