Effects Of Nelson Bay Orthoreovirus Replication On Cell Autophagy And Research Of Related Nonstructural Proteins

Objective: Nelson Bay orthoreoviruses(NBV)were originally isolated from fruit bats in Australia.They existed independently for more than 40 years and were not associated with any disease.However,several strains of NBV have recently been confirmed to be pathogens of human respiratory infections,and pathogens isolated from some patients with acute respiratory diseases have shown that NBV has evolved into a zoonotic form and can be transmitted in humans.Pathogenic NBV can cause acute respiratory and intestinal inflammation in humans,and it is gradually spreading in Southeast Asia.In2007,Japanese scholar TAKAHIRO and others used the reverse genetics system of RNA virus for the first time to successfully prepare recombinant NBV,laying a foundation for further research on the pathogenesis of NBV.Autophagy is a physiological metabolic compensation process performed by eukaryotic cells to maintain cell homeostasis.Autophagy is an evolutionarily conserved behavior of cell self-rescue,which is of great significance for maintaining cell homeostasis and cell survival under stress conditions.Autophagy is an evolutionarily conserved process in eukaryotic cells,degrades intracellular substrates,participates in a variety of physiological processes,and maintains cell homeostasis.In addition,autophagy also plays an important role in removing bacteria and viruses from infected cells.Normally,the autophagy program is at a basal level,but is activated by various intracellular and extracellular stimuli.However,some viruses are clearly not affected by autophagy,and many other viruses have evolved to evade or use this mechanism to promote their survival and replication in various ways.The role of autophagy in the infection of the host by the virus varies from virus to virus.It is reported that autophagy can promote the replication of mammalian orthoreovirus(MRV)and avian orthoreovirus(ARV).Another study found that Hepatitis B virus(HBV),Several viruses such as Rotavirus(RV)and Hepatitis C virus(HCV)can induce autophagy on the one hand,and autophagy can also play a role in the life cycle and pathogenesis of the virus Important role.However,so far,there have been no reports about NBV infection and cell autophagy.Due to the previous studies,either one or two nonstructural proteins of each virus were known to be required for forming the viral factories(VF,also known as cytoplasmic structures).The results of studies on MRV and ARV show that the VF formed by the non-structural protein ?NS,which is separately expressed in cells under a light microscope,is similar to the VF formed in infected cells.Studies have shown that mammalian or avian reovirus virus non-structural proteins,?NS and ?NS play a key role in the formation of viral inclusions and the recruitment of other viral proteins and RNAs required for replication and assembly.Based on these studies,we know that reovirus replication and recombination occur in VF.But so far,no reports on ?NS and ?NS of NBV have been published,and the specific functions of ?NS and ?NS proteins during NBV replication are unclear.Based on the NBV reverse genetic technology developed by TAKAHIRO,this subject first successfully co-transformed plasmids or obtained NBV recombinant strains(named NBV-MB to distinguish it from wild strains)and tested them to provide materials for further research on the pathogenic mechanism of NBV.It plays a certain role in the process,so this topic studies the relationship between cell autophagy and viral infection and replication,in order to reveal the role of autophagy in the pathogenesis of NBV in vivo;In addition,?NS is a major non-structural component of NBV Protein,its role may be related to the formation of inclusion bodies of the virus,and found in the study of autophagy,?NS protein may be involved in NBV-MB infection of cells and enhance the process of cell autophagy.Therefore,this study further investigated the expression and biological characteristics of?NS and ?NS proteins.The experimental results confirmed the interaction between ?NS and ?NS protein,and basically determined the binding region of ?NS protein,which laid the foundation for further research on the pathogenic mechanism of reovirus.Methods: 1.Recovery of virus and assay: BKH(Baby hamster kidney,BHK)cells and mouse fibroblast L929 cells were cultured,and the reverse genetic system was used to transfect BKH with 10 NBV virus gene cDNA plasmids and a plasmid encoding T7 polymerase protein pcDNA3.1-T7RNAPO1 Cells to prepare recombinant virus NBV-MB,and passaged through L929 cells for expansion;Viral plaque detection method to detect the titer of recombinant virus;Detection of recombinant NBV-MB byimmunofluorescence,western blotting,and viral RNA vertical electrophoresis;2.Study on virus infection and cell autophagy: After the recombinant virus infected BHK cells,the amount of autophagy related LC3-II protein LC3-II protein in BHK cells was detected by western blotting to evaluate the effect of NBV-MB on autophagy;NBV-MB was infected after transfection of cells treated with autophagy inducer rapamycin(RAPA),autophagy inhibitor 3-methyladenine(3-MA),and chloroquine(CQ)or GFP-LC3 plasmids,and detected Changes in LC3 gene expression and LC3-II protein expression in BHK cells;After treatment of BHK cells with autophagy inducers and inhibitors or transfection with plasmids pCAGM3 and pCAGS3 of the virus non-structural proteins?NS and ?NS,respectively,the changes in virus titers were measured by the plaque method to detect the effects of autophagy inducers and inhibitors treatment or transfection on virus replication;3.Studies on the biological characteristics of ?NS and?NS proteins: BHK cells were cultured and transfected with pCAG M3 and pCAGS S3 plasmids respectively,and the expression of ?NS and ?NS proteins in cells was detected by immunofluorescence;After co-transfection of pCAG M3 and pCAGS S3 plasmids into BHK cells,the expression of ?NS and ?NS proteins in cells was detected by immunofluorescence,and the expression of ?NS and ?NS proteins in infected cells was detected by western blotting;Construct a plasmid with a deletion of 1-60 amino acid residues at the N-terminus of ?NS,verify the sequencing and detect the protein expression of each plasmid transfected with BHK cells by western blotting;Detection of the co-localized binding regions of ?NS and ?NS proteins by immunofluorescence;Finally,yeast two-hybrid experiments were used to verify the binding region of ?NS and?NS protein interaction.Results: 1.Recovery of virus and assay: Recombinant virus NBV-MB was successfully prepared and passaged by L929 cells;A plaque detection method for reovirus was established,and the titer after amplification of the recombinant virus reached 108 PFU/ml;The results of immunofluorescence and western blotting showed that the non-structural protein ?NS or ?NS was expressed in NBV-MB infected cells,but not in the uninfected control group;Viral RNA vertical electrophoresis results showed that compared with wild strains,the distribution and number of NBV-MB RNA bands were consistent;2.Viral infection can promote cell autophagy: NBV-MB infection can induce autophagy inhost cells,and virus replication can promote autophagy;Activation of autophagy increases virus replication,and ?NS protein can promote cell autophagy during NBV-MB infection;3.?NS and ?NS proteins interact: Subcellular localization results showed that ?NS of NBV would form inclusion body structures without other viral proteins,while ?NS was diffusely distributed throughout the cytoplasm;?NS can co-localize in viral inclusions of infected cells by interacting with ?NS;Both immunofluorescence detection and yeast two-hybrid detection confirmed that the basic region of ?NS interacting with ?NS was located within the 60 amino acid residue region of the N-terminal region of ?NS.Conclusion: In this subject,through the reverse genetic technology of NBV virus,this project successfully prepared and tested the infectious recombinant virus NBV-MB,so that the recombinant virus can meet the requirements of subsequent experiments;The experimental results show that NBV-MB infection promotes the occurrence of autophagy,and that virus replication is the key to inducing autophagy;Cell autophagy can also promote NBV replication;This study demonstrated that ?NS of NBV can co-localize in virus inclusions of infected cells by interacting with ?NS;?NS can co-localize in virus inclusions of infected cells by interacting with ?NS,and the interacting ?NS binding region is basically determined.