Classical swine fever (CSF),caused by classical swine fever virus (CSFV),is a highly contagious infectious swine disease,causing huge economic losses every year.CSFV is a member of the Pestivirus genus within the Flaviviridae family and harbors a single-strand positive RNA genome containing a single large open reading frame (ORF),which encodes a 3898-amino acid polyprotein.This polyprotein is processed by viral and host proteases to produce 12 mature proteins,including 4 structural proteins (C,Erns,E 1,E2) and 8 non-structural proteins (Npro,p7,NS2,NS3,NS4A,NS4B,NS5A and NS5B) (Lindenbach and Rice,2007).Glycoprotein Erns of classical swine fever virus (CSFV) is a virion-associated and secreted protein containing RNase activity and was identified as a critical factor for viral pathogenesis.Previous studies show that Erns translation is regulated by a C-terminal signal peptide of Core protein (C),but its post-translational processing remains unknown.In present study,swine testicular cell lines (ST) expressing CSFV C strain Erns protein with or without core protein signal peptide at its N-terminal were constructed to elucidate the role of the signal peptide in the biosynthesis and release of Erns protein.In the presence of signal peptide,Erns protein expression was significantly enhanced compared to that without signal peptide.After translation,the signal peptide can direct translocation of the nascent Erns protein to endoplasmic reticulum (ER) for the N-glycosylated modification.The glycosylated Erns protein was subsequently secreted into culture medium,but not the Erns protein without signal peptide or glycosylation,and an extracellular ultra-glycosylated Erns protein was observed in comparison with the intracellular form.In addition,mutagenesis experiments suggest that the highly conserved residues 251LLAW254 are critical motif in the signal peptide for biosynthesis of Erns protein.Taken together,these results suggest that the N-terminal signal peptide play a pivotal role in the expression,processing and release of Erns protein.