De novo RNA synthesis by a recombinant classical swine fever virus RNA‐dependent RNA polymerase

Classical swine fever virus nonstructural protein 5B (NS5B) encodes an RNA‐dependent RNA polymerase, a key enzyme of the viral replication complex. To better understand the initiation of viral RNA synthesis and to establish an in vitro replication system, a recombinant NS5B protein, lacking the C‐terminal 24‐amino acid hydrophobic domain, was expressed in Escherichia coli . The truncated fusion protein (NS5BΔ24) was purified on a Ni‐chelating HisTrap affinity column and demonstrated to initiate either plus‐ or minus‐strand viral RNA synthesis de novo in a primer‐independent manner but not by terminal nucleotidyle transferase activity. De novo RNA synthesis represented the preferred mechanism for initiation of classical swine fever virus RNA synthesis by RNA‐dependent RNA polymerase in vitro . Both Mg 2+ and Mn 2+ supported de novo initiation, however, RNA synthesis was more efficient in the presence of Mn 2+ than in the presence of Mg 2+ . De novo initiation of RNA synthesis was stimulated by preincubation with 0.5 m m GTP, and a 3′‐terminal cytidylate on the viral RNA template was preferred for de novo initiation. Furthermore, the purified protein was also shown, by North‐Western blot analysis, to specifically interact with the 3′‐end of both plus‐ and minus‐strand viral RNA templates.