The Study On The Interaction Of Porcine Circovirus Type 2 And Nucleolin In Host Cell

Porcine circovirus type 2(PCV2)is the main etiological agent of PCV2-associated diseases(PCVAD).Since been discovered in the 90 's,PCV2 virus spread and expand rapidly across the global porcine industry.In 2000,Lang reported the presence of the virus in China for the first time.PCV2 as a pathogen can mainly cause post-weaning multisystemic wasting syndrome(PMWS),reproductive disturbance of sows,porcine respiratory disease complex(PRDC),porcine dermatitis and nephropathy syndrome(PDNS)and other related chronic diseases in pigs.After infection,PCV2 tend to propagate in lymphoblastoid cells,monocytes,and other immune cells.The alveolar macrophage is the main target cells.At the early stage of infection,virus particles will cause slight damages to the alveolar macrophages in the function of antigen presentation and processing.The combination of ligand with receptor is the key step of virus infection into host cells,and the receptor recognition is the first step of virus adhesion to the surface of target cells that plays a critical role in viral pathogenesis,transmission and proliferation.Therefore,identifying the PCV2 receptors that mediate viral binding and entry to the target cells is essential to eventually reveal the mechanisms that underlie PCV2 infection and pathogenesis.Although some broad-spectrum receptors of PCV2 have been found,but how do interactions happen after the adhesion of PCV2 to the target cells,how do PCV2 particles get through cell membranes and enter the target cells,and last how do replication and release of PCV2 viral particles in the host cells,these mechanisms are not well known to us,thus it is very necessary for us to deeply research on the interactions between PCV2 and the host cells.In this study,PCV2 capsid protein was expressed in E.coli prokaryotic expression system and purified by Ni+ affinity chromatography,virus-like particles(VLPs)were assembled in vitro successfully.As a pseudovirus,VLPs were composed of main structural proteins of virus,and almost do not contain any genetic materials that have a similar immunogenicity with natural virus.By using Indirect immunofluorescence(IFA)techniques,we found that PCV2 VLPs have the ability of invasion of PK15 cells,3D4/2 cells and Hela cells as well as wild-type virus.Besides we used co-immunoprecipitation(Co-IP)and mass spectrometry(MS)to do proteomics research of 3D4/2 cell lysates after PCV2 VLPs infection,46 proteins showed significant change were identified,and most of these protein are located in the cytoplasm and organelles that have binding activity and catalytic activity.Compared with the protein database,we found a special protein(I3LRH2)which has various biological functions and its gene sequence is highly identical with nucleolin(NCL,human)that conserved in many species.To determine whether overexpress NCL on the host cell membrane can favor PCV2 binding,infection,and production,hela cells were transfected with a vector containing the NCL gene tagged with FLAG.Two cell clones,pcDNA-TOPO-FNCL and pcDNA-TOPO-NCLF,were established.The result of real-time PCR showed that overexpression of NCL significantly increased viral yield in Hela cells.To determine the influence of NCL deficiency on PCV2 infection,the expression of NCL in Hela cells was knocked down with siRNA.Decreased PCV2 mRNA level was observed by real-time PCR analysis.All of these results suggest that the nucleolin has a certain influence against PCV2 infection and replication,and it is indispensable to uncover the mechanisms that underlie virus infection and replication in host cells.