Effects Of The Nucleoprotein-Phosphoprotein Interaction Of Human Parainfluenza Virus Type3on The Viral Replication And The Formation Of Cytoplasmic Inclusion Bodies

Human Parainfluenza Virus Type3(HPIV3) belongs to the nonsegmented negative-strand virus family, which include the respiratory syncytial virus, rabies virus and measles virus. It is the main cause of severe respiratory tract diseases such as bronchiolitis and pneumonia in infants and young children. There is no effective antiviral therapy or vaccine available so far, thus, it is useful to explore the mechanism of the replication and transcription of HPIV3for the development of novel therapeutic approaches.The nucleoprotein (N) encapsulates the RNA genome of HPIV3to form an N-RNA complex which serves as a template for replication and transcription. Phosphoprotein (P) is the cofactor of the RNA-dependent RNA polymerase (RdRp) complex. Both N and P protein, together with the large protein (L), play important roles in the replication and transcription, and the interaction between N and P protein having been the hot issue for the study of the mechanism of HPIV3replication and transcription. It has been reported that the N-terminus of the N protein is the critical region for its self-assembly, and it is also the RNA binding site for the N-RNA complex formation, while the C terminus is important for the binding of the N-RNA template and the P protein. According to sequence alignment and structural analysis of the P protein, the sites for N0-P interaction are located in the40aa of the N terminus of the P protein, and the20aa in the C terminus is responsible for the interaction of the N-RNA and the P protein.Although the N-P interaction had been extensively investigated in the past few years, many questions remain to be unfold, such as the precise sites for the N-P interaction in the N protein, the relationships of the N0-P and N-RNA-P interaction. In order to answer the above questions, we detected an alanine mutant in Leu478(NL478A) of the N protein which lost the minigenome RNA synthesis activity by the In vivo HPIV3minigenome assay. By virtue of In vivo coimmunoprecipitation assay, we confirmed that the NL478A lost the ability to interact with the P protein. To further distinguish the N0-P and N-RNA-P interactions, In vitro coimmunoprecipitation assay was performed using the N-RNA purified by CsCl gradient centrifugation and the lysates of the P protein or the P mutants. Finally, we came to a conclusion that the NL478A maintained NL478A0-P interaction but was defective in NL478A-RNA-P interaction. Furthermore, we evaluated the localization of the NL78A and the N protein with the P protein. And we found that coexpression of the N protein and the P protein supported the formation of cytoplasmic inclusion bodies, which might be the center for the transcription and replication of HPIV3. Neither the inclusion bodies formation nor the recombinant virus recovery by reverse genetics was supported by the NL478A. Both the N0-P interaction and N-RNA-P interaction were proved to be indispensable for the inclusion bodies formation by the study of the coexpression the N protein with the P mutations.In summary, we are the first to characterize the N-P interaction in a paramyxovirus and to explore the precise motif in N protein for the formation of the inclusion bodies. Our results provide new theoretical and experimental basis for rational antiviral approaches and may be inspirations for other NNS virus.