Classical swine fever virus nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase, a key enzyme of the viral replication complex. To better under-stand the initiation of viral RNA synthesis and to establish anin vitroreplication system, a recombinant NS5B protein, lacking the C-terminal 24-amino acid hydrophobic domain, was expressed inEscherichia coli. The truncated fusion protein (NS5BD24) was purified on a Ni-chelating HisTrap affinity column and demonstrated to initiate either plus-or minus-strand viral RNA synthesisde novoin a primer-independent manner but not by terminal nucleotidyle transferase activity
