Comparative Proteomic Analysis Of Rabies Virus-infected N2a Cells And Functional Analysis Of Chaperonin CCTγ

Rabies virus (RV), is a member of the Rhabdovirus family, Lyssavirus genus, with high neurotropism and lethality. The genome is a single-stranded, negative-sense RNA of approximately12kb that encodes five structural proteins, glycoprotein(G), nucleoprotein(N), matrix protein(M), phosphoprotein(P) and large protein(L). The pathogenic mechanism of RV is still unclear, and the research on interaction of the host cell and virus is relatively limited. In this study, the differential protein expression profiles of host cells infected by RV were analysed, to understand the pathogenesis of RV infection on host cells and find potential drug targets for treatment.Monoclonal antibody is a powerful weapon for disease diagnosis, prevention, treatment and study of the immune system, in the current prevention and control of viral diseases and basic research, there are no commercial monoclonal antibodies available for the structural proteins of rabies virus, except glycoprotein. In this study, recombinant rabies virus nucleoprotein expression vector pET32a-NP and glycoprotein recombinant expression vector pET32a-GP were induced, purified under denaturing conditions, and immunized BALB/c mice, nine monoclonal anti-RV-N antibodies and one anti-RV-G monoclonal antibodies were successfully prepared. On the other side, immunized mice with the suspension of rabies virus infected suckling mouse brain tissue antigen,7monoclonal antibodies against rabies virus were identified to be anti-P monoclonal antibodies.In order to explore the interaction of virus-host cell, this study choose mouse neuroblastoma N2a cells as cell model, differential protein expression profiles of N2a cells infected with RV at24,48and96h post infection (p.i.) were analyzed by two-dimensional gel electrophoresis combined with mass spectrometry. Software PDQuest analysis showed that RV infected N2a cells resulting in a total of97differential protein spots, and significant differences of these protein points are mainly found in the48h and96h p.i., and most of the protein spots were up-regulated. Fifty-three proteins, corresponding to49kinds of different proteins were identified successfully by MALDI-TOF/TOF tandem mass spectrometry. The functions of the differential proteins are involved in protein synthesis and processing, energy metabolism, signal transduction, stress response/chaperone protein, gene regulation, immune response and ubiquitin-proteasome pathway. Specific primers were designed, and the changes of27differentially expressed proteins corresponded gene transcripts were verified through quantitative PCR, confirmed the reliability of the mass spectrometry results at some extent. Western blot experiments demonstrated HSP90and CCTy upregulation of the infected group compared with the control. For the differentially expressed proteins, by IPA Ingenuity biological network analysis, the protein interaction network of these differential proteins was drawed. All the information about differentially expressed proteins and protein interaction networks provide important proteomic information for further resolve the pathogenesis of rabies.On the base of comparative proteomics information, subcellular distribution of CCTα, CCTβ, CCTγ, HSP90, PFDN1and DLC8were analyzed by indirect immunofluorescence double-labeling. The results showed that viral infection caused the highly recruitments of cellular protein CCTa and CCTy and partially recruitments of PFDN1and DLC8to the position of Negri bodies, CCTa and CCTy colocalized with the viral N, P protein well; however, CCTβ and HSP90are not recruited to the Negri bodies. RV N, P eukaryotic expression vectors co-transfected cells can form Negri body like structure within the cell, similar to the RV infected cells, CCTγ, CCTα, PFDN1and DLC8were also gathered to the Negri body-like structures, and colocalized with the viral N, P protein of the Negri body-like structures. In the cells mono-transfected N or P plasmid are also showing varying degrees of CCTy and CCTa co-localization with viral proteins N or P. Quantitative results showed that DLC8, CCTy and HSP90were showed up-regulation in the infected cells; mono-tranfection of P plasmid or co-transfected with N plasmid in N2a cells caused the up-regulation of DLC8, and other proteins has no significant difference. All this shows that these host cell proteins by way of active or passive participation in the virus life activities.In order to clarify the exact function of CCTγ and CCTα in the life cycle of rabies virus, this study using lentivirus-mediated shCCTγ, shCCTα and nonsense sequence shcoo2v transduction, established stable expression of shCCTγ RNA interference cell lines, through virus seeding, TCID50determination and quantitative PCR analysis, we found that knockdown of CCTγ and CCTα significantly reduced both the replication and viral gene transcription of rabies virus, indicating that CCTγ and CCTα are key host factors for rabies virus.