Influenza replicase/RNA polymerase is essential for a productive influenza infection. Using its RNA-dependent replicase activity, this enzyme generates new copies of the influenza RNA that are packaged into new virions. Additionally, this enzyme acts as an RNA-dependent RNA polymerase to synthesize mRNAs that code for the various influenza proteins. In order to generate mRNA, the enzyme first steals a capped primer from cellular mRNAs, elongates the primer, copies the coding portion of the viral RNA, and then polyadenylates the newly generated viral mRNA. The presence of multiple activities essential for survival and replication of influenza suggests that the replicase/RNA polymerase would be an ideal target for novel therapeutics. Additional support for the idea of attacking influenza replicase/RNA polymerase is the observation that the vast majority of all antiviral agents directed against other viruses target the viral RNA or DNA polymerase. The goal of this application is to develop a high-throughput assay for identifying novel inhibitors of the influenza replicase/RNA polymerase. In order to accomplish this goal, two specific aims will be addressed; i) develop a baculovirus system for producing multi-mg amounts of the enzyme, and; ii) design and demonstrate an assay that will serve as a quantitative measure of the potency with which compounds inhibit either the replicase or RNA polymerase functions. Importantly, assays of RNA polymerase activity will detect inhibition of any of the 3 essential functions associated with the RNA polymerase; cap snatching, RNA synthesis, and polyadenylation. A high throughput assay for inhibition of influenza replicase/RNA polymerase activity will provide a means to screen thousands of compounds, thereby providing a powerful platform for the discovery of novel influenza therapeutics.

R01AI071338

2006-02-01

2008-07-31