Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE- vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE- replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs