Assay for influenza virus endonuclease using DNA polymerase extension of a specific cleavage product.
The synthesis of influenza virus mRNA requires primers generated by cleavage of host cell transcripts 10-13 nucleotides from the 5' end by a virally encoded endonuclease. This novel enzyme is an attractive target for the development of antiviral agents. An assay for the influenza virus endonuclease has been developed that monitors the substrate cleavage reaction only at the correct position in the sequence, thereby discriminating against nonspecific RNA cleavage products. The influenza endonuclease assay is sensitive enough to detect 200 amol of product. The assay employs a DNA polymerase-catalyzed extension of the endonuclease cleavage product using radiolabeled dGTP and a DNA template containing a 3' region complementary to the product joined to a 5' region consisting of 10 dC residues. The influenza endonuclease assay does not involve gel electrophoretic separation and is amenable to high volume screening of potential inhibitors. The assay may also be employed to determine the site of influenza endonucleolytic cleavage in the substrate.
Cole
J L
JL
Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Kuo
L C
LC
Olsen
D B
DB
D001483:Base Sequence; D002384:Catalysis; D004259:DNA-Directed DNA Polymerase; D004720:Endonucleases / Q000032:analysis*; D016014:Linear Models; D008969:Molecular Sequence Data; D009975:Orthomyxoviridae / Q000201:enzymology*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013696:Temperature
C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D004720:Endonucleases