Study On Apoptosis Induced By Endoplasmic Reticulum Stress Response During Newcastle Disease Virus Infection

Newcastle Disease Virus?NDV?is a spherical,enveloped paramyxovirus,also highly contagious avian pathogen.Its genome is single-stranded,negative-stranded,non-segmented RNA.NDV has been identified as an important oncolytic virus,which selectively infects and kills human cancer cells by apoptosis.Recently,it has been reported that NDV infection induces both extrinsic and intrinsic apoptosis,and the extrinsic apoptosis is mediated by induction of death ligands,such as TNF-?and TRAIL.However,there is not systematic and specific report on how NDV infection causes endogenous apoptosis.Under different physiological and/or pathological conditions,such as virus infection,lack of nutrition,disorder of calcium ion balance,large amounts of proteins enter the ER,unfolded or misfolded proteins accumulate in the ER lumen and induce ER stress.For survival,cell will activate several signaling pathways collectively termed as ER stress response,also known as the unfolded protein response?UPR?.Three transmembrane ER stress sensors have been identified,including protein kinase RNA?PKR?-like ER kinase?PERK?,activating transcription factor 6?ATF6?andInositol-Requiring Protein 1 alpha?IRE1??.During RNA virus infection,large amount of viral proteins are synthesized,which usually activate PERK.Meanwhile,another eIF2?kinase,PKR,binds with viral double stranded RNA?dsRNA?and is activated by auto-phosphorylation.Activation of PERK and PKR are responsible for promotingphosphorylation of eIF2?,resulting in preferential translation of ATF4,which translocate into thenucleus and transcriptionally upregulates CHOP?known as GADD153 or DDIT3?.The IRE1?-XBP1 branch is the most evolutionarily conserved in Eukarya.Upon ER stress,IRE1?is released from Bip and undergoes homo-oligomerization,autophosphorylation,and activation.The activated IRE1?harbors the kinase activity and endoribonuclease activity.The endoribonuclease leads to unconventional enzymatic splicing of XBP1u mRNA into XBP1s mRNA by removing 26 nucleotide introns,and the spliced mRNA is then translated into an active transcription factor XBP1s.XBP1s enters into the nucleus and controls the uphospho-regulation of the UPR related genes,which enhance the ER protein folding capacity and ERAD.IRE1?also degrades ER-associated mRNAs,including ER-localized mRNAs,ribosome RNA and microRNA,named as regulated IRE1?-dependent decay?RIDD?,to reduce protein load in the ER.Activated IRE1?post-transcriptionally stabilizes the mRNA encoding the thioredoxin-interacting protein?TXNIP?by reducing miR-17 in favor of the expression of TXNIP.TXNIP interacts with thioredoxin?Trx?and blocks the interactions of Trx with ASK1.Releasing ASK1 is phosphorylated and activated,inducing apoptosis by activating the pathway of JNK and p38.Activated IRE1?also interacts with TNF receptor-associated factor 2?TRAF2?,an adaptor protein,which recruits apoptosis signal-regulating kinase 1?ASK1?.The complex induces apoptosis by activation of the pro-apoptotic ASK1-c-Jun amino-terminal kinase?JNK?pathway.There are many reports that viruses induce apoptosis mediated by UPR.To explore whether NDV also induces apoptosis by UPR,what is the mechanism of UPR inducing apoptosis,how UPR affects the replication of NDV,we have researches as followed:?1?NDV infection can activate pathways of CHOP and IRE1?.In order to study whether NDV infection can activate CHOP and IRE1?signaling pathways,human cervical cancer cell line HeLa cells were infected by NDV virulent strain Herts/33,and immunofluorescence and Western blot were used to detect their activation.The results showed that the expression of CHOP was significantly elevated in NDV-infected cells at 12-24 h;Meanwhile,CHOP signal was intensified and mainly localized within the nucleus in NDV infected cells;IRE1?was activated by phosphorylation,and its downstream product,XBP1s,was also obviously expressed.XBP1 was diffused in cytoplasm in mocked-infected cells,and partially entered into nucleus during NDV infection.In order to further confirm that CHOP is mediated by the PERK pathway,or the PKR pathway?Detection of CHOP changes after treatment with the PERK/PKR inhibitor GSK2606414,results showed that NDV infection induced CHOP expression by the signaling pathway of PKR-eIF2?.These data suggest that NDV infection can activate signaling pathways of CHOP and IRE1?.?2?The relationship between CHOP signaling pathway,and NDV replication and apoptosis.In order to investigated the effect of CHOP pathway on NDV infection and apoptosis,we use NDV infection to induce CHOP expression,interference