Screening And Identification Of ?NS Interaction Proteins Of Human Pathogenic Reovirus

Objective:The localization of ?NS protein in Nelson Bay virus(NBV)-infected cells was observed by immunofluorescence technique using confocal microscopy.The inclusion bodies of ?NS protein in cells transfected with M3 plasmid alone was observed by immunofluorescence technique using confocal microscopy.The molecular interaction analysis was used to screen and analyze the host interaction protein of human pathogenic reovirus ?NS protein,and the amino acid sequence and structure of unknown protein were clarified,its role in viral infection replication was inferred.This study provides a theoretical basis for in-depth study of Nelson's Gulf virus and positive reovirus for human pathogenesis.Methods : In this study,the localization of ?NS protein in NBV-infected cells was observed by immunofluorescence under confocal microscopy.In order to determine the location of the ?NS protein in the viral inclusion body-like structure,human pathogenic reovirus was infected with L929 cells at a multiplicity of infection(MOI)of 0.1 PFU/cell,incubated for 24 hours,The ?NS protein was immunostained with ?NS polyclonal antibody and Alexa 488 goat anti-rabbit antibody,and stained with confocal microscopy after immunostaining.The ?NS protein was immunostained with ?NS polyclonal antibody and Alexa 488 goat anti-rabbit antibody,and stained with confocal microscopy after immunostaining.In order to demonstrate that ?NS can form viral inclusion body-like structure without other NBV viral protein expression,this study used pEFHAMBM3 plasmid(an expression vector containing the M3 gene from NBV virus)to transfect L929 cells,and observed the distribution of ?NS in cells by immunofluorescence under confocal microscopy.In order to obtain the muNS protein of human pathogenic reovirus and its interaction protein,the plasmid pEFHAM3 carrying the M3 gene fragment was transfected into 293 T cells,and the pEFHA empty plasmid was used as a control.The ?NS protein encoded by the M3 gene fragment and the host protein which may interact with it were obtained by immunoprecipitation,and the protein of the experimental group and the control group were separately subjected to protein qualitative detection(Shotgun) to analyze the differential protein.Results: After immunostaining,the confocal microscopy showed that the ?NS protein was expressed in a small dot-like dense structure in human pathogenic reovirus-infected cells.This small point-like dense structure is the inclusion body,suggesting that the ?NS protein is localized in the inclusion body-like structure.After immunostaining the cells transfected with M3 plasmid alone,the point-like dense structure with the inclusion body-like structure in the human pathogenic reovirus-infected cells was observed by confocal microscopy,suggesting that the ?NS protein can form Inclusion body alone.Protein qualitative tests were performed on the experimental and control protein samples,and 9 different proteins were obtained after comparison: serine/threonine protein kinase,arginine synthase,keratin 14,mitochondrial Rho GTPase 1,histone H2 B,40S ribose Somatic protein S4,ribosomal protein L26,Actin-2,uncharacterized protein.Conclusions : The muNS protein of human pathogenic reovirus is localized to the inclusion body structure of virus-infected cells.The muNS protein of human pathogenic reovirus can be independently expressed in the M3 plasmid alone transfected cells and has the ability to form inclusion body-like structures without any other viral protein expression.One or more of the following nine proteins may be involved in the proliferation of reovirus by interacting with the ?NS protein: serine/threonine protein kinase,arginine synthase,keratin 14,mitochondrial Rho GTPase 1,Histone H2 B,40S ribosomal protein S4,ribosomal protein L26,Actin-2,uncharacterized protein.