The Ca2+-dependent phosphatase calcineurin dephosphorylates TBK1 to suppress antiviral innate immunity.

Tumor necrosis factor receptor-associated factor family member-associated NF-κB activator-binding kinase 1 (TBK1) plays a key role in the induction of the type 1 interferon (IFN-I) response, which is an important component of innate antiviral defense. Viruses target calcium (Ca2+) signaling networks, which participate in the regulation of the viral life cycle, as well as mediate the host antiviral response. Although many studies have focused on the role of Ca2+ signaling in the regulation of IFN-I, the relationship between Ca2+ and TBK1 in different infection models requires further elucidation. Here, we examined the effects of the Newcastle disease virus (NDV)-induced increase in intracellular Ca2+ levels on the suppression of host antiviral responses. We demonstrated that intracellular Ca2+ increased significantly during NDV infection, leading to impaired IFN-I production and antiviral immunity through the activation of calcineurin (CaN). Depletion of Ca²+ was found to lead to a significant increase in virus-induced IFN-I production resulting in the inhibition of viral replication. Mechanistically, the accumulation of Ca2+ in response to viral infection increases the phosphatase activity of CaN, which in turn dephosphorylates and inactivates TBK1 in a Ca2+-dependent manner. Furthermore, the inhibition of CaN on viral replication was counteracted in TBK1 knockout cells. Together, our data demonstrate that NDV hijacks Ca2+ signaling networks to negatively regulate innate immunity via the CaN-TBK1 signaling axis. Thus, our findings not only identify the mechanism by which viruses exploit Ca2+ signaling to evade the host antiviral response but also, more importantly, highlight the potential role of Ca2+ homeostasis in the viral innate immune response.IMPORTANCEViral infections disrupt intracellular Ca2+ homeostasis, which affects the regulation of various host processes to create conditions that are conducive for their own proliferation, including the host immune response. The mechanism by which viruses trigger TBK1 activation and IFN-I induction through viral pathogen-associated molecular patterns has been well defined. However, the effects of virus-mediated Ca2+ imbalance on the IFN-I pathway requires further elucidation, especially with respect to TBK1 activation. Herein, we report that NDV infection causes an increase in intracellular free Ca2+ that leads to activation of the serine/threonine phosphatase CaN, which subsequently dephosphorylates TBK1 and negatively regulates IFN-I production. Furthermore, depletion of Ca2+ or inhibition of CaN activity exerts antiviral effects by promoting the production of IFN-I and inhibiting viral replication. Thus, our results reveal the potential role of Ca2+ in the innate immune response to viruses and provide a theoretical reference for the treatment of viral infectious diseases.

Oncolytic Newcastle disease virus induced degradation of YAP through E3 ubiquitin ligase PRKN to exacerbate ferroptosis in tumor cells.

Ferroptosis, a form of programmed cell death characterized by iron-dependent lipid peroxidation, has recently gained considerable attention in the field of cancer therapy. There is significant crosstalk between ferroptosis and several classical signaling pathways, such as the Hippo pathway, which suppresses abnormal growth and is frequently aberrant in tumor tissues. Yes-associated protein 1 (YAP), the core effector molecule of the Hippo pathway, is abnormally expressed and activated in a variety of malignant tumor tissues. We previously proved that the oncolytic Newcastle disease virus (NDV) activated ferroptosis to kill tumor cells. NDV has been used in tumor therapy; however, its oncolytic mechanism is not completely understood. In this study, we demonstrated that NDV exacerbated ferroptosis in tumor cells by inducing ubiquitin-mediated degradation of YAP at Lys90 through E3 ubiquitin ligase parkin (PRKN). Blocking YAP degradation suppressed NDV-induced ferroptosis by suppressing the expression of Zrt/Irt-like protein 14 (ZIP14), a metal ion transporter that regulates iron uptake. These findings demonstrate that NDV exacerbated ferroptosis in tumor cells by inducing YAP degradation. Our study provides new insights into the mechanism of NDV-induced ferroptosis and highlights the critical role that oncolytic viruses play in the treatment of drug-resistant cancers.IMPORTANCEThe oncolytic Newcastle disease virus (NDV) is being developed for use in cancer treatment; however, its oncolytic mechanism is still not completely understood. The Hippo pathway, which is a tumor suppressor pathway, is frequently dysregulated in tumor tissues due to aberrant yes-associated protein 1 (YAP) activation. In this study, we have demonstrated that NDV degrades YAP to induce ferroptosis and promote virus replication in tumor cells. Notably, NDV was found to induce ubiquitin-mediated degradation of YAP at Lys90 through E3 ubiquitin ligase parkin (PRKN). Our study reveals a new mechanism by which NDV induces ferroptosis and provides new insights into NDV as an oncolytic agent for cancer treatment.