CHOP by special siRNA and exogenous CHOP,Western blot results reflected that NDV infection down-regulated expression of BCL-2 and MCL-1,activated AKT and MAPKs signaling pathways;Knock down CHOP greatly reduced the release of virus progeny,increased BCL-2 and MCL-1 expression,decreased PARP cleavage and MAPKs activation;Over expression CHOP could up-regulated expression of viral protein,but the virus yield in culture medium was not obviously increased,inhibited activation of AKT,and expression of BCL-2 and MCL-1,but markedly activated JNK and enhanced the cleavage of PARP.These data indicate that the NDV infection induced CHOP promotes apoptosis via regulation of BCL-2 family proteins,MAPKs signaling,and AKT signaling.In addition to the pro-apoptotic role,CHOP is also essential for NDV proliferation.?3?The relationship between IRE1?-XBP1 signaling pathway,and NDV replication and apoptosis.In order to research whether activated IRE1?has effect on NDV infection and cell apoptosis,we down regulation IRE1?by interference,detection of the effects of siIRE1?on virus replication and apoptosis by Western blot and qRT-PCR.Results indicated that deficiency IRE1?reduced the cleavage of caspase-3 and PARP,obviously decreased the transcription and translation of viral protein NP,and reduced NDV-induced transcription of the ER chaperones and components of ERAD:p58^IPK,ERdj4 and EDEM1 genes.While transfection of IRE1?plasmid resulted in more phospho-IRE1?,increased caspase 3 cleavage and PARP cleavage,and more viral NP protein production.To further investigation whether IRE1?can regulate viral replication and apoptosis via the XBP1 pathway,we transfected IRE1?siRNA into HeLa cells,depletion of XBP1s resulted in decrease of PARP cleavage and NP synthesis.These results suggest that IRE1?is beneficial to viral proliferation and promotes NDV-induced apoptosis,through regulating the transcription and expression of XBP1s and upregulating the transcription of UPR related genes EDEM1,ERdj4 and p58^IPK.?4?The relationship between IRE1?-TXNIP signaling pathway,and NDV replication and apoptosis.To investigate the effect of NDV infection on cell apoptosis mediated by IRE1?-TXNIP signaling pathway,NDV infected HeLa cells,Western blot and IFA detected TXNIP changes.Results demonstrate that NDV infection controls the expression of TXNIP by activating IRE1?.Experimental results of interference TXNIP by special siRNA and exogenous expression of TXNIP indicate that TXNIP is not benefit to virus proliferation.Meanwhile,qRT-PCR detected IFN-?,TNF-?,IL-6 and IL-8,results show that TXNIP down-regulates transcription of cytokines.To sum up,NDV infection regulates apoptosis and virus replication by activating IRE1?-TXNIP signaling pathway.?5?The relationship between NDV infection and JNK signaling pathway;NDV infection could activate JNK by phosphorylation,allowing it to translocate the nucleus.To analysis how to regulate the JNK signaling pathway during NDV infection,we transfected IRE1?siRNA into HeLa cells.si IRE1?decreased the phosphorylation of JNK,as well as the treatment of IIKK16;But overexpression IRE1?enhanced activation of JNK.These data indicate that NDV infection can active JNK by signaling pathways of IRE1?and NF-?B.JNK is an important Serine/threonine-protein kinase,which involved in various physiological processes such as inflammation,cell proliferation,differentiation and programmed cell death.To explore the role of JNK in NDV-induced apoptosis,JNK kinase activity was inhibited by SP600125.Results showed that SP600125 treatment inhibited the phosphorylation of JNK and reduced the cleavage of PARP.Meanwhile,inhibition of JNK kinase activity decreased NDV NP expression.NP mRNA was also decreased by SP600125 treatment.Moreover,the transcription of IFN-?,TNF-?,IL-6and IL-8 mRNA was markedly reduced in SP600125-treated cells.Consistent with these,depletion of JNK by siRNA knock down significantly reduced the cleavage of PARP and viral NP expression.Moreover,NP,TNF-?,IL-6 and IL-8 m RNA was also reduced by JNK knock down Collectively,above results demonstrate that activation of JNK promotes apoptosis and inflammation during NDV infection,also is involved in virus proliferation.In this study,we focused on the ER stress related apoptosis in NDV infected tumor cells,found that virus induced apoptosis by regulation or activation of several UPR stimulated transcription factors:such as CHOP and XBP1s,and apoptotic MAPK signaling.NDV infection can promote apoptosis by inducing CHOP expression and activating the IRE1?-XBP1s/JNK pathway.Moreover,activation of these transcription factors and signaling helped virus proliferation.