NS3 of pestiviruses contains a protease domain and a RNA helicase/NTPase domain. 
Contradictory results have been reported regarding NS3 in RNA synthesis. To 
investigate the effect of NS3 on classical swine fever virus (CSFV) NS5B 
RNA-dependent RNA polymerase activity (RdRp) activity and NS3-NS5B interaction, 
RdRp reactions, GST-pull-down assays and co-immunoprecipitation analyses 
containing NS5B and either of NS3 protein and the different truncated NS3 
mutants were performed, respectively. We found that NS3 stimulated NS5B RdRp 
activity in a dose-dependent manner by binding to NS5 through a NS3 protease 
domain. Furthermore, mapping important regions of the NS3 protease domain was 
carried out by deletion mutagenesis, associated with RdRp reactions, 
GST-pull-down assays and co-immunoprecipitation analyses. Results showed that 
stimulation of CSFV NS5B RdRp activity was obtained by NS3 binding to NS5B 
through a 31-amino acid fragment at the N-terminal end of NS3 protease domain, 
which mediated a specific NS3-NS5B interaction.