Newcastle Disease Virus Manipulates Mitochondrial MTHFD2-Mediated Nucleotide Metabolism for Virus Replication.

Viruses require host cell metabolic reprogramming to satisfy their replication demands; however, the mechanism by which the Newcastle disease virus (NDV) remodels nucleotide metabolism to support self-replication remains unknown. In this study, we demonstrate that NDV relies on the oxidative pentose phosphate pathway (oxPPP) and the folate-mediated one-carbon metabolic pathway to support replication. In concert with [1,2-13C2] glucose metabolic flow, NDV used oxPPP to promote pentose phosphate synthesis and to increase antioxidant NADPH production. Metabolic flux experiments using [2,3,3-2H] serine revealed that NDV increased one-carbon (1C) unit synthesis flux through the mitochondrial 1C pathway. Interestingly, methylenetetrahydrofolate dehydrogenase (MTHFD2) was upregulated as a compensatory mechanism for insufficient serine availability. Unexpectedly, direct knockdown of enzymes in the one-carbon metabolic pathway, except for cytosolic MTHFD1, significantly inhibited NDV replication. Specific complementation rescue experiments on small interfering RNA (siRNA)-mediated knockdown further revealed that only a knockdown of MTHFD2 strongly restrained NDV replication and was rescued by formate and extracellular nucleotides. These findings indicated that NDV replication relies on MTHFD2 to maintain nucleotide availability. Notably, nuclear MTHFD2 expression was increased during NDV infection and could represent a pathway by which NDV steals nucleotides from the nucleus. Collectively, these data reveal that NDV replication is regulated by the c-Myc-mediated 1C metabolic pathway and that the mechanism of nucleotide synthesis for viral replication is regulated by MTHFD2. IMPORTANCE Newcastle disease virus (NDV) is a dominant vector for vaccine and gene therapy that accommodates foreign genes well but can only infect mammalian cells that have undergone cancerous transformation. Understanding the remodeling of nucleotide metabolic pathways in host cells by NDV proliferation provides a new perspective for the precise use of NDV as a vector or in antiviral research. In this study, we demonstrated that NDV replication is strictly dependent on pathways involved in redox homeostasis in the nucleotide synthesis pathway, including the oxPPP and the mitochondrial one-carbon pathway. Further investigation revealed the potential involvement of NDV replication-dependent nucleotide availability in promoting MTHFD2 nuclear localization. Our findings highlight the differential dependence of NDV on enzymes for one-carbon metabolism, and the unique mechanism of action of MTHFD2 in viral replication, thereby providing a novel target for antiviral or oncolytic virus therapy.

Chicken-derived MERTK protein inhibits Newcastle disease virus replication by increasing STAT1 phosphorylation in DF-1 cells.

