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Purification and enzymatic characterization of west nile virus RNA dependent RNA polymerase
Author(s) -
Mattheus Alexandra,
Coleman Liana,
Temple Jeffrey S.,
Doughty Michael B.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a903-b
Subject(s) - polymerase , rna dependent rna polymerase , rna polymerase , virology , rna , enzyme , biology , chemistry , biochemistry , gene
West Nile Virus is a virulent member of the Flaviviridae family of viruses. Infection occurs through viral transfer from mosquitoes to birds with some human and equine infections. Since the summer of 2002 there have been over 16,000 human cases with a mortality rate of 3.8%. The WNV genome contains approximately 11,000 base pairs encoding ten viral proteins, three of which are structural proteins (C, prM, and E), while the other seven are non‐structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The NS5 region is of particular interest to this project since it encodes the RNA dependent RNA polymerase (RdRp). It is believed that the polymerase uniquely replicates the RNA genome during the replication process. The coding region for RNA polymerase has been amplified via Polymerase Chain Reaction and cloned into an E. coli expression system. The enzyme has been partially purified by affinity chromatography and gel filtration chromatography and an assay for the enzyme activity has been developed. Presented here are the results of the purification scheme and enzyme assay.