Newcastle disease virus (NDV) causes severe infectious disease in poultry and selectively kills tumor cells, by inducing apoptosis and cytokines secretion. In this report, we study the mechanisms underlying NDV-induced apoptosis by investigating the unfolded protein response (UPR). We found that NDV infection activated all three branches of the UPR signaling (PERK-eIF2α, ATF6, and IRE1α) and triggered apoptosis, in avian cells (DF-1 and CEF) and in various human cancer cell types (HeLa, Cal27, HN13, A549, H1299, Huh7, and HepG2). Interestingly, the suppression of either apoptosis or UPR led to impaired NDV proliferation. Meanwhile, the inhibition of UPR by 4-PBA protected cells from NDV-induced apoptosis. Further study revealed that activation of PERK-eIF2α induced the expression of transcription factor CHOP, which subsequently promoted apoptosis by downregulating BCL-2/MCL-1, promoting JNK signaling and suppressing AKT signaling. In parallel, IRE1α mediated the splicing of XBP1 mRNA and resulted in the translation and nuclear translocation of XBP1s, thereby promoting the transcription of ER chaperones and components of ER-associated degradation (ERAD). Furthermore, IRE1α promoted apoptosis and cytokines secretion via the activation of JNK signaling. Knock down and overexpression studies showed that CHOP, IRE1α, XBP1, and JNK supported efficient virus proliferation. Our study demonstrates that the induction of eIF2α-CHOP-BCL-2/JNK and IRE1α-XBP1/JNK signaling cascades promote apoptosis and cytokines secretion, and these signaling cascades support NDV proliferation.

IFN regulatory factor (IRF) 3 has been identified as the most critical regulator of both RNA and DNA virus–induced IFN production in mammals. However, ambiguity exists in research on chicken IRFs; in particular IRF3 seems to be missing in chickens, making IFN regulation in chickens unclear. In this study, we comprehensively investigated the potential IFN-related IRFs in chickens and showed that IRF7 is the most critical IFN-β regulator in chickens. With a chicken IRF7 (chIRF7) knockout DF-1 cell line, we conducted a series of experiments to demonstrate that chIRF7 is involved in both chicken STING (chSTING)- and chicken MAVS (chMAVS)-mediated IFN-β regulation in response to DNA and RNA viral infections, respectively. We further examined the mechanisms of chIRF7 activation by chSTING. We found that chicken TBK1 (chTBK1) is indispensable for chIRF7 activation by chSTING as well as that chSTING interacts with both chIRF7 and chTBK1 to function as a scaffold in chIRF7 activation by chTBK1. More interestingly, we discovered that chSTING mediates the activation of chIRF7 through a conserved SLQxSyS motif. In short, we confirmed that although IRF3 is missing in chickens, they employ IRF7 to reconstitute corresponding IFN signaling to respond to both DNA and RNA viral infections. Additionally, we uncovered a mechanism of chIRF7 activation by chSTING. The results will enrich and deepen our understanding of the regulatory mechanisms of the chicken IFN system.

Viral infections result in cellular stress responses, which can trigger protein translation shutoff via phosphorylation of eukaryotic initiation factor 2 alpha (eIF2a). Newcastle disease virus (NDV) causes severe disease in poultry and selectively kills human tumour cells. In this report, we determined that infection of HeLa human cervical cancer cells and DF-1 chicken fibroblast cells with NDV maintained protein at early infection times, 0-12 h post-infection (p.i.), and gradually inhibited global protein translation at late infection times, 12-24 h p.i. Mechanistic studies showed that translation inhibition at late infection times was accompanied by phosphorylation of eIF2a, a checkpoint of translation initiation. Meanwhile, the eIF2a kinase, PKR, was upregulated and activated by phosphorylation and another eIF2a kinase, PERK, was phosphorylated and cleaved into two fragments. Pharmacological inhibition experiments revealed that only PKR activity was required for eIF2a phosphorylation, suggesting that recognition of viral dsRNA by PKR was responsible for translation shutoff. High levels of phospho-eIF2a led to preferential translation of the transcription factor ATF4 and an increase in GADD34 expression. Functionally, GADD34, in conjunction with PP1, dephosphorylated eIF2a and restored protein translation, benefiting virus protein synthesis. However, PP1 was degraded at late infection times, functionally counteracting the upregulation of GADD34. Taken together, our data support that NDV-induced translation shutoff at late infection times was attributed to sustaining phosphorylation of eIF2a, which is mediated by continual activation of PKR and degradation of PP1.

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The stimulator of interferon genes (STING) protein has been shown to play a pivotal role in response to both cytosolic RNA and dsDNA to elicit interferon (IFN) production in mammals. However, the role of duck STING (DuSTING) in antiviral innate immunity, especially in anti-RNA virus infection, has yet to be elucidated. In this study, the function of DuSTING in IFN induction and its role in anti-RNA virus infections were studied. DuSTING was amplified via reverse transcription-polymerase chain reaction (RT-PCR) from Pekin duck, showing that its cDNA sequence contains an open reading frame (ORF) of 1,149 bp and encodes 382 amino acids (aa). Sequence alignment showed that DuSTING protein shares 71.1, 43.4, and 33.3% identity with chickens, humans, and zebra fish, respectively. Overexpression of DuSTING in duck embryo fibroblasts (DEFs) strongly activated IFN-β promotor activity. Deletion mutant analysis revealed that the first 42 aa containing the first transmembrane (TM) domains and the last 32 aa containing a part of the C-terminal tail (CTT) are essential for its IFN-β activation. In vitro experiments showed that the mRNA levels of DuSTING and IFNs were all upregulated when the DEFs were infected with H9N2 avian influenza virus (AIV) SH010, while overexpression of DuSTING inhibited the replication of this virus. In vivo studies showed that DuSTING mRNA was widely expressed in different tissues, and was up-regulated in the spleen and lung of ducks challenged with SH010. In conclusion, our results indicate that DuSTING is an essential IFN mediator and plays a role in anti-RNA virus innate immunity.

