RNA-dependent RNA polymerase activity of Classical swine fever virus NS5B protein expressed in natural host cells.
The NS5B gene, cloned from Classical swine fever virus (CSFV) genome, was expressed in porcine kidney cells PK-15, natural host of CSFV. In purifying cytoplasmic extracts from these cells by means of different concentrations of salt, glycerol and detergent four fractions, namely crude supernatant (SC) and different purified supernatants (S1, S2 and S3) were obtained. Using Western blot analysis the NS5B protein was found in all these fractions, showing that it was soluble in both higher and lower concentrations of salt, glycerol and detergent. The NS5B protein present in the four different fractions exhibited RNA-dependent RNA polymerase (RdRp) activity, but it was unable to complete the whole process of RNA synthesis. Site-directed mutation analysis showed that Thy216 and Cyt228 were essential for RNA synthesis while Cyt219 was not, suggesting that CSFV RdRp was template-specific. We conclude that initiation of RNA synthesis by CSFV RdRp includes also template priming.
Xiao
M
M
Biology Department, College of Life and Environment Sciences, Shanghai Teachers' University, Shanghai, 200234 P.R. China. xiaoming88@263.net
Chen
J
J
Li
B
B
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006692:Classical Swine Fever Virus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D016679:Genome, Viral; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000032:analysis / Q000378:metabolism*; D013552:Swine; D013698:Templates, Genetic; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis*
C096041:NS5 protein, flavivirus; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase