The Intracellular Interaction Between IgA And Measles Virus

The mucosal immunity is the front line for the body to defense against pathogens,and the important effector in the mucosal immunity is secretory IgA(S-IgA)antibodies.Unlike serotype IgG and other antibodies,secretory IgA can be transcytosized from the basal side of the mucosal epithelial cells to lamina propria by polymeric immunoglobulin receptor(pIgR),and S-IgA has the opportunity to interact with pathogens in cells.It has been known that S-IgA can specifically interact with various components of the virus and effectively inhibit the replication of the virus inside epithelial cells.Therefore,the intracellular neutralization represents a unique way for S-IgA to defense viral infections.However,hard to understand about the molecular mechanism and the effect of the interaction between S-IgA and viral components.In this study,the hybridoma technique and the gradient dilution method were used to screen anti-P 1D11-IgA and anti-P 7F1-IgA specific IgA antibodies against measles virus phosphoprotein(P protein).Polarized cells culture in polycarbonate membrane Transwell system was used to investigate the interaction between the two monoclonal IgA antibodies and measles virus intracellularly.The two IgA antibodies were first found to be able to specifically bind to the viral P protein in MV-infected-Vero-pIgR cells in Transwell system.However,only anti-P 1D11-IgA antibody could neutralize virus in Vero-pIgR cells,while anti-P 7F1-IgA antibody could not neutralize virus in the same interferon-deficient Vero-pIgR cells,but could significantly inhibit virus replication in Caco-2 cells.This suggests that two specific IgA antibodies against the same target antigen inhibit virus by different mechanisms.Therefore,we have further studied the molecular mechanisms of the interaction between the two IgA antibodies and the virus respectively.Firstly,we confirmed that the key binding site of anti-P 1D11-IgA antibody on measles virus P protein was the N-terminal proline residue(Pro23)of P protein.Therefore,we speculated that anti-P 1D11-IgA antibody could disrupt the binding of the N-terminal ?-molecule recognition element(?-MoRE)of P protein to measles virus nucleoprotein(N protein).It was confirmed by immunoprecipitation test that the structure of P-N complex could be destroyed by anti-P 1D11-IgA antibody.Since the stability of the structure and function of P-N complex is an important basis for the function of RNA-dependent RNA polymerase(RdRp)in the genome transcription and replication of measles virus,we have found that anti-P 1D11-IgA antibody can reduce the synthesis of virus genome and mRNA in MV-infected cells,and ultimately reduce the generation of virus.Unlike anti-P 1D11-IgA antibody,the specific binding site of anti-P 7F1-IgA antibody locates at amino acid residues 77-86 of N-terminus of measles virus P protein.Anti-P 7F1-IgA antibody was found to be more effective to neutralize measles virus than anti-P 1D11-IgA in Caco-2 cells.ELISA and RT-PCR showed that anti-P 7F1-IgA antibody could promote the production of IFN-? and the expression of IFN-stimulating genes.Therefore,we speculated that anti-P 7F1-IgA antibody could neutralize the virus by blocking the immune escape of the virus from type?IFN.It was found that the interaction between anti-P 7F1-IgA antibody and measles virus P protein hindered the binding of P protein to JAK1 protein and STAT1 protein.Further Western Blot assay was used to monitor protein expression in infected cells and it was confirmed that anti-P 7F1-IgA antibody could induce the earlier phosphorylation of JAK1 and STAT1 in infected cells.Confocal result also showed that while added in anti-P 7F1-IgA antibody,STAT1 protein in MV-infected cells could perform nuclear translocation more effectively.Finally,through identifying the interactions in proteins in 293 T cells,it was indicated that anti-P 7F1-IgA antibody could block the binding of measles virus P protein with JAK1 protein and STAT1 protein,activate the transduction of type?interferon signaling pathway,and inhibit the virus replication by endowing host cells with more effective innate immune function.Although targeting the same antigen P protein of measles virus,the two specific IgA antibodies can resist the pathogen by destroying the structure of the key components in the viral genome transcription and replication or by preventing the virus from escaping from the host's innate immunity.This suggests that the direct antigen-antibody binding is not the only functional explanation of IgA antibodies in the infected cells.Due to the binding specificity of IgA antibody with the virus,IgA antibody can first target the key structural and functional P protein in the virus replication.IgA antibodies could also neutralize viruses by targeting the proteins or complexes involved in the viral genome transcription and replication or the assembling core connexins of viral particles.On the other hand,IgA antibody can coordinate the interaction between virus and cells,inhibit the immune escape ability of the virus,enhance the innate immunity of cells,and ultimately play the antiviral role.In a word,the study of the molecular mechanism of the interaction between IgA antibodies and viruses provides an exploration for whether virus or cell components can be used as drug targets,and also provides a theoretical basis for antiviral vaccines.