Signal Peptide-mediated Post-translational Processing And Release Of CSFV E^rns

Classical swine fever（CSF）,caused by classical swine fever virus（CSFV）, is a highly contagious infectious disease with the characteristic of immunosuppression and high mortality.Under the natural conditions, only hog and sus scrofa at different age can be infected with CSFV. CSF epidemic worldwide represents one of the leading threats to the pig industry yearly, thus it is listed as one of notifiable diseases of Office International Des Epizooties（OIE）. CSFV is a single strand positive sence RNA containing a single large open reading frame（ORF）, which serves as m RNA to directly translate into polyprotein for viral propagation. CSFV consists of the envelope proteins E^rns、E1、E2 and Core protein. The heavily glycoprotein E^rns only exists in pestivirus and exhibits RNase activity to degrade viral single strand and double strand RNA that inhibit the production of type I interferon, thus influences the host immune response to natural antiviral. Moreover, it should be notified that partial E^rns protein exists as a soluble form in the extracellular space and binds to the surface receptor of host cell to induce the adsorption and entry of CSFV. It can induces apoptosis in T-cells of several species without cell membrane damage, thus influences the host immune response to natural antiviral.In eukaryotic cells, signal peptides are commonly N-terminal extensions directing nascent or completed proteins from the cytosol to translocation sites in the membrane of the endoplasmic reticulum（ER） and cleaved from the precursor protein by a membrane-bound signal peptidase. The ER lumen provides a distinct folding environment for fulfilling the multitude of post-translational modifications, including asparagine-linked glycosylation and intermolecular disulfide bonds. The N-glycans in the modified protein contribute to folding processes and secretion of proteins. E^rns protein represents a virulence factor and exerts multiple function in viral infection and pathogenesis of CSFV, extracellular secretion may be the prerequisite for its function realization, thus, uncovering the factors regulating the biosynthesis of E^rns protein is necessary.In this study, to express E^rns protein of CSFV and prepare its polyclonal antibody（p Ab）, the E^rns gene from CSFV Shimen strain was amplified and cloned into the prokaryotic expression vector p ET-28 a, which was transformed into the prokaryotic expression strain E.coli BL21（DE3）. A recombinant protein E^rns about 25 k Da was expressed with IPTG induction mainly in the inclusion body by SDS-PAGE analysis. Under the conditions of denaturation, the E^rns protein in the inclusion body were successively purified by affinity chromatography and gel slice. The purified recombinant E^rns protein specially reacted with the hyper-immune serum against CSFV. In additon, BALB/c mice were immunized four times with the purified recombinant E^rns protein for the polyclonal antibody. Indirect ELISA result showed that the antibody titers in the serum of immunized mice was at 1:50000. Moreover, the obtained anti-E^rns p Ab can specifically react with the E^rns protein expressed in virus-infected cells and E^rns-EGFP-expressing cells.Glycoprotein E^rns plays multiple functions in CSFV life cycle and be secreted outside of infected cells, while the mechanism and role of its extracellular secretion has not yet identified. In this study, a 20-amino acid signal peptide sequence at the C-termini of CSFV Core protein, preceding E^rns protein, was predicted by online programs Signal P. To study its role in the posttranslational processing and release of E^rns protein, the swine testicle（ST） cell lines stably expressing the E^rns and ΔE^rns（signal peptide deleted） proteins of HCLV strain were established by screening with puromycin. As a result, higher fluorescence signal of E^rns-EGFP was observed in comparison with that of ΔE^rns-EGFP, and both of them were predominantly distributed in the perinuclear region. N-glycosylation digestion assay and Western blot analysis revealed that the intracellular expression of the EGFP-tagged target proteins were enhanced by the signal peptide, which also determines the glycosylation of E^rns protein. Meanwhile, the intracellular distribution of E^rns protein deleted carboxyl terminal membrane anchor sequence were subjected for a confocal laser scanning microscopy and the result showed that the signal peptide directed translocation of the nascent E^rns protein to endoplasmic reticulum（ER） for an N-glycosylated modification. Moreover, to study the role of signal peptide in the release of E^rns protein, the secreted fusion proteins were detected by quantifing green fluorescent intensity of the culture medium. As expected, the signal peptide induced the secretion of E^rns protein into culture medium and ΔE^rns protein can not be glycosylated and released without the signal peptide. Moreover, some the exosome about diameter 30 nm were observed by transmission electron microscope.The secreted E^rns protein has higher degree glycosylation than its intracellular form. In addition, the E^rns protein may be released as an insoluble form since it can be isolated with Total Exosome Isolation Reagents.Multiple alignment showed that there were several different residues in the signal peptide sequences derived from the vaccine strain HCLV, virulent Shimen strain and field isolate GD53/2011. The EGFP-tagged ΔE^rns and E^rns fusion proteins of different CSF viruses were transiently expressed and detected by western blot. The result showed that signal peptides also regulated the glycosylation of E^rns proteins of CSFV strains Shimen and GD53/2011, but the molecular mass of the latter was a little higher than that derived from Shimen and HCLV. To identify the crucial amino acid residues in the signal peptide of C-strain associated with the biosynthesis and release of E^rns protein, multiple alignment of amino acids of signal peptides was performed and the amino acid residues 251LLAWA255 in the hydrophobic core of the signal peptide were highly conserved among different CSFV genotypes. Then a series of E^rns mutant proteins with truncated or mutated signal peptides were transiently expressed. Interestingly, the dual substitutions of 251LL252 and 253AW254 with NN or 251LLAW254-deleted（ΔLLAW） mutants completely blocked the intracellular glycosylation of E^rns protein and the extracellular secretion was also significantly reduced. Futher, E^rns proteins with mutated or deleted signal peptides were found to be mainly distributed in the nucleus when the C-terminal membrane anchor sequences were deleted. Meanwhile, the point mutation of 7Trp with asparagine and ternary mutations of 251L252L254 W substituted with A（alanine） markedly reduced the expression and secretion of E^rns mutants. Collectively, it indicated that the highly conserved residues 251LLAW254 in signal peptide are critical for the glycosylation and secretion of E^rns protein.In conclusion, the recombinant E^rns protein was successfully expressed and purified by by affinity chromatography and gel slice, and polyclonal antibody against E^rns protein with a high antibody titers was obtained, which are useful for future studies on the functions and immunogenicity of E^rns protein. Moreover, the signal peptide was found to be a determinator in the intracellular expression, N-glycosylated modification and release of CSFV E^rns protein. In comparison with the intracellular form, extracellular ultra-glycosylated E^rns protein was observed and it may be secreted as an insoluble form. In addition,the highly conserved residues 251LLAW254 in the signal peptide were identified as the critical motif for the biosynthesis of E^rns protein. Overall, this study provides a basis for future studies on the molecular mechanism of intracellular processing and extracellular secretion of CSFV E^rns protein.