Identification A Putative E2-binding Cellular Receptor Of Classical Swine Fever Virus

Classical swine fever virus (CSFV) is classified within the genus Pestivirus in the family Flaviviridae. It is an enveloped, non-segmented, positive-stranded RNA virus encoding four structural proteins: C, Erns (formerly known as E0), E1, and E2. Erns and E2 reside on the outer surface of CSFV virion and transmembrane glycoprotein E1 is present as an E1-E2 heterodimer by disulfide linkage. Glycoprotein E2 is the most immunogenic protein of CSFV and glycoprotein Erns is the second target for neutralizing antibodies. Glycoproteins, which present in the viral envelope, are involved in the attachment to susceptible cells and mediate the CSFV entry. Interaction of Erns with heparin sulfate (HS) immobilizes CSFV at the cell surface but E2 also associates with cellular receptor(s) by an as yet unknown mechanism.Anti-idiotype antibodies (anti-Ids) elicited by an antibody molecule are directed against antigenic determinants in or close to the antibody's combing site. Besides using anti-Ids as immunogens for a protective response against a particular infectious agent, Ab2βinternal image antibodies have been used as probes for receptors by mimicking viral epitopes that bind to cell receptors.Polyclonal anti-Ids were produced in New Zealand White rabbits by immunizing monoclonal antibody (MAb) 6E10 via subcutaneous injection. Characterization of anti-Ids, which can mimic an epitope on the CSFV glycoprotein E2, was performed by competitive ELISA. In immunohistochemistry assay and Western blot analysis, a 54 kDa cell surface protein (p54) was detected by the anti-Ids from PBMC. Microchemical analysis showed that the most similar molecule of p54 was IgG-like. To identify the structure and function of p54, we searched a mass of literatures and found that the molecular masses between p54 and CD46 are similar. To verify our hypothesis, the CD46 gene of pig was cloned and expressed in vectors pET32a and pcDNA3.1. Polyclonal rabbit anti-CD46 sera were produced and they could block CSFV infection in porcine kidney cells. We also found that p54 and CD46 were different proteins because anti-Ids could not recognize CD46 and p54 could not be recognized by anti-CD46 serum. In further study, BHK-21 cells were transfected with the expression plasmid pcDNA-CD46. Stable CD46-expressing cell lines (BHK-CD46) were selected by adding G418 to the culture medium after transfection. CSFV infection on BHK-CD46 cells expressing CD46 was detected by antigen-capture ELISA and laser confocal light microscopy. Co-IP showed that CSFV E2 could precipitate CD46 from PK15 cells. Our data showed that the expression of CD46 did not confer full susceptibility to non-permissive cells but binding. This study indicates that CD46 is a cellular receptors that is involved in attachment to susceptible cells of CSFV.