Screening And Validating The Interacted CellProteins With NS3 Of TMUV

The NS3 protein is a member of the non-structural protein of the duck Tembusu virus(TMUV),which contains three domains,each of which has serine protease,nucleotide triphosphatase,and RNA helicase activities respectively.It performs a variety of biological functions and acts as a multifunctional protein involved in the regulation of the viral life cycle and host immune response.Based on the Yeast two-hybrid System,we successfully transformed pGBKT7-NS3 into Y2 H Gold and tested it to prove that it has no self-activation and toxicity,which indicates that the bait yeast strain has been prepared for the following screening.Then hybridized with the prey yeast strain of the Duck embryo fibroblast(DEF)cDNA library to screen,ultimately,the hybrid diploid yeast cells coexisting with the bait and the prey plasmid were obtained.In the next part,different stringency levels were screened on auxotrophic medium.First,more well-grown yeasts with no phenotype were obtained on the SD/-Trp/-Leu culture agar plate with low screening intensity.A one-to-one transfer was performed,and blue positive clones were screened using DDO/X/A with higher screening strength.Next,turn all the blue positive clones to QDO/X/A for high-level screening.Select positive clones and then inoculate them into QDO/X/A with repeat screening measures.After high-stringency selected,positives alignment with the NCBI database revealed seven potential interactive proteins:MGST1,ERCC4,WIF1,WDR75,ACBD3,PRDX1 and RPS7.Peroxiredoxin(PRDX1),which is widely distributed and highly expressed in cells,is in high affinity with reactive oxygen species(ROS).Its enzymatic activity can regulate the level of oxidative stress mediated by ROS in cells,and able to be a cofactor participating in the reduction process of many transcription factors and signal factors.Therefore,the utmost interested one PRDX1 was selected for subsequent verification test.In vivo validation using yeast two-hybrid return experiments,the full-length NS3 protein and the three-domain S7,DEXDc,and HELICc proteins of NS3 itself were grouped and subsequently verified with duck and human PRDX1.The regressive verification results proved that both HELICc and NS3 were positive.Further in vitro interactions were verified by GST pull-down assay,thefinal results indicate that PRDX1 indeed interact with the HELICc domain of NS3.Human and duck-derived PRDX1 were positive in the interaction results as well.Although there were differences in amino acid homology between them,by reason of the interaction sites were similar,indicating that further study on the function of PRDX1 and its involved mechanisms could be transformed into the HEK293 cell model for subsequent researches.Laser confocal microscopy technology was used to localize PRDX1 and HELICc in cells,showing co-localization in parts of the cytoplasm,with the possibility of interaction.The HELICc domain of the TMUV NS3 involves the RNA unwinding process of viral replication.At this stage,it requires mitochondria to accelerate the hydrolysis of ATP to provide a large amount of energy and aggravate the mitochondrial workload.Therefore,excess metabolism of mitochondria produces a large number of by-products,freely distributed in the cytoplasm in cells,especially ROS.Excessive ROS cause disruption of cellular homeostasis,which activates ASK1 and mediates the P38/MAPK pathway to induce apoptosis.Perhaps,HELICc domain binding partner PRDX1 protein can reduce ROS levels,thereby alleviating intracellular oxidative stress levels,allowing the virus to evade host immune responses.The results showed that the expression of HELICc protein can up-regulate the transcription level of PRDX1 and down-regulate the transcriptional level and phosphorylation level of the regulatory factors P38 in the relevant pathways,and finally inhibit the occurrence of apoptosis and escape the antiviral immune response in vivo.