The receptor tyrosine kinases TYRO3, AXL, and MERTK (TAM) are transmembrane proteins associated with the regulation of the innate immune response. In this study, the role of the chicken-derived MERTK protein (chMertk) in the regulation of the type I interferon (IFN) signaling pathway and its antiviral effect were investigated in vitro. Newcastle disease (ND) caused by the Newcastle disease virus (NDV) is able to widely spread in chickens and give rise to massive losses in the poultry industry around the world. We found that the overexpression of the exogenous chMertk upregulated the STAT1 phosphorylation and the expression of IFN-stimulated gene IFITM3 and significantly reduced the NDV titer (p < 0.05). A mutation assay showed that three tyrosine residues (Y739, Y743, and Y744) in chMertk promoted STAT1 phosphorylation and inhibited NDV replication. However, the chicken-derived E3 ubiquitin ligase CBL significantly negatively regulated chMertk expression, thus attenuating STAT1 phosphorylation. chMertk function was restored by the ubiquitin-proteasome inhibitor MG132, demonstrating that chMertk was controlled by Casitas B-lineage proto-oncogene (CBL) ubiquitination and degradation. Together, these results suggested that chMertk participated in regulating the immune responses to NDV infection, and that CBL significantly downregulated the expression of chMertk through its ubiquitination and degradation, to maintain cellular homeostasis. Overall, our study provided new insights into the role of chMertk in regulating the innate immune response and its anti-NDV activity.

Classification, replication, and transcription of Nidovirales.

Nidovirales is one order of RNA virus, with the largest single-stranded positive sense RNA genome enwrapped with membrane envelope. It comprises four families (Arterividae, Mesoniviridae, Roniviridae, and Coronaviridae) and has been circulating in humans and animals for almost one century, posing great threat to livestock and poultry，as well as to public health. Nidovirales shares similar life cycle: attachment to cell surface, entry, primary translation of replicases, viral RNA replication in cytoplasm, translation of viral proteins, virion assembly, budding, and release. The viral RNA synthesis is the critical step during infection, including genomic RNA (gRNA) replication and subgenomic mRNAs (sg mRNAs) transcription. gRNA replication requires the synthesis of a negative sense full-length RNA intermediate, while the sg mRNAs transcription involves the synthesis of a nested set of negative sense subgenomic intermediates by a discontinuous strategy. This RNA synthesis process is mediated by the viral replication/transcription complex (RTC), which consists of several enzymatic replicases derived from the polyprotein 1a and polyprotein 1ab and several cellular proteins. These replicases and host factors represent the optimal potential therapeutic targets. Hereby, we summarize the Nidovirales classification, associated diseases, "replication organelle," replication and transcription mechanisms, as well as related regulatory factors.

SLC1A3 facilitates Newcastle disease virus replication by regulating glutamine catabolism.

As obligate intracellular parasites, viruses rely completely on host metabolic machinery and hijack host nutrients for viral replication. Newcastle disease virus (NDV) causes acute, highly contagious avian disease and functions as an oncolytic agent. NDV efficiently replicates in both chicken and tumour cells. However, how NDV reprograms host cellular metabolism for its efficient replication is still ill-defined. We previously identified a significantly upregulated glutamate transporter gene, solute carrier family 1 member 3 (SLC1A3), during NDV infection via transcriptome analysis. To investigate the potential role of SLC1A3 during NDV infection, we first confirmed the marked upregulation of SLC1A3 in NDV-infected DF-1 or A549 cells through p53 and NF-κB pathways. Knockdown of SLC1A3 inhibited NDV infection. Western blot analysis further confirmed that glutamine, but not glutamate, asparagine, or aspartate, was required for NDV replication. Metabolic flux data showed that NDV promotes the decomposition of glutamine into the tricarboxylic acid cycle. Importantly, the level of glutamate and glutaminolysis were reduced by SLC1A3 knockdown, indicating that SLC1A3 propelled glutaminolysis for glutamate utilization and NDV replication in host cells. Taken together, our data identify that SLC1A3 serves as an important regulator for glutamine metabolism and is hijacked by NDV for its efficient replication during NDV infection. These results improve our understanding of the interaction between NDV and host cellular metabolism and lay the foundation for further investigation of efficient vaccines.

Newcastle disease virus degrades SIRT3 via PINK1-PRKN-dependent mitophagy to reprogram energy metabolism in infected cells.

