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 (NS5Bdelta24) 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 Mg2+ and Mn2+ supported de novo initiation, however, RNA synthesis was more efficient in the presence of Mn2+ than in the presence of Mg2+. De novo initiation of RNA synthesis was stimulated by preincubation with 0.5 mm 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.
Yi
Guang-Hui
GH
Institute of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China.
Zhang
Chu-Yu
CY
Cao
Sheng
S
Wu
Hai-Xiang
HX
Wang
Yi
Y
D020413:3' Untranslated Regions; D015152:Blotting, Northern; D015153:Blotting, Western; D006692:Classical Swine Fever Virus / Q000201:enzymology*; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature
D020413:3' Untranslated Regions; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D008345:Manganese; D012313:RNA; D012324:RNA-Dependent RNA Polymerase; D008274:Magnesium