Asp-Glu-Ala-Asp (DEAD)-box polypeptide 3 X-linked (DDX3X) is an ATP-dependent RNA helicase, In addition to involvement of eukaryotic gene expression regulation, mammalian DDX3X has recently been found to regulate IFN-β production via the adaptor MAVS mediated cascade signaling. In our studies, we demonstrated that chicken DDX3X (chDDX3X) is also involved in the IFN-β regulation, and demonstrated that chDDX3X regulated IFN-β via an essential adaptor chicken stimulator of IFN genes (chSTING). We found that chDDX3X overexpression in DF-1 cells induced expression of IFN-β and inhibited avian influenza virus (AIV) or Newcastle disease virus (NDV) replication. Knockdown of chDDX3X decreased the production of IFN-β induced by RNA analog polyinosinic-polycytidylic acid and increased viral yield. Furthermore, chDDX3X was identified as a potential chSTING-interacting protein by co-immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). And exogenous Co-IP in transfected cells with or without virus-stimulations further confirmed the interaction between chDDX3X and chSTING. With the gene overexpression and RNA interference studies, the chDDX3X was confirmed to be located upstream of chSTING and activate IFN-β via the chSTING-chTBK1-chIRF7-IFN-β signaling axis. In brief, our results suggest that chDDX3X is an important IFN-β mediator and is involved in RNA- and RNA virus-mediated chDDX3X-chSTING-IFN-β signaling pathway.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and severe hereditary skin disease, caused by mutations in the COL7A1. However, whether non‐invasive prenatal testing (NIPT) can be used for this monogenic genodermatosis remains unknown. Accordingly, we conducted a study in which one couple at high risk of having a fetus with RDEB were recruited and tested by haplotyping‐based NIPT. Next‐generation sequencing‐based multi‐gene panel testing was carried out in this couple and their first child as proband who was affected with RDEB. We deduced parental haplotypes via single nucleotide polymorphism (SNP)‐based haplotype linkage analysis. Then the maternal plasma cell‐free DNA was also sequenced to determine the fetal haplotypes using a parental haplotype‐assisted hidden Markov model (HMM) analysis. Results show that the fetus was only a heterozygous mutation carrier in COL7A1 and the identical results were obtained after birth. These results demonstrate that haplotyping‐based NIPT is a feasible method for NIPT of RDEB.

Newcastle disease virus (NDV) causes severe infectious disease in poultry, and selectively kills tumor cells by inducing apoptosis. In this report, we revealed the mechanisms underlying NDV-induced apoptosis via investigation of endoplasmic reticulum (ER) stress-related unfolded protein response (UPR) in HeLa cells. We found that NDV infection induced the expression of pro-apoptotic transcription factor CHOP via PKR-eIF2α pathway. Knock down and exogenous expression studies showed that CHOP promoted cell apoptosis by down-regulation of anti-apoptotic protein BCL-2 and MCL-1, promotion of pro-apoptotic JNK and p38 signaling, and suppression of pro-survival AKT signaling. Meanwhile, CHOP facilitated NDV proliferation. Furthermore, virus infection activated IRE1α, another ER stress sensor, thereby promoting the mRNA splicing of XBP1 and resulting in the translation of transcription factor XBP1s. XBP1s entered into cell nucleus, promoted the expression of ER chaperones and components of ER associated degradation (ERAD). Exogenous expression of XBP1s helped IBV proliferation, and silence of XBP1s reduced virus proliferation. Meanwhile, exogenous expression and knock down studies demonstrated that IRE1α activated pro-apoptotic JNK signaling, promoted apoptosis and inflammation. In conclusion, our current study demonstrates that the induction of CHOP and activation of IRE1α-XBP1/JNK signaling cascades promote apoptosis and benefit NDV proliferation.IMPORTANCEIt is well known that NDV kills host animal and tumor cells by inducing cell apoptosis. Although several studies investigate the apoptotic phenomena in NDV-infected tumor cells, the molecular mechanisms underlying this oncolytic virus induced apoptosis is not well understood yet. In this study, we focus on characterization of the ER stress responses in NDV-infected tumor cells, and find that virus induces apoptosis by up-regulation or activation of several unfolded protein responses (UPR) related transcription factors and signaling: such as ATF4, CHOP and XBP1s, and pro-apoptotic kinases (IRE1α, JNK, p38). Moreover, activation of these transcription factors and signaling cascades helps virus proliferation. Our study dissects the UPR induced apoptosis in NDV-infected tumor cells, and provides the evidence that UPR favors NDV proliferation.