Yuan Zhan

Autophagy Benefits the Replication of Newcastle Disease Virus in Chicken Cells and Tissues

ABSTRACT Newcastle disease virus (NDV) is an important avian pathogen. We previously reported that NDV triggers autophagy in U251 glioma cells, resulting in enhanced virus replication. In this study, we investigated whether NDV triggers autophagy in chicken cells and tissues to enhance virus replication. We demonstrated that NDV infection induced steady-state autophagy in chicken-derived DF-1 cells and in primary chicken embryo fibroblast (CEF) cells, evident through increased double- or single-membrane vesicles, the accumulation of green fluorescent protein (GFP)-LC3 dots, and the conversion of LC3-I to LC3-II. In addition, we measured autophagic flux by monitoring p62/SQSTM1 degradation, LC3-II turnover, and GFP-LC3 lysosomal delivery and proteolysis, to confirm that NDV infection induced the complete autophagic process. Inhibition of autophagy by pharmacological inhibitors and RNA interference reduced virus replication, indicating an important role for autophagy in NDV infection. Furthermore, we conducted in vivo experiments and observed the conversion of LC3-I to LC3-II in heart, liver, spleen, lung, and kidney of NDV-infected chickens. Regulation of the induction of autophagy with wortmannin, chloroquine, or starvation treatment affects NDV production and pathogenesis in tissues of both lung and intestine; however, treatment with rapamycin, an autophagy inducer of mammalian cells, showed no detectable changes in chicken cells and tissues. Moreover, administration of the autophagy inhibitor wortmannin increased the survival rate of NDV-infected chickens. Our studies provide strong evidence that NDV infection induces autophagy which benefits NDV replication in chicken cells and tissues.

Activation of the PKR/eIF2α signaling cascade inhibits replication of Newcastle disease virus

BackgroundNewcastle Disease virus (NDV) causes severe and economically significant disease in almost all birds. However, factors that affect NDV replication in host cells are poorly understood. NDV generates long double-stranded RNA (dsRNA) molecules during transcription of single-stranded genomic RNA. Protein kinase R (PKR) is activated by dsRNA. The aim of this study was to elucidate the role of PKR in NDV infection.ResultsNDV infection led to the activation of dsRNA-dependent PKR and phosphorylation of its substrate, translation initiation factor eIF2α, in a dose-dependent manner by either the lentogenic strain LaSota or a velogenic strain Herts/33. PKR activation coincided with the accumulation of dsRNA induced by NDV infection. PKR knockdown remarkably decreased eIF2α phosphorylation as well as IFN-β mRNA levels, leading to the augmentation of extracellular virus titer. Furthermore, siRNA knockdown or phosphorylation of eIF2α or okadaic acid treatment significantly impaired NDV replication, indicating the critical role of the PKR/eIF2α signaling cascade in NDV infection.ConclusionPKR is activated by dsRNA generated by NDV infection and inhibits NDV replication by eIF2α phosphorylation. This study provides insight into NDV-host interactions for the development of candidate antiviral strategies.

Rescue of virulent class I Newcastle disease virus variant 9a5b-D5C1

BackgroundThe virulent class I Newcastle disease virus (NDV) variant 9a5b was generated from a nonvirulent NDV isolate Goose/Alaska/415/91 via nine consecutive passages in the chicken air sac, followed by five passages in the chick brain. The evolutionary mechanism of virulence in the class I NDV isolate is not fully understood. To elucidate this evolutionary mechanism, a reverse genetics manipulation specific for class I NDV is indispensable.ResultsA full-length cDNA clone of 9a5b and the helper plasmids pCI-NP, pCI-P, and pCI-L were constructed from segments of cDNA. After these plasmids were co-transfected into BSR T7/5 cells, infectious viral particles were obtained. The rescued viruses were genetically and biologically identical to the parental strain and showed similar pathogenicity in chickens.ConclusionA stable recovery method for class I NDV was established. Reverse genetics of the class I NDV variant 9a5b allowed for the generation of genetically altered and virulent NDV, and can be used as a foundation for research on the evolution of virulence in class I NDV isolates.

Newcastle disease virus induces stable formation of bona fide stress granules to facilitate viral replication through manipulating host protein translation.