Lacking a self-contained metabolism network, viruses have evolved multiple mechanisms for rewiring the metabolic system of their host to hijack the host's metabolic resources for replication. Newcastle disease virus (NDV) is a paramyxovirus, as an oncolytic virus currently being developed for cancer treatment. However, how NDV alters cellular metabolism is still far from fully understood. In this study, we show that NDV infection reprograms cell metabolism by increasing glucose utilization in the glycolytic pathway. Mechanistically, NDV induces mitochondrial damage, elevated mitochondrial reactive oxygen species (mROS) and ETC dysfunction. Infection of cells depletes nucleotide triphosphate levels, resulting in elevated AMP:ATP ratios, AMP-activated protein kinase (AMPK) phosphorylation, and MTOR crosstalk mediated autophagy. In a time-dependent manner, NDV shifts the balance of mitochondrial dynamics from fusion to fission. Subsequently, PINK1-PRKN-dependent mitophagy was activated, forming a ubiquitin chain with MFN2 (mitofusin 2), and molecular receptor SQSTM1/p62 recognized damaged mitochondria. We also found that NDV infection induces NAD+-dependent deacetylase SIRT3 loss via mitophagy to engender HIF1A stabilization, leading to the switch from oxidative phosphorylation (OXPHOS) to aerobic glycolysis. Overall, these studies support a model that NDV modulates host cell metabolism through PINK1-PRKN-dependent mitophagy for degrading SIRT3.Abbreviations: AMPK: AMP-activated protein kinase; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ECAR: extracellular acidification rate; hpi: hours post infection LC-MS: liquid chromatography-mass spectrometry; mito-QC: mCherry-GFP-FIS1[mt101-152]; MFN2: mitofusin 2; MMP: mitochondrial membrane potential; mROS: mitochondrial reactive oxygen species; MOI: multiplicity of infection; 2-NBDG: 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxyglucose; NDV: newcastle disease virus; OCR: oxygen consumption rate; siRNA: small interfering RNA; SIRT3: sirtuin 3; TCA: tricarboxylic acid; TCID50: tissue culture infective doses.

Newcastle-disease-virus-induced ferroptosis through nutrient deprivation and ferritinophagy in tumor cells.

A number of new cell death processes have been discovered in recent years, including ferroptosis, which is characterized by the accumulation of lipid peroxidation products derived from iron metabolism. The evidence suggests that ferroptosis has a tumor-suppressor function. However, the mechanism by which ferroptosis mediates the response of tumor cells to oncolytic viruses remains poorly understood. The Newcastle disease virus (NDV) can selectively replicate in tumor cells. We show that NDV-induced ferroptosis acts through p53-SLC7A11-GPX4 pathway. Meanwhile, the levels of intracellular reactive oxygen species and lipid peroxides increased in tumor cells. Ferritinophagy was induced by NDV promotion of ferroptosis through the release of ferrous iron and an enhanced Fenton reaction. Collectively, these observations demonstrated that the NDV can kill tumor cells through ferroptosis. Our study provides novel insights into the mechanisms of NDV-induced ferroptosis and highlights the critical role of viruses in treating therapy-resistant cancers.

The RNA helicase DDX5 promotes viral infection via regulating N6-methyladenosine levels on the DHX58 and NFκB transcripts to dampen antiviral innate immunity.

Multi-functional DEAD-box helicase 5 (DDX5), which is important in transcriptional regulation, is hijacked by diverse viruses to facilitate viral replication. However, its regulatory effect in antiviral innate immunity remains unclear. We found that DDX5 interacts with the N6-methyladenosine (m6A) writer METTL3 to regulate methylation of mRNA through affecting the m6A writer METTL3-METTL14 heterodimer complex. Meanwhile, DDX5 promoted the m6A modification and nuclear export of transcripts DHX58, p65, and IKKγ by binding conserved UGCUGCAG element in innate response after viral infection. Stable IKKγ and p65 transcripts underwent YTHDF2-dependent mRNA decay, whereas DHX58 translation was promoted, resulting in inhibited antiviral innate response by DDX5 via blocking the p65 pathway and activating the DHX58-TBK1 pathway after infection with RNA virus. Furthermore, we found that DDX5 suppresses antiviral innate immunity in vivo. Our findings reveal that DDX5 serves as a negative regulator of innate immunity by promoting RNA methylation of antiviral transcripts and consequently facilitating viral propagation.

