ASSAY FOR INFLUENZA-VIRUS ENDONUCLEASE USING DNA-POLYMERASE EXTENSIONOF A SPECIFIC CLEAVAGE PRODUCT

The synthesis of influenza virus mRNA requires primers generated by cl eavage of host cell transcripts 10-13 nucleotides from the 5' end by a virally encoded endonuclease. This novel enzyme is an attractive targ et for the development of antiviral agents. An essay for the influenza virus endonuclease has been developed that monitors the substrate cle avage reaction only at the correct position in the sequence, thereby d iscriminating 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 endonuc lease cleavage product using radiolabeled dGTP and a DNA template cont aining a 3' region complementary to the product joined to a 5' region consisting of 10 dC residues. The influenza endonuclease assay does no t involve gel electrophoretic separation and is amenable to high volum e screening of potential inhibitors. The assay may also be employed to determine the site of influenza endonucleolytic cleavage in the subst rate. (C) 1995 Academic Press, Inc.