Identification Of Cellular Proteins Interacting With E2of Classical Swine Fever Virus By Shotgun

Classical Swine fever (CSF) is a highly contagious infectious disease caused byclassical swine fever virus (CSFV) with high morbility and mortality. CSF is anotifiable disease of World organization for animal health (OIE). Outbreak of CSF leads tosignificant economic losses to the pig industry worldwide. As a glycoprotein of CSFV, E2isthe major protective antigen and can induce neutralization antibody in CSFV-infectedanimals. Furthermore, E2is also the major protein of Pestivirus to recognize and infect hostcell, and it is the pathogenic determinant of CSFV. Endothelial cell (EC) are the majortargets of CSFV, infection of EC by CSFV causes severe widespread haemorrhages inpigs and represents the major cause of pigs’ death. PK-15cell line is the mostfrequently used cell line for the study of CSFV.In this study, E2protein of CSFV was selected as the reaserch object, and ECand PK-15cell lines expressing E2protein were constructed, named EC-E2andPK-E2, respectively. Co-immunoprecipitation (co-IP)-shotgun was used to separateand identify host proteins interacting with E2in CSFV-infected EC, EC-E2andPK-E2. Results showed that25,16and95host proteins were identified inCSFV-infected EC, EC-E2and PK-E2, respectively. Elongation factor1-gamma(EF-1γ), ubiquitin-60S ribosomal protein L40,CUE domain-containing protein1-likewere simultaneously identified in CSFV-infected EC,EC-E2and PK-E2. uvealautoantigen with coiled-coil domains and ankyrin repeats-like, Myosin lightpolypeptide6, ADP/ATP translocase3were were identified both in CSFV-infectedEC and EC-E2. Betaine-homocysteine S-methyltransferase,Glutathione S-transferasealpha M14, sequestosome-1-like were obtained from EC-E2and PK-E2. InCSFV-infected EC and PK-E2, leucyl-tRNA synthetase (cytoplasmic-like) wassimultaneously identified. Western Blotting indicated that EF-1γ interact with E2inCSFV-infected EC, EC-E2and PK-E2, these were identical to the result ofco-IP-shotgun.To classify the obtained124proteins interacting with E2, Gene ontology (GO) analysis was performed through cellular component, molecular function andbiological process. According to molecular function of the obtained proteins, theybelongs to12groups, such as binding, catalytic activity, transporter activity,structural molecule activity, signal transducer activity, enzyme regulator activity,motor activity, transcription regulator activity, antioxidant activity, translationregulator activity, protein tag, obsolete molecular function. These results suggest thecomplicated CSFV-host interaction, which provide a basis for further unravel themolecular mechanism of CSFV pathogenesis.It has been demonstrated that elongation factor1-alpha (EF1-), EF1-γ, TCP-1βand interferon stimulated gene15(ISG15) play a pivotal role in the translation andreplication of many virus. ISG15and CYR61were identified to be involved inestablishing persistent infection and apoptosis pathway, respectively. Currently, it isnot clear whether the above proteins are also involved in the replication and infection ofCSFV. Furthermore, work is ongoing about the role of17kDa myosin light chain (LC17)å'Œ20kDa myosin light chain（LC20）in pathogenesis of hemorrhage of CSFV, and the effect ofGTP-binding nuclear protein Ran (Ran) and ISG15in Host antiviral pathwaytargeting CSFV.Overall, EC and PK-15cell line expressing E2of CSFV were successfullyconstructed, and cellular proteins interacting with E2in these cell lines andCSFV-infected E2and PK-15were screened by co-IP-shotgun and some of themwere identified to interact with E2by western blot. The resultant cell lines and theindentified proteins will provide insight fot the study of the pathogenesis of CSFV.