Safety and Efficacy Evaluation of Recombinant Marek's Disease Virus with REV-LTR.

Recently, chickens vaccinated with the CVI988/Rispens vaccine showed increased tumor incidence. Moreover, many strains of Marek's disease virus (MDV) that were naturally integrated with the long terminal repeat (LTR) of the avian reticuloendotheliosis virus (REV) have been isolated, which means it is necessary to develop a new vaccine. In this study, two LTR sequences were inserted into Rispens to construct a recombinant MDV (rMDV). Then, the safety and efficacy of rMDV were evaluated separately in chickens. The growth rate curves showed that the insertion of REV-LTR into MDV enabled a faster replication in vitro than Rispens. Chickens immunized with high or repeated dose rMDV had no MD clinical signs. Further, no tumor, tissue lesions, or evident pathological changes were observed in the chicken organs. Polymerase chain reaction (PCR) and virus isolation revealed that rMDV had the ability to spread horizontally to non-immunized chickens and had no impact on the environment. After five passages in chickens, there were no obvious lesions, and the LTR insertion was stable. There were also no deletions or mutations, which indicates that rMDV is safe in chickens. In addition, rMDV has an advantage over Rispens against vvMDV Md5 at low doses. All results demonstrate that the transgenic strain of rMDV with REV-LTR can be used as a live attenuated vaccine candidate.

Newcastle Disease virus infection activates PI3K/Akt/mTOR and p38 MAPK/Mnk1 pathways to benefit viral mRNA translation via interaction of the viral NP protein and host eIF4E.

Newcastle disease virus (NDV), a member of the Paramyxoviridae family, can activate PKR/eIF2α signaling cascade to shutoff host and facilitate viral mRNA translation during infection, however, the mechanism remains unclear. In this study, we revealed that NDV infection up-regulated host cap-dependent translation machinery by activating PI3K/Akt/mTOR and p38 MAPK/Mnk1 pathways. In addition, NDV infection induced p38 MAPK/Mnk1 signaling participated 4E-BP1 hyperphosphorylation for efficient viral protein synthesis when mTOR signaling is inhibited. Furthermore, NDV NP protein was found to be important for selective cap-dependent translation of viral mRNAs through binding to eIF4E during NDV infection. Taken together, NDV infection activated multiple signaling pathways for selective viral protein synthesis in infected cells, via interaction between viral NP protein and host translation machinery. Our results may help to design novel targets for therapeutic intervention against NDV infection and to understand the NDV anti-oncolytic mechanism.

ATM-mediated DNA double-strand break response facilitated oncolytic Newcastle disease virus replication and promoted syncytium formation in tumor cells.

Deoxyribonucleic acid (DNA) damage response (DDR) is the fundamental cellular response for maintaining genomic integrity and suppressing tumorigenesis. The activation of ataxia telangiectasia-mutated (ATM) kinase is central to DNA double-strand break (DSB) for maintaining host-genome integrity in mammalian cells. Oncolytic Newcastle disease virus (NDV) can selectively replicate in tumor cells; however, its influence on the genome integrity of tumor cells is not well-elucidated. Here, we found that membrane fusion and NDV infection triggered DSBs in tumor cells. The late replication and membrane fusion of NDV mechanistically activated the ATM-mediated DSB pathway via the ATM-Chk2 axis, as evidenced by the hallmarks of DSBs, i.e., auto-phosphorylated ATM and phosphorylated H2AX and Chk2. Immunofluorescence data showed that multifaceted ATM-controlled phosphorylation markedly induced the formation of pan-nuclear punctum foci in response to NDV infection and F-HN co-expression. Specific drug-inhibitory experiments on ATM kinase activity further suggested that ATM-mediated DSBs facilitated NDV replication and membrane fusion. We confirmed that the Mre11-RAD50-NBS1 (MRN) complex sensed the DSB signal activation triggered by NDV infection and membrane fusion. The pharmacological inhibition of MRN activity also significantly inhibited intracellular and extracellular NDV replication and syncytia formation. Collectively, these data identified for the first time a direct link between the membrane fusion induced by virus infection and DDR pathways, thereby providing new insights into the efficient replication of oncolytic NDV in tumor cells.

