Screening And Identification Of A Novel Duck Reovirus(NDRV-TH11) ?C Protein And Duck TRAM1 Protein Interaction

The novel duck reovirus(NDRV)belongs to the reoviridae family and is a reovirus genus.After the identification of the NDRV-TH11 strain in 2011,it was found that it has a broader host spectrum and stronger pathogenicity than the previously reported avian reovirus,causing serious economic losses to the duck industry.The NDRV aC protein is a structural protein with an adsorption function that initiates the viral infection process by recognizing the host cell.The host protein TRAM 1(Translocation-associated membrane protein 1)is a protein involved in the breakdown of type I membrane proteins,which can increase the content to relieve the stress of the endoplasmic reticulum(ER)under stress.The completion of self-replication of a virus must depend on the host system,and the antagonistic and defense mechanisms produced by the host in the face of viral invasion depend on the interaction of the virus and the host protein.In this paper,we screened host factors that may interact with virus ?C,and studied the effects of the interaction between the two on viral replication,providing a theoretical basis for understanding the pathogenesis of NDRV,and providing a reference for the prevention and treatment of the disease.The main contents are as follows:(1)Using CO-IP technology combined with mass spectrometry,silver staining experiments screened all host factors that might interact with NDRV-TH11 ?C protein and performed biological analysis.100 host protein factors that may interact with ?C were screened.Host factors are closely related to the metabolic process of proteins and participate in the process of protein translation and replication.(2)We further studied the TRAM1 gene and amplified the TRAM1 gene using Duck embryo fibroblast cells(DEFs),sequenced it and submitted the results to GenkBanK.Subsequent biological analysis of the TRAM1 gene and establishment of a fluorescence quantitative assay for the TRAM1 gene.The results showed that the duck source TRAM1 gene was similar to the avian TRAM1 gene and similar to mammals.At the same time,a fluorescence quantitative method for detecting duck source TRAM1 was successfully established.(3)Subsequently,using immunoprecipitation(CO-IP),GST PULL-DOWN,and laser confocal experiments to identify whether ? C and TRAM I have an interaction relationship.The results show that ? C and TRAM1 have an interaction relationship and co-localization Phenomenon,co-localization in the cytoplasm.(4)In order to further study the effect of ?C and TRAM1 interaction on viral replication,we overexpress TRAM1 and inhibit the expression of TRAM1 by siRNA technology,and explore its effects by fluorescence quantification and plaque assay.The results showed that inhibition of TRAM1 by siRNA promoted viral replication,whereas overexpression of TRAM1 inhibited viral replication.This result suggests that interaction between ?C and TRAM1 will inhibit viral replication.