Studies on Classical Swine Fever Virus structural and non-structural proteins: Immunogenicity of envelope proteins and NTPase activity of NS4B protein

Classical swine fever (CSF) is a highly contagious and often fatal disease that affects swine throughout various regions of the world. The etiological agent, CSF virus (CSFV), is a positive-sense single-stranded RNA virus that belongs to the genus Pestivirus within the family Flaviviridae. The CSFV genome encodes a single polyprotein precursor that is co- and post-translationally processed to produce four structural (C, Erns, E1, E2) and eight non-structural (NS) proteins (N pro, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Each of the three envelope proteins (Erns, E1, and E2) contains highly conserved glycosylation sites. We investigated the role of glycosylafion in the immunogenicity and protective efficacy of CSFV baculovirus-expressed nonglycosylated and glycosylated strain Brescia (BICv) envelope proteins following intramuscular injection of 10-12 week-old pigs. Immunization with nonglycosylated or glycosylated E1 didn\u27t confer protection against BICv. Complete removal of carbohydrate moieties of Erns and E2 impaired the ability of these proteins to induce protection in pigs. Only glycosylated Erns and E2 generated strong antibody response and protected animals against challenge. Analysis of the immunogenicity and protective efficacy of single site glycosylafion mutant Erns and E2 revealed that all mutant Ems and E2 induced protection in pigs, however, inoculation with ErnsN3, E rnsN7, E2N1, and E2N2 did not prevent clinical manifestation of CSF. These results indicate that glycosylation of E1 and E2 is critical for the development of protection against CSFV and its modulation affects the immunogenicity of the two proteins. We also produced polyclonal anti-E1 serum in rabbits inoculated with E. coli-expressed E1 which was used to investigate the relative electrophoretic mobility of E1 in lysates of swine kidney cells infected with different parental and mutant viruses missing one or more E1 glycosylation sites.^ In addition, we identified conserved sequence elements observed in nucleotide-binding motifs (NBM) that hydrolyze NTPs within the CSFV NS4B. Expressed NS4B hydrolyzes both ATP and GTP. Substitutions of critical residues within the identified NBM Walker A and B motifs significantly impair the ATPase and GTPase activities of expressed proteins. These results have important implications for developing novel antiviral strategies against CSFV.