Characterization and functional analysis of chicken APOBEC4.

The APOBEC proteins play significant roles in the innate and adaptive immune system, probably due to their deaminase activities. Because APOBEC1 (A1) and APOBEC3 (A3) are absent in the chicken genome, we were interested in determining whether chicken APOBEC4 (A4) possessed more complex functions than its mammalian homologs. In this study, chicken A4 (chA4) mRNA was identified and cloned for the first time. Based on bioinformatics analyses, the conserved zinc-coordinating motif (HXE … PC(X)2-6C) was identified on the surface of chA4 and contained highly conserved His97, Glu99, Pro130, Cys131 and Cys138 active sites. The highest expression levels of constitutive chA4 were detected in primary lymphocytes and bursa of Fabricius. Newcastle Disease (ND) is one of the most serious infectious diseases in birds, causing major economic losses to the poultry industry. In vitro, Newcastle Disease Virus (NDV) early infection induced significant increases in chA4 expression in the chicken B cell line, DT40, the macrophage cell line, HD11 and the CD4+ T cell line, MSB-1, but not the fibroblast cell line, DF-1. In vivo, the expression levels of chA4 were up-regulated in several tissues from NDV-infected chickens, especially the thymus, testicles, duodenum and kidney. The high level expression of exogenous chA4 displayed inhibitory effects on NDV and reduced viral RNA in infected cells. Taken together, these data demonstrate that chA4 is involved in the chicken immune system and may play important roles in host anti-viral responses.

eIF2α-CHOP-BCl-2/JNK and IRE1α-XBP1/JNK signaling promote apoptosis and inflammation and support the proliferation of Newcastle disease virus.

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.

Newcastle Disease Virus V Protein Degrades Mitochondrial Antiviral Signaling Protein To Inhibit Host Type I Interferon Production via E3 Ubiquitin Ligase RNF5.

Paramyxovirus establishes an intimate and complex interaction with the host cell to counteract the antiviral responses elicited by the cell. Of the various pattern recognition receptors in the host, the cytosolic RNA helicases interact with viral RNA to activate the mitochondrial antiviral signaling protein (MAVS) and subsequent cellular interferon (IFN) response. On the other hand, viruses explore multiple strategies to resist host immunity. In this study, we found that Newcastle disease virus (NDV) infection induced MAVS degradation. Further analysis showed that NDV V protein degraded MAVS through the ubiquitin-proteasome pathway to inhibit IFN-ß production. Moreover, NDV V protein led to proteasomal degradation of MAVS through Lys362 and Lys461 ubiquitin to prevent IFN production. Further studies showed that NDV V protein recruited E3 ubiquitin ligase RNF5 to polyubiquitinate and degrade MAVS. Compared with levels for wild-type NDV infection, V-deficient NDV induced attenuated MAVS degradation and enhanced IFN-ß production at the late stage of infection. Several other paramyxovirus V proteins showed activities of degrading MAVS and blocking IFN production similar to those of NDV V protein. The present study revealed a novel role of NDV V protein in targeting MAVS to inhibit cellular IFN production, which reinforces the fact that the virus orchestrates the cellular antiviral response to its own benefit.IMPORTANCE Host anti-RNA virus innate immunity relies mainly on the recognition by retinoic acid-inducible gene I and melanoma differentiation-associated protein 5 and subsequently initiates downstream signaling through interaction with MAVS. On the other hand, viruses have developed various strategies to counteract MAVS-mediated signaling. The mechanism for paramyxoviruses regulating MAVS to benefit their infection remains unknown. In this article, we demonstrate that the V proteins of NDV and several other paramyxoviruses target MAVS for ubiquitin-mediated degradation through E3 ubiquitin ligase RING-finger protein 5 (RNF5). MAVS degradation leads to the inhibition of the downstream IFN-ß pathway and therefore benefits virus proliferation. Our study reveals a novel mechanism of NDV evading host innate immunity and provides insight into the therapeutic strategies for the control of paramyxovirus infection.