Mammalian cells respond to various environmental stressors to form stress granules (SGs) by arresting cyto‐plasmic mRNA, protein translation element, and RNA binding proteins. Virus‐induced SGs function in different ways, depending on the species of virus; however, the mechanism of SG regulation of virus replication is not well understood. In this study, Newcastle disease virus (NDV) triggered stable formation of bona fide SGs on HeLa cells through activating the protein kinase R (PKR)/eIF2α pathway. NDV‐induced SGs contained classic SG markers T‐cell internal antigen (TIA)‐1, Ras GTPase‐activating protein‐binding protein (G3BP)‐1, eukaryotic initiation factors, and small ribosomal subunit, which could be disassembled in the presence of cycloheximide. Treatment with nocodazole, a microtubule disruption drug, led to the formation of relatively small and circular granules, indicating that NDV infection induces canonical SGs. Furthermore, the role of SGs on NDV replication was investigated by knockdown of TIA‐1 and TIA‐1‐related (TIAR) protein, the 2 critical components involved in SG formation from the HeLa cells, followed by NDV infection. Results showed that depletion of TIA‐1 or TIAR inhibited viral protein synthesis, reduced extracellular virus yields, but increased global protein translation. FISH revealed that NDV‐induced SGs contained predominantly cellular mRNA rather than viral mRNA. Deletion of TIA‐1 or TIAR reduced NP mRNA levels in polysomes. These results demonstrate that NDV triggers stable formation of bona fide SGs, which benefit viral protein translation and virus replication by arresting cellular mRNA. —Sun, Y., Dong, L., Yu, S., Wang, X., Zheng, H., Zhang, P., Meng, C., Zhan, Y., Tan, L., Song, C., Qiu, X., Wang, G., Liao, Y., Ding, C. Newcastle disease virus induces stable formation of bona fide stress granules to facilitate viral replication through manipulating host protein translation. FASEB J. 31, 1337–1353 (2017) www.fasebj.org

Newcastle Disease Virus V Protein Targets Phosphorylated STAT1 to Block IFN-I Signaling.

Newcastle disease virus (NDV) V protein is considered as an effector for IFN antagonism, however, the mechanism remains unknown. In this study, the expression of STAT1 and phospho-STAT1 in cells infected with NDV or transfected with V protein-expressing plasmids were analyzed. Our results showed that NDV V protein targets phospho-STAT1 reduction in the cells depends on the stimulation of IFN-α. In addition, a V-deficient genotype VII recombinant NDV strain rZJ1-VS was constructed using reverse genetic technique to confirm the results. The rZJ1-VS lost the ability to reduce phospho-STAT1 and induced higher expression of IFN-responsive genes in infected cells. Furthermore, treatment with an ubiquitin E1 inhibitor PYR-41 demonstrated that phospho-STAT1 reduction was caused by degradation, but not de-phosphorylation. We conclude that NDV V protein targets phospho-STAT1 degradation to block IFN-α signaling, which adds novel knowledge to the strategies used by paramyxoviruses to evade IFN.

Newcastle disease virus employs macropinocytosis and Rab5a-dependent intracellular trafficking to infect DF-1 cells

Oncolytic Newcastle disease virus (NDV) reportedly employs direct fusion of the viral envelope with the plasma membrane and caveolae-dependent endocytosis to enter cells. Here, we show that macropinocytosis and clathrin-mediated endocytosis are involved in NDV entry into a galline embryonic fibroblast cell line. Upon specific inhibition of clathrin assembly, GTPase dynamin, Na+/H+ exchangers, Ras-related C3 botulinum toxin substrate 1, p21 activated kinase 1 or protein kinase C, entry of NDV and its propagation were suppressed. NDV entry into cells triggers Rac1-Pak1 signaling and elicits actin rearrangement and plasma membrane ruffling. Moreover, NDV internalization within macropinosomes and trafficking involve Rab5a-positive vesicles. This is the first report demonstrating that NDV utilizes clathrin-mediated endocytosis and macropinocytosis as alternative endocytic pathways to enter cells. These findings shed new light on the molecular mechanisms underlying NDV entry into cells, and provide potential targets for NDV-mediated therapy in cancer.

