The Role Of V Protein On Newcastle Disease Virus-mediated Type I Interferon Sigaling Antagonism

Newcastle disease(ND), an acute, deadly, septicaemic and highly contagious infectious avian disease, is caused by virulent Newcastle disease virus(NDV), also named as avian paramyxovirus type I(APMV-1), belonging to the genus of Avulavirus, Paramyxovirinae, Paramyxoviridae, Mononegavirales. The Viral genome of NDV is composed of a single negative stranded RNA, encoding nucleocapsid protein(NP), phosphoprotein(P), matrix protein(M), fusion protein(F), haemagglutinin-Neuraminidase protein(HN) and large protein(L) in the order of 3’→5’. In addition, two extra proteins V and W protein were encoded from P gene via the mechanism of "RNA editing ". In the process of P gene transcription, one or two guanines(G) were inserted in the specific position of some of the P gene transcripts, and introduced P protein open reading frame +1 or +2 shift. The P/V/W proteins share a same amino terminal portion and differ at carboxyl portion. P protein is a crucial subunit for NDV RNA-dependent RNA polymerase(RdRp); and P protein play an important role in NP protein wrapping genomic RNA. The structure of the C terminal domains of V protein are similar in all paramyxoviruses, containing a zinc finger motif composed of 7 cysteine residues with two zinc atoms. It is reported that NDV V protein degradated STAT1 protein to antagonize IFN signaling pathway; however, recent reported revel that V protein can coexist with STAT1 protein in certain cell lines, suggesting V protein may not mediate STAT1 protein immediately and directly. The detailed mechanism of V protein antagonising IFN is still unclear. In order to explore the mechanism of V protein mediated IFN signalling antagonism, we perfromed a series of experiments.1. Preparation of monoclonal antibody against P/V/W proteinsIn order to detect the expression of P/V/W protein in cells transfected with P/V/W plasmids or infected with NDV, the monoclonal antibody against P/V/W protein was prepared firstly. The V protein open reading frame(ORF) was inserted into the pET28 a vector, and the V protein was expressed in BL21(DE3) at 37℃ with 1mM IPTG for 18 h. Prepared V protein was taken as immunogen and used for 4 times immunizations of 6 week-old BALB/c mice every 3 weeks. After cell fusion, the supernatant of hybridoma cells in each well were detected in ELISA assay, coating with LaSota viruses or prepared V proteins. At last, 5 hybridoma cell lines against P/V/W proteins were screened out, which displayed high reactivity in IFA assay, WB test and ELISA with all genotypes of NDV, they were named 2F4, 3D5, 4A8, 5C9 and 4E35, respectively. This study provided an important reference for the development of rapid broad-sprectrum NDV detection technology.2. Dosage-dependent effect IFN on NDV V protein-mediated IFN antagonismIt is reported that NDV V protein degraded STAT1 protein to block IFN signaling. However, latest reports revealed that V protein did not lead to STAT1 decrease in various cells, such as DF1. In previous study of our lab, we have proved that NDV V protein did not give rise to the STAT1 degradation in V-expressing A549, HeLa and Vero cells in WB assay. In order to figure out function discrepancies of V protein in different cell lines, we performed further studies. Firstly, we confirmed the co-existence of V protein and STAT1 protein in a cell with IFA assay. Further IFA and Western Blot assay displayed that V protein targeted the phosphorylated form of STAT1 protein and IFN stimulation played a key role in this process. When the dosage of IFN and MOI of NDV were compared, the dosage-related effect of V protein mediated STAT1 degradation was determined. In the mechanism involved, we detected the expression of IFN-I and downstream related genes by using ELISA kit and Real-time PCR. The results showed that V proteins indeed effectively down-regulate the expression of IFN-I, IFIT1, OAS1, ISG15 and so on, even though NDV infection will inevitably induce high level of IFN-I and downstream related genes. In sum, NDV V protein mediated the downregulation of pSTAT1 to block IFN-I signaling, which was a IFN dosage-dependent relationship. These findings increased our understanding about strategies of paramyxovirus immune escape.3. NDV V protein targeted phosphorylated STAT1 for degradation to block IFN-I signalingIn above-mentioned study, we have found that NDV V protein mediated the down-regulation of pSTAT1 to block IFN-I signaling. To explore related mechanism, ubiquitin inhibitors assays were performed, which revealed that V protein-mediated pSTAT1 decrease was blocked when the cellular ubiquitin proteasome system was inhibited by the ubiquitin E1 inhibitor. In following study, two eukaryotic expression plasmids were constructed, expressing wild-type STAT1 protein or deficient STAT1, which has a Y/F mutation at 701 phosphorylation site. The NDV infection mediated the decrease of total STAT1 but not STAT1-Y701 F, suggestion pSTAT1 was degraded rather than dephosphorylated in this process. To explore detailed mechanism, plasmids expressing STAT2, STAT1, ROC1, DDB1 and so on were constructed for IP and Co-IP assays. We found direct interaction of V protein with STAT2 protein but not STAT1, ROC1 or DDB1, suggesting the mechanism of NDV V mediated STAT1 degradation may be quite different from those found in Simian Virus 5. After capturing cellular protein with V protein in IP test, mass spectrum was performed and identified various proteins, which may play a role in V-STAT1 reaction. This provided a new way for the researches on virus immune escape, and was contributive to the studies on the species-specific pathogenesis of RNA virus.