Functional Study Of Key Amino Acids Of Nucleoprotein And Phosphoprotein Of Vesicular Stomatitis Virus

Many viruses that can cause human diseases belong to nonsegmented negative strand RNA (NNS RNA) virus, such as measles virus, rabies virus, respiratory syncytial virus and human parainfluenza virus, etc. Vesicular stomatitis virus (VSV) is a model virus for the studying of NNS RNA virus. As we known, the replication and transcription of VSV only occur in the cytoplasm and depend on the polymerase complex and template carried by it. The polymerase complex of VSV is constituted by three proteins: nucleoprotein (N), phosphoprotein (P) and large protein (L). Nucleoprotein binds with viral genomic RNA forming N-RNA template for the replication and transcription. It can also protect the viral genomic RNA from being digested by nuclease. P protein is highly phosphorylated. In the life cycle of VSV, P protein helps nucleoprotein recognize and bind with the viral genomic RNA specifically. In this study, we try to identify the key amino acids on N and P protein, and try to reveal the role of these amino acids in viral replication and transcription.Recent studies demonstrated that N-terminal arm of N protein was involved in the interaction between N-N in the N-RNA complex. When the polymerase moves along the N-RNA complex during the replication and transcription of VSV, the N-terminal arm will be temporarily dissociated from the body of the N-RNA template. But the role of the key amino acids in the N-terminal arm is still unclear. In the complex of polymerase of VSV, P plays a very important role. Phosphorylation is very important for the function of P. Previous study has demonstrated that P without the phosphorylation in N-terminal couldn’t support the transcription of VSV. But the reason for this phenomenon is still unknow.In our study, VSV minigenome system and the reverse genetic system were used. Our study found N protein with mutations of4-6A3,7-9A3,13-15A3and R7A fail to support the expression of the reporter gene of CAT minigenome. Synthesized RNA levels supported by these N mutants have different degrees of decline in replication and transcription. N4.6A3, N7.9A3, N13-15A3and NR7A maintained the interaction of N-N, N-P and N-RNA. However, these mutant N proteins are unable to protect genomic RNA from being digested by nuclease and can’t support the formation of inclusion bodies. Growth curve of the recombinant viruses with mutation of R7A decreased significantly. These results indicated that the amino acids in N-terminal of the N protein are involved in protecting the viral genomic RNA from being digested by nuclease to form a functional template for transcription and replication. In the studying of P, mutant P (P3A), the N-terminal phosphate acceptor sites are replaced with alanines (S60/A, T62/A, and S64/A), failed to support the synthesized RNA of replication and transcription. But the interaction of P-P, N-P and P-L haven’t been affected by P3A. Furthermore, we found that P3A abolish the ability to help N recognize and bind with viral genomic RNA specifically. Thus, the forming of functional N-RNA template was affected, which lead to the decrease of synthesized RNA levels of transcription and replication.