Study On The Functional Domain Of Newcastle Disease Virus V Protein Binding MDA5 To Inhibit Interferon β Production In DF-1 Cells

Newcastle disease(ND), which is caused by Newcastle disease virus(NDV), is a serious infectious viral disease in avian species. NDV belongs to the genus Avulavirus in family Paramyxoviridae. It possesses a negative-sense, single-stranded RNA genome with approximately 15 kb in length. With the infection and host range of NDV expanding constantly in recent years, new challenges for the prevention and control of ND ariose. Interferon plays a significant role in defensing host against virus infection. Many recent progresses have demonstrated that V protein, encoded by most paramyxoviruses can be acted as an interferon antagonist in viral immune escape, no exception with NDV. Its V protein is expressed by RNA-editing event during transcription of the P gene. Studies showed that NDV V protein not only bound the pattern recognition receptors MDA5 to inhibit interferon β production directly, but also blocked the JAK / STAT signaling pathway of type I interferon. However, the MDA5-binding domain of V protein is only located on the cysteine-rich region of the C-terminal. It is unclear whether each cysteine residue or other conserved residues is required for V protein binding to MDA5. Somer studies indicated that the N-terminal of V protein might play a role in the V protein function.In order to illustrate the specific MDA5-binding functional domains of NDV V protein, on the basis of analysis of the C-terminal domain of Paramyxoviruses V proteins, single or multiple residues of NDV V protein were mutated in eukaryotic expression vector. Using reporter gene assay, real time PCR, ELISA and Co-immunoprecipitation methods, the expression levels of interferon β were investigated in DF-1 cells at post co-transfection with wide-type or mutants of V and MDA5. This study containing three different parts is presented in the following.1. Effects of amino acids of zinc finger structure of C-terminal domain on NDV V protein interacting with MDA5In order to investigate the role of the histidine and seven cysteine residues in NDV V protein C-terminal interacting with MDA5, schematic diagram of NDV V protein was modeled by using parainfluenza virus type 5(PIV5) V protein as a reference. Eight amino acids were selected to construct V protein mutations in eukaryotic expression vector. Reporter gene assay, ELISA and real time PCR were applied to detect the expression levels of interferon β in DF-1 cells at post co-transfection with wide-type or mutants of NDV V proteins with MDA5. The interaction of V mutants and MDA5 was verified by co-immunoprecipitation assay. The results showed that the expression levels of V protein and mutants were comparable to each other in DF-1 cells. NDV V protein was able to suppress the activity of IFN-β promoter induced by poly(I:C). The larger finger loops L1(H177, C196, C221 and C224) are not required for NDV V protein binding to MDA5. However, the residues of the smaller finger loops L2(C200, C212, C214 and C217) were essential for the V-MDA5 binding.2. Effects of other conversed amino acids of C-terminal domain on NDV V protein interacting with MDA5There are a series of conserved amino acids in paramyxovirus V protein C-terminal domain except the eight main amino acids in the zinc finger structure. In order to understand the effect of these residues on NDV V protein binding to MDA5, fifteen amino acids were mutated. Reporter gene assay, ELISA and real time PCR were applied to detect the expression levels of interferon β in DF-1 cells at post transfection with wide-type or mutants V proteins and MAD5, and determine the effect of critical residues on V protein interacting with MDA5. The results showed that the expression level of mutants was consistent with the wild type V protein. The residues R178 and I183 were required for the V protein inhibiting IFN-β production in DF-1 cells. The arginine and isoleucine mutants were unable to bind to MDA5. The E180 mutant still supports V protein interacting with MDA5. The remaining 12 residues were nonessential for V binding to MDA5. The mutants did not affect the interaction between V protein and MDA5. 178 arginine changed to lysine and 180 glutamic acid mutated to aspartic acid were also maintained the ability of V binding to MDA5, suggesting that basic residue at 178, acidic residue at 180 and isoleucine at 183 were necessary for MDA5 and V protein interaction.3. Effects of amino acids of N-terminal domain on NDV V protein interacting with MDA5To study the role of certain residues of N-terminal of NDV V protein in interaction between V and MDA5, seven residues were select for site-directed mutagenesis. Luciferase reporter gene system, real time PCR and ELISA were carried out for detect the interferon β expression levels after transfection with mutants and MDA5. Co-immunoprecipitation was carried out to detect the interaction between V mutants and MDA5. The results showed that the residue N65 of N-terminal of V protein was slightly affected the interaction between V protein and MDA5. The IFN-β expression level was increased after transduction of mutation in DF-1 cells, but it was not significant. The results of promoter activation, transcriptional and protein expression assays were inconsistent with co-immunoprecipitation data, suggesting that the N-terminal amino acids have slightly affected the V protein inhibiting IFN-β production.In summary, this study illustrates the role of main functional domain of NDV V protein, which inhibits the host cell IFN-β production. The results have shown that different amino acids of V protein play a diverse role in V protein binding MDA5 to inhibit IFN-β production. It would be helpful for further exploring the mechanism of viral immune escape.