The Role Of The E2 Protein In The Internalization Of Classical Swine Fever Virus

Classical swine fever?CSF?is a kind of highly contagious and economically important viral disease with the characteristic of high fever,hemorrhage and high mortality,caused by classical swine fever virus?CSFV?.CSF is listed by the World Organization for Animal Health?OIE?as a disease that needs to be declared and it is also classified as 1st animal contagion in China.CSFV is small enveloped virus with a single-strand RNA genome,belongs to the member of the Pestivirus genus within the Flaviviridae family.E^rns,E1 and E2 are the glycoproteins on the surface of virions,composed the virus structural proteins together with C protein.The virus genome also codes eight nonstructural proteins.All the proteins of virions play roles in the CSFV life cycle.It has been shown that the entry of CSFV into host cells is mediated by clathrin-mediated endocytosis.However,the specific entry mechanism still unknown,viral protein involved in this stage remains to be elucidated.This study aimed to clarify the role of the E2 protein in the internalization of CSFV.A 293 suspension cell line stably expressing the E2 protein was established with lentivirus system,and the recombinant E2?rE2?protein was purified by affinity chromatography.The expression level and reactivity of the rE2 protein was identified by SDS-PAGE and Western blotting.The effects of the rE2 protein on CSFV infection,attachment and internalization were determined by infection,attachment and internalization inhibition assay,respectively.Anti-E2 polyclonal antibodies were prepared by immunizing BALB/c mice with the rE2 protein.Attachment and internalization assays were performed using the prepared polyclonal antibodies.SDS-PAGE and Western blotting results showed that 293 suspension cell line stably expressing the E2 protein was established,and the rE2 protein could be recognized by the anti-E2 monoclonal antibody WH303.Under the optimized expression condition,the concentration of the rE2 protein in the supernatants of the established cell line was up to 5.84?g/mL,soluble protein blocking assay results showed that the expressed rE2 protein exerted antiviral activity against CSFV infection.CSFV infection was significantly inhibited by the rE2protein treated in the entry step;the anti-E2 polyclonal antibodies could neutralize the CSFV infection;attachment and internalization assays demonstrated that CSFV internalization could be inhibited by the rE2 protein and viral attachment was blocked by the polyclonal antibodies.In conclusion,the established suspension 293 cell line could stably express the rE2protein,and the rE2 protein clarified that the E2 protein involved in the internalization of CSFV.