Identification and functional analysis of phosphorylation in Newcastle disease virus phosphoprotein

Newcastle disease virus (NDV) encodes a highly phosphorylated P protein; however, the phosphorylation sites have not been identified, and the relationship between phosphorylation and protein function is still unclear. In this study, we bioinformatically predicted 26 amino acid residues in the P protein as potential phosphorylation sites. Furthermore, we treated infected cells with kinase inhibitors to investigate NDV propagation and found that protein kinase C (PKC) is involved in the NDV life cycle and that PKC-activated phosphorylation functions in NDV replication. Using an NDV minigenome assay, we found that expression of a reporter protein decreased when the minigenome system contained P mutants lacking T44, S48, T271, S373 and especially T111. The phosphorylation status of S48, T111, S125 and T271 was determined by Phos-tag SDS-PAGE analysis. Coimmunoprecipitation assays showed that the binding activity of NP and the P-T111A mutant was stronger than that of NP and the wild-type P, suggesting that P-T111 is involved in NP-P interaction. This study sheds light on the mechanism by which P protein phosphorylation affects NDV replication and transcription.

Infectious bronchitis virus poly-epitope-based vaccine protects chickens from acute infection.

Infectious bronchitis virus (IBV) causes major losses in the poultry industry. The safe and effective vaccine to control IBV spread is imperative. In the present study, we developed IBV S1 glycoprotein poly-epitope-based DNA vaccine pV-S1B+S1T consisting of SH1208 and Holte strain BF2-restricted T cell epitopes and Australian T strain dominant B cell neutralization epitopes. Specific pathogen-free chickens were vaccinated with pV-S1B+S1T and control plasmids twice to elicit strong humoral and cellular immune response, as indicated by viral neutralization titers and results of CD8+ T cell proliferation assays. A lethal dose of IBV SH1208 strain used for protection and challenge experiments at two weeks post-booster immunization following challenge protection and virus shedding reverse transcription quantitative PCR assay, indicated that pV-S1B+S1T protected against IBV and significantly reduced viral excretion. These results demonstrated that the IBV poly-epitope-based vaccine effectively prevents infection and represents a potential IBV vaccine.

Newcastle disease virus infection induces activation of the NLRP3 inflammasome.

Inflammatory responses are important aspects of the innate immune system during virus infection. We found that Newcastle disease virus can induce inflammasome activation in the human macrophage-like cell line THP-1. Viral replication was required for inflammasome activation, and small hairpin RNA knockdown experiments indicated that IL-1β secretion was mediated by the NLRP3 inflammasome. We also verified the results in LPS-primed bone marrow-derived macrophages (BMDMs) from NLRP3-deficient and wild type mice. NDV is considered to be a promising oncolytic virus. Stimulating the immune system has been proposed as a key mechanism of oncolytic specificity, and the inflammasome appears to be an important mechanism by which NDV is controlled. Knockdown of inflammasome components or chemical inhibition of caspase-1 activity shows that cell survival was augmented and benefited NDV replication. This study shows that NLRP3 inflammasome activation is an innate cellular response to NDV infection and offers insights into the oncolytic specificity of NDV.

The LXR ligand GW3965 inhibits Newcastle disease virus infection by affecting cholesterol homeostasis

Newcastle disease (ND) is a contagious disease that affects most species of birds. Its causative pathogen, Newcastle disease virus (NDV), also exhibits considerable oncolytic activity against mammalian cancers. A better understanding of the pathogenesis of NDV will help us design efficient vaccines and novel anticancer strategies. GW3965, a widely used synthetic ligand of liver X receptor (LXR), induces the expression of LXRs and its downstream genes, including ATP-binding cassette transporter A1 (ABCA1). ABCA1 regulates cellular cholesterol homeostasis. Here, we found that GW3965 inhibited NDV infection in DF-1 cells. It also inhibited NF-κB activation and reduced the upregulation of proinflammatory cytokines induced by the infection. Further studies showed that GW3965 exerted its inhibitory effects on virus entry and replication. NDV infection increased the mRNA levels of several lipogenic genes but decreased the ABCA1 mRNA level. Overexpression of ABCA1 inhibited NDV infection and reduced the cholesterol content in DF-1 cells, but when the cholesterol was replenished, NDV infection was restored. GW3965 treatment prevented cholesterol accumulation in the perinuclear area of the infected cells. In summary, our studies suggest that GW3965 inhibits NDV infection, probably by affecting cholesterol homeostasis.