NEW MOTILITY MECHANISM AND HIGH SPEED, IN VIVO, IMAGING OF MOTOR PROTEIN DYNAMICS - DESCRIPTION (provided by applicant): Live cell fluorescence microscopy will be used to investigate anomalously rapid actin-dependent motility of poliovirus that is 5-10 times faster than the fastest known actin-dependent translocation of cargo. Three aspects of this behavior will be investigated. First, the hypothesis that a myosin motor protein is responsible for the motility will be tested by means of various drugs, fluorescent protein myosin analogs, and siRNA. Myosin specific drugs or siRNA will be tested for inhibition of the rapid motion and various fluorescent protein myosin analogs will be tested for colocalization with poliovirus during transport. Second, the novel motility will be characterized in vivo using imaging-based, high speed and high accuracy particle tracking with the goal of dissecting the motion to the level of individual motor protein steps. Careful analysis of particle tracking data will be used to gain insight into the mechanochemical properties that give rise to the rapid motion. Third, the role of the motility to the infection pathway of poliovirus will be evaluated. Cells which lack an endogenous poliovirus receptor will be transfected with a fluorescent protein poliovirus receptor analog in order to directly visualize whether the rapid motion of poliovirus represents the active recruitment of poliovirus receptors. A three-dimensional particle tracking method, in combination with a dual fluorescent labeling scheme in which poliovirus capsids are labeled with one dye and poliovirus RNA is labeled with a different dye, will be used to determine whether the motion functions to transport viruses to a site of genome release. Poliovirus is a model system for nonenveloped RNA viruses