Exosomes Carry microRNAs into Neighboring Cells to Promote Diffusive Infection of Newcastle Disease Virus.

Newcastle disease virus (NDV), an avian paramyxovirus, was shown to prefer to replicate in tumor cells instead of normal cells; however, this mechanism has not been fully elucidated. Exosomes play a crucial role in intercellular communication due to the bioactive substances they carry. Several studies have shown that exosomes are involved in virus infections. However, the effect that exosomes have on NDV-infected tumor cells is not known. In this study, we focus on the role of exosomes secreted by NDV-infected HeLa cells in promoting NDV replication. Three miRNA candidates (miR-1273f, miR-1184, and miR-198) embraced by exosomes were associated with enhancing NDV-induced cytopathic effects on HeLa cells. Furthermore, luciferase assays, RT-qPCR, and enzyme-linked immunosorbent assay (ELISA) all demonstrated that these miRNAs could suppress interferon (IFN)-ß gene expression. Enhanced NDV replication in HeLa cells was identified by Western blot and plaque assays. Based on these results, we speculate that NDV employed exosomes entry into neighboring cells, which carry miRNAs, resulting in inhibition of the IFN pathway and promotion of viral infection. To our knowledge, this is the first report on the involvement of NDV-employed exosomes in tumor cells, and as such, it provides new insights into the development of anti-tumor therapies.

Production, characterization, and epitope mapping of a monoclonal antibody against genotype VII Newcastle disease virus V protein.

Newcastle disease virus (NDV) V protein is crucial for viral interferon (IFN) antagonism and virulence, determining its host range restriction. However, little information is available on the B cell epitopes of V protein and the subcellular movement of V protein in the process of NDV infection. In this study, the monoclonal antibody (mAb) clone 3D7 against genotype VII NDV V protein was generated by immunizing mice with a purified recombinant His-tagged carboxyl-terminal domain (CTD) region of V protein. Fine epitope mapping analysis and B-cell epitope prediction indicated that mAb 3D7 recognized a linear epitope 152RGPAELWK159, which is located in the V protein CTD region. Sequence alignment showed that the mAb clone 3D7-recognized epitope is highly conserved among Class II genotype VII NDV strains, but not among other genotypes, suggesting it could serve as a genetic marker to differentiate NDV genotypes. Furthermore, the movement of V protein during NDV replication in infected cells were determined by using this mAb. It was found that V protein localized around the nucleus during virus replication. The establishment of V protein-specific mAb and identification of its epitope extend our understanding of the antigenic characteristics of V protein and provide a basis for the development of epitope-based diagnostic assays.

Recombinant oncolytic Newcastle disease virus displays antitumor activities in anaplastic thyroid cancer cells.

