Mechanism Of Newcastle Disease Virus P Gene Encoded Proteins Blocking Type â…  Interferon Signalling Pathways

Newcastle disease (ND) is a kind of fulminating and highly contagious infections avain disease which results from Newcastle disease virus (NDV) velogenic strains. The disease is prevalent worldwide and causes severe economic losses in the poultry industry. NDV possesses a negative-sense, single-stranded continuous RNA genome, which contains six genes in the order of3’-NP-P-M-F-HN-L-5’. In common with other paramyxoviruses, NDV produces two additional proteins, V and W, from the P gene by alternative mRNAs that are generated by RNA editing. All three P gene-derived proteins are amino coterminal but vary at their carboxyl terminus in length and amino acid composition. The V protein of NDV is considered as one of the virulent factor, and it was shown that the V protein is responsible for blocking the antiviral action of IFN, as well as oncolytic property of NDV.There are few researches on the specific mechanism of NDV V protien antagonizing IFN signal. It is demonstrated that V protein can block the IFN signal pathway by targeting the signal transducer and activator of transpriction1(STAT1) for proteasome-mediated degradation, whereas another study finds that in V protein overexpressed and NDV infected cells, STAT1can coexist with the V protein, which suggest that other mechanism of IFN signal supression may exist. For further investigating how V protein act as an IFN antagonist, in this study, by using different NDV strains and kinds of cell lines, as well as NDV protein plasmid transfection techniques, we detected the expression and phosphorylation modification level of key cytokines in interferon signal pathways. Subsequently, we identified the virus proteins which may interact with the cytokines, and ultimately, the results were verified by using the mutant ZJ1-Vs lacking V protein. The results of this study provided new insights into the role of P gene encoded protein P/V/W on blocking of type I interferon signalling pathways.1. Construction of expression plasmids and preparation of polyclonal antibodies against P/V/W proteins from different NDV strains.In order to obtain the antibodies which can used to detect the expression of V protein, the N-terminal of phosphoprotein (PNT) and the C-terminal of V protein (CTD) of NDV strain9a5b as well as the CTD domain of NDV strain ZJ1were amplified. Then the V genes were cloned into the prokaryotic vector pET-28a (+) or pCold TF. After the recombinant plasmid was transduced into E.coli BL21cells, the expression of V protein was induced by IPTG The antisera against Class I and Class II NDV V proteins were generated by immunizing BABL/c mice with purified V proteins. Meanwhile, eukaryotic expression plasmid of V and W protein were constructed through overlapping PCR. It was shown that the antibodies worked well and the plasmids were successfully generated. Host differences of V protein expression were identified in NDV infected mammal cells and avian cells. There was more expression of the V protein in avian cells than that in mammal cells. These findings indicate that the expression of NDV V protein is affected by cell types.2. Factor analysis on the differences of NDV V protein-mediated degradation of STAT1.For purpose of verifying the function of STAT1in interferon signal pathways, HeLa or Vero cells were infected with NDV9a5b,La Sota, or ZJ1and transfected with V proteins. The experssion level of relevant proteins were detected by western-blot. Then we compared the expression level of STAT1in NDV infected and V protein transfected cells, or cells under the combined effects, respectively. There was no obvious degradation of STAT1in NDV infected HeLa and Vero cells, and same results were obtained in cells transfected with V proteins. Interestingly, only in cells transfected with V protein and then co-infected with NDV, STAT1degraded apparently, and the degradation degree differs between the different NDV genotypes. These results indicate that the V protein mediated STAT1degradation may be influenced by strain differences, changes in the biological activity of cells after infection, or other viral proteins.3. Effects of P gene encoded proteins on STAT1phosphorylation level and verification of reverse genetic strains According to the above results, we hypothesized that V protein might induce STAT1degradation through other ways indirectly. As we all know, phosphorylation of STAT1is an essential part of the IFN signalling pathways. To verify whether V protein interacted with phosphorylated STAT1, methods including exogenous IFN stimulation, NDV infection and protein transfection and ubiquitin inhibitor treatment were applied. Ultimately, with the verification of the recombinant virus ZJ1-Vs without expression of V protein. It was domenstrated that the V protein of NDV inhibited interferon signalling by targeting STAT1for proteasome-mediated degradation. Furthermore, the viral P and W protein of certain strains also blocked type Ⅰ IFN-induced activation of STATl in transfection experiments. In addition, results of IFN-γ stimulation showed that unlike IFN-a, IFNy-induced activation of STAT1was not blocked by NDV, indicating that NDV can inhibit type Ⅰ interferon signalling whereas has no effect on type Ⅱ interferon system.In this study, the specific mechanism of NDV inhibiting type Ⅰ IFN signalling was elucidated, and for the first time, we discovered that NDV P gene encoded proteins P/V/W had similar functions. All these findings may provide new perspectives and insights into unveiling NDV viral strategies of innate immune evasion.