Screening And Identification Of BmCPV Candidate Receptor Proteins And Co-receptors

Virus binding to the host cells and then entering into cells is a multi-factor participation process.Receptors play a key role in the infection of virus but virus-invading cells usually utilized more than one receptor.It is important for the understanding of virus invasion and the development of new antiviral drugs to verify virus receptors.Bombyx mori cytoplasmic polyhedrosis virus was a model of Cypo virus,Reoviridae.BmCPVinfected midgut cells of Bombyx mori which may lead to the occurrence of silkworm midgut jaundice and reduce the production of silk industry.At present,most of the researches on BmCPV focused on the morphological structure of the virus,the function of the viral gene and the response of the silkworm to BmCPV infection.But the receptors of BmCP V and the molecular mechanisms of infection were poorly understood.To clarify the entry of BmCPV into cells,co-immunoprecipitation(Co-IP),Virus overlay protein-binding assay(VOPBA)and Virus overlay protein-binding assay were applied to screen candidate receptor proteins and some proteins that may interact with BmCPV.In order to further explore the role of these proteins during infection,RNAi assay was applied to down-regulate the expression of 13 proteins,includingDown syndrome cell adhesion molecule-1(Dscam),epidermal growth factor receptor(EGFR),vascular endothelial growth factor receptor 1(VEGFR),vascular endothelial growth factor receptor 1(VEGFR),nAChR subunit beta 2(nAChR b2),transmembrane 7 superfamily member 3(TM7SF3),sortilin-related receptor(SOR),heat shock 70 kD protein cognate(HSC70),receptor expression-enhancing protein 4(REEP4),scavenger receptor class B member 1(SCRB),scavenger receptor class B member 1(SCRB),and tight junction protein Zonula occludens-1 isoform X1(BmZO-1).The results showed that the infectivity of BmCPV was reduced at different levels by the silencing of 13 proteins.The effect ofBmZO-1,VEGFR,HSC70 and BmZO-1silencing on BmCPV infection was greatest.A part of the above five genes were cloned by prokaryotic expression.The polyclonal anti-body was prepared by immunizing mice with recombinant protein.The block of five polyclonal antibody reduced the infection of BmCPV,which mean that these proteins may be a member of the BmCPV receptor complex or a member of interaction protein that play an important role in the process of BmCPV infectio.Previous studies revealed that tyrosine-protein kinase TPK Src64B-like(Src)and Integrin?subunit 1(Integrin)may play an important role in the infection of Reovirus.Reovirus utilized Integrin ?1 to internalize into cells through clathrin-mediated endocytosis pathway.Receptor for activated protein kinase C,RACK1,interacted with Src64B and Integrin.A comlex of Src,RACK and Integrin may play a part in infection of BmCPV In the present study,the interaction between BmCPV and two proteins was detected by immunofluorescence.The result revealed colocalizations of Integrin and BmCPV.Src and Integrin overexpressed cells were infected with BmCPV virons.The result showed that the up-regulation of Src and Integrin could enhance the infectivity of BmCPV.The antibody blocking assay showed that the block of Integrin prevent the infection of BmCPV into cells,but the block of Src had no obvious influence.The above results showed that Src and Integrin could participated in the infection of BmCPV and interacted with virons.Gangliosides on the cell surface were reported to act as a coreceptor of Reovirus and Rotavirus.But it is unclear if glycans act as a coreceptor of BmCPV.In this study,BmN cells were treated with neuraminidase and the infection of BmCPV into cells was detected by Real-time PCR and immunofluorescence.The result showed that the infection of BmCPV into cells was inhibited by the treatment of neuraminidase.BmCPV virions were preincubated with different concentrations GM1,GM2 and GM3,the infectivity of virons was decreased by the preincubation of GM2.The antibody blocking assay of GM2showed the same result.The above results showed that GM2 could interact with BmCPV virions and act as a coreceptor of BmCPV.Previous studies used Co-IP,VOPBA and pull-down combined with SDS-PAGE to identify the receptor protein of BmCPV.This method had low efficiency and large workload.In the process of infection,BmCPV attach to the brush border membrane of midgut.In this study,brush border membrane vesicle,bbmv,of Bombyx mori midgut were extracted and separated by dimensional electrophoresis combined with VOPBA.Combined with bioinformatics analysis by mass spectrometry,10 kinds of candidate receptor proteins were initially screened.transferrin-like(BmTF),general odorant-binding protein 71(OBP71)and ABC transporter G family member 23-like(BmABCG23)had transmembrane structure.serpin-19,CAP-Gly domain-containing linker protein(CLIP),neurocalcin homolog(BmNCA),imaginal disk growth factor(BmIDGF),sterol carrier protein x(SCP-X),tektin-B1 and synaptotagmin-C-like(BmSytC)were proteins ouside the membrane.OBP71 may be a member of B1CPV receptor complex.BmABCG23 and SCP-X may regulate the infection of BmCPV by mediating the transport of steroids.CLIP and tektin-B1 may participate in intracellular transport of BBMV.NCA and BmSytC maybe involved in the calcium ion transport during BmCPV endocytosis.BmTF was associated with endocytosis of BmCPV as a structural component of BBMV.