BACKGROUND: Anaplastic thyroid cancer (ATC) is one of the most aggressive of all solid tumors for which no effective therapies are currently available. Oncolytic Newcastle disease virus (NDV) has shown the potential to induce oncolytic cell death in a variety of cancer cells of diverse origins. However, whether oncolytic NDV displays antitumor effects in ATC remains to be investigated. We have previously shown that the oncolytic NDV strain FMW (NDV/FMW) induces oncolytic cell death in several cancer types. In the present study, we investigated the oncolytic effects of NDV/FMW in ATC. METHODS: In this study, a recombinant NDV expressing green fluorescent protein (GFP) was generated using an NDV reverse genetics system. The resulting virus was named after rFMW/GFP and the GFP expression in infected cells was demonstrated by direct fluorescence and immunoblotting. Viral replication was evaluated by end-point dilution assay in DF-1 cell lines. Oncolytic effects were examined by biochemical and morphological experiments in cultural ATC cells and in mouse models. RESULTS: rFMW/GFP replicated robustly in ATC cells as did its parent virus (NDV/FMW) while the expression of GFP protein was detected in lungs and spleen of mice intravenously injected with rFMW/GFP. We further showed that rFMW/GFP infection substantially increased early and late apoptosis in the ATC cell lines, THJ-16 T and THJ-29 T and increased caspase-3 processing and Poly (ADP-ribose) polymerase (PARP) cleavage in ATC cells as assessed by immunoblotting. In addition, rFMW/GFP induced lyses of spheroids derived from ATC cells in three-dimensional (3D) cultures. We further demonstrated that rFMW/GFP infection resulted in the activation of p38 MAPK signaling, but not Erk1/2 or JNK, in THJ-16 T and THJ-29 T cells. Notably, inhibition of p38 MAPK activity by SB203580 decreased rFMW/GFP-induced cleavage of caspase-3 and PARP in THJ-16 T and THJ-29 T cells. Finally, both rFMW/GFP and its parent virus inhibited tumor growth in mice bearing THJ-16 T derived tumors. CONCLUSION: Taken together, these data indicate that both the recombinant reporter virus rFMW/GFP and its parent virus NDV/FMW, display oncolytic activities in ATC cells in vitro and in vivo and suggest that oncolytic NDV may have potential as a novel therapeutic strategy for ATC.

Newcastle disease virus induces G0/G1 cell cycle arrest in asynchronously growing cells.

The cell cycle, as a basic cellular process, is conservatively regulated. Consequently, subversion of the host cell replication cycle is a common strategy employed by many viruses to create a cellular environment favorable for viral replication. Newcastle disease virus (NDV) causes disease in poultry and is also an effective oncolytic agent. However, the effects of NDV infection on cell cycle progression are unknown. In this study, we showed that NDV replication in asynchronized cells resulted in the accumulation of infected cells in the G0/G1 phase of the cell cycle, which benefitted the proliferation of NDV. Examination of various cell cycle-regulatory proteins showed that expression of cyclin D1, was significantly reduced following NDV infection. Importantly, the decreased expression of cyclin D1 was reversed by inhibition of CHOP expression, indicating that induction of the PERK-eIF-2a-ATF4-CHOP signaling pathway was involved in the G0/G1 phase cell cycle arrest observed following NDV infection.

Vitamin E Supplementation Ameliorates Newcastle Disease Virus-Induced Oxidative Stress and Alleviates Tissue Damage in the Brains of Chickens.

Newcastle disease (ND), characterized by visceral, respiratory, and neurological pathologies, causes heavy economic loss in the poultry industry around the globe. While significant advances have been made in effective diagnosis and vaccine development, molecular mechanisms of ND virus (NDV)-induced neuropathologies remain elusive. In this study, we report the magnitude of oxidative stress and histopathological changes induced by the virulent NDV (ZJ1 strain) and assess the impact of vitamin E in alleviating these pathologies. Comparative profiling of plasma and brains from mock and NDV-infected chicken demonstrated alterations in several oxidative stress makers such as nitric oxide, glutathione, malondialdehyde, total antioxidant capacity, glutathione S-transferase, superoxide dismutase, and catalases. While decreased levels of glutathione and total antioxidant capacity and increased concentrations of malondialdehyde and nitric oxide were observed in NDV-challenged birds at all time points, these alterations were eminent at latter time points (5 days post infection). Additionally, significant decreases in the activities of glutathione S-transferase, superoxide dismutase, and catalase were observed in the plasma and brains collected from NDV-infected chickens. Intriguingly, we observed that supplementation of vitamin E can significantly reduce the alteration of oxidative stress parameters. Under NDV infection, extensive histopathological alterations were observed in chicken brain including neural inflammation, capillary hyperemia, necrosis, and loss of prominent axons, which were reduced with the treatment of vitamin E. Taken together, our findings highlight that neurotropic NDV induces extensive tissue damage in the brain and alters plasma oxidative stress profiles. These findings also demonstrate that supplementing vitamin E ameliorates these pathologies in chickens and proposes its supplementation for NDV-induced stresses.