High Throughput Assays for Ion Channel Activities of Influenza A & B Viruses

Project: Research project

Project Details


DESCRIPTION (provided by applicant): Influenza contributes substantially to worldwide morbidity and mortality. Influenza A virus has caused all human pandemics and influenza B virus infections occur annually and in some recent years they have constituted 50% of total influenza virus infections, with high infection rates in nursing homes, hospitals, boarding schools and prisons. Influenza is a select agent. The M2 ion channel protein of influenza A virus is the target of the antiviral drug amantadine and the BM2 ion channel protein is a potential target for antiviral drugs because the ion channel activity of each protein is essential for replication of the virus. There is a need to develop more effective antiviral drugs against the A/M2 ion channel because escape mutants arise in patients taking the drug and there are no presently known antivirals against the BM2 ion channel. To screen compounds for antiviral activity the community needs high-throughput methods that can reliably detect decreases in ion channel activity. We propose to develop and validate two high-throughput screening assays for this purpose. The assays are based on the high rate of proton conduction of both of these ion channels and involve measurement of either intracellular pH changes in mammalian cells that express the ion channel or pH changes in liposomes reconstituted to contain the recombinant ion channel protein. Both of these assays are now used routinely in our laboratories for mechanistic studies of these ion channels but neither of the assays has been developed for high-throughput screening. We propose to develop robotic methods to perform assays in 596 well plates using both methods. Our goals will be (1) to identify the experimental variables that give rise to variation in the readings from well-to-well and minimize this variation such that the coefficient of variation is less than 10% and (2) to reduce the baseline activity for A/M2 in the presence of amantadine to less than 2% of the control value. The methods developed will be disseminated to others in three ways. First, we will publish our results in the peer-reviewed scientific literature; second, we will post the results and a detailed method on our laboratory web-sites; third, we will offer a minicourse that will be open to members of the community and will give both theoretical and practical aspects of performing the assays, reducing variability and interpretation of results. These assays will make it possible to perform efficient searches for antiviral drugs against the M2 ion channels.
Effective start/end date3/1/068/30/07


  • National Institute of Allergy and Infectious Diseases (1 R01 AI071342-01 (year 2))


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