Expression Of Classical Swine Fever Virus Proteins In Host Cells And Its Regulation Of Viral Infection

In this study, we focused on the expression of CSFV proteins in host cells, which either were infected with CSFV or transfected with eukaryotic expression systems for expressing CSFV proteins. In addition, influences of CSFV proteins, which were expressed with eukaryotic expression systems, on host cells as well as virus infection were investigated.In order to observate CSFV proteins in infected cells, the recombinant proteins by prokaryotic expression system were used to immunize Kunming mice for preparation of polyclonal antibody against CSFV proteins. Eleven recombinant CSFV proteins were successfully recovered except P7, and a series of sera, which contained high titer antibodies against CSFV proteins, were obtained. The target viral proteins were detected in infected cells by Western-Blotting, and proteins including E0, E1, E2, and NS5A were also checked as polymeric proteins. Indirect immunofluorescence assay (IFA) revealed that Npro is located at nucleus, and C, EO and NS2both in cytoplasm and at nucleus, while the remaining proteins in cytoplasm. Unusually, Erns was also distributed on cellular membrane. These findings implied that proteins distributed at nucleus were related to a function of modulating cellar life cycle or function, while those in cytoplasm were implicated in viral replication, particle assembly, and proliferation.Eukaryotic expression vectors containing protein genes of CSFV C-strain were conducted with pEGFP-Nl vector to investigate the expressions of CSFV protein in ST cells. The results of PCR detection showed that the target protein genes were successfully transfected into ST cells, and the expression of recombinant CSFV proteins was observed under fluorescent microscope except pEGFP-E1cells. Taken EGFP as flag, there exited differences of the location and intensity among CSFV protein cells:EGFP was detected in cytoplasm of all positive cells, while also detected at nucleus of pEGFP-N1, pEGFP-Npro, pEGFP-C and pEGFP-NS2cells. Enrichment of recombinant protein surround the nucleus was observed in pEGFP-EO and pEGFP-NS3cells. Additionally, EO-EGFP alone can secrete out and distribute on cellular membrane. Eleven cell lines, including Npro, C,△EO, E0, E2, P7, NS2, NS4A, NS4B, NS5A, and NS5B, were obtained, while El and NS3failed. As the thesults of Western-Blotting revealed, recombinant proteins in pEGFP-Npro, pEGFP-C, pEGFP-NS2and pEGFP-NS5B cell lines were cleaved into viral proteins and EGFP completely, and those in pEGFP-AEO, pEGFP-EO, and pEGFP-E2cleaved partly, while in pEGFP-NS4A and pEGFP-NS4B detected to be un-cleaved. These results demonstrated as followed:NS4A and NS4B were cleaved by viral protease during CSFV infection; and the self protease is responsible for cleaving Npro-EGFP and NS2-EGFP; and C-EGFP and NS5B-EGFP was digested by a cellular protease, which occurred at the3-terminal of C or NS5B; while△EO-EGFP, EO-EGFP, E2-EGFP and NS5A-EGFP were partly cleaved by cellular protease, however the mechanism of regulation remains un-known. Cell proliferation detection by CCK-8kit demonstrated that, compared with ST cells, the proliferation of pEGFP-NS4B cells was obviously inhibited, and pEGFP-NS2, pEGFP-NS5A and pEGFP-P7cells slightly lagged behind, and pEGFP-N1, pEGFP-Npro, pEGFP-E0, pEGFP-E2and pEGFP-NS4A cells equally, however pEGFP-NS5B cells were equally in early stage then backward at late. These results illustrated the cellular growth can be regulated by some CSFV proteins with an inhibition. Cell cycle arrest at S-phase was observed in pEGFP-NS4B and pEGFP-NS5B cells based on cell cycle analysis by flow cytometry, and it can be concluded that both CSFV NS4B and NS5B have a capability to modulate cell cycle by prolonging cellular S-phase.The CSFV protein cell lines were separately infected with CSFV C-strain or HNLY-2011isolate, and the viral yield was detected by quantitative real-time RT-PCR, and then the regulation of viral proliferation by CSFV proteins was studied. A SYBR-Green I based quantitative real-time RT-PCR, which taken β-Actin as an internally control, was successfully developed for detecting CSFV yield. According to the effect on viral proliferation, these cell lines could be classified as three types:pEGFP-Npro, pEGFP-EO and pEGFP-NS5B cell lines have a promoting action (pEGFP-EO cells especially), and pEGFP-C, pEGFP-E2, pEGFP-NS2, pEGFP-NS4B and pEGFP-NS5A exhibit a negative effect, while pEGFP-P7and pEGFP-NS4A present positive and negative feedback control. Additionally, based on the difference of proliferation between CSFV C-strain and HNLY-2011strain, CSFV proteins were further clustered into three groups:C and NS4B presented as specific action, E0, E2, P7, NS2and NS4A as non-specific action, meanwhile Npro, NS5A and NS5B as relatively specific action. So it can get a conclusion that the viral proliferation can be modulated by both viral-viral and viral-celllular protein interaction networks, which may be favourable for viral intracellular survival.The reconbinant protein EO-EGFP was observed on the cellular manbrane of pEGFP-EO cell line, and also secreted into cells’culture, which can be precipated by centrifugation. The result of Western-blotting revealed that the signal peptide is crucial for processing EO mature and glycosylation, and the glycosylated EO has a property of polymerization to form protein aggregates and secretion out of cells. The further experiment revealed that CSFV virions can adsorb each other and aggregate into virosomes, which can be precipated from culture by centrifugation same as EO. Taken together, these results demonstrated that CSFV EO has a function of clustering virions to assemble virus clumps, and then virus clumps can be secreted out of infected cells through Golgi apparatus by means of EO secretion. This may laid a foundation for further studies of CSFV release.