The Mechanism Of Porcine CD46 Protein In Classical Swine Fever Virus Infection

Viruses,as strict intracellular parasitic organism,rely on a variety of host proteins to complete their life cycle.The binding between virus and cellular receptors is the first sterp for virus invasion.As a portal for virus to invade hosts,the structure and function of cellular receptors that mediates viral invasion has been the hotspot of virology research.Screening and identification of cellular receptors are helpful to elucidate the pathogenesis of viruses and provide an important scientific basis for prevention and control of viral diseases.Classical swine fever(CSF),caused by classical swine fever virus(CSFV),is a highly contiguous and fatal pig disease.The disease is worldwide distributed with different degree of prevalence,causing serious economic losses in the pig production.CSFV is an enveloped virus containing singe-stranded and positive RNA genome,belonging to the genus Pestivirus of the family Flaviviridae.Firstly,CSFV binds to cellular surface through the interaction with attachment receptor(s),and then invades host cell via clathrin-mediated endocytosis.Host cellular factors are required for the virus attachment and penetration.Previous studies have shown that membrane-associated heparan sulfate(HS)and laminin receptor mediate attachment for CSFV.In addition,it has been demonstrated that anti-CD46 antibodies can efficiently suppressed the CSFV replication,thus concluding that membrane cofactor protein(CD46)acts as an attachment factor.However,additional experiments are required to validate the critical roles of CD46 during CSFV infection.The aim of this study was to elucidate the role of CD46 in the CSFV life cycle.The main contents are shown as follows:1.CD46 promotes replication of CSFV in PK-15(porcine kidney)cells.Small interfering RNA(si RNA)molecule-mediated knockdown CD46 significantly inhibited the proliferation of CSFV in PK-15 cells;Lentiviral system-mediated overexpression of CD46 significantly enhanced the replication of CSFV.In addition,CRISPR/Cas9-mediated knockout CD46 suppressed CSFV replication.2.CD46 interacts with E2 protein.CD46 was identified to interact with E2 by coimmunoprecipitation,and colocalization of CD46 and E2 was observed on the cellular membrane by using laser confocal microscopy assay.In addition,the results of MST showed that CD46 has a binding affinity to E2 protein with a KD value of about 1.74 μΜ.Furthermore,the interaction between CD46 and E2 was confirmed by surface plasmon resonance(SPR)analysis,and the KD value was 2.95 μΜ.3.CD46 facilitates CSFV attachment.Soluble CD46 ectodomain protein or anti-CD46monoclonal/polyclonal antibodies efficiently suppressed the CSFV infection in PK-15 cells in a dose-dependent manner.CD46 was identified to facilitate CSFV attachment in PK-15 cells.Attachment assay using knockout or overexpression cell lines demonstrated that CD46 mediates CSFV attachment.Taken together,our results indicate that CD46 acts as a critical host fastor in the attachment of CSFV to host cells.This study outlines a foundation for further elucidation of the invasion mechanism of CSFV,and provides a theoretical basis for further revealing the pathogenesis and antiviral mechanism of CSFV.