Characterization of NS3, NS5A and NS5B of classical swine fever virus through mutation and complementation analysis

In order to get further insight into the organization of the pestiviral replication machinery, characterization of NS3, NS5A and NS5B of classical swine fever virus (CSFV) through mutation and complementation analysis was performed. Mutation analysis in genomic replicons and subgenomic replicons indicated importance of the GDD motif in NS5B, the DEYH motif in NS3 and the conserved sequence C2717-C2740-C2742-C2767 in the NS5A for CSFV recover and viral RNA synthesis. Complementation experiments were performed between subgenomic replicons, between RNA replicons or between RNA replicon and expressed nonstructural protein. Rescue of virus and recover of viral RNA synthesis were examined in these complementation experiments. Results showed that mutations within NS5A, neither NS5B nor NS3, can be trans-complemented, strongly suggesting that NS5B and NS3 function in cis mode for regulation of replication. We assumed that the necessary membrane association of CSFV NS5B and NS3 could occur only when they are being translated and originated from an identical translation template, with the exception of NS5A whose membrane association might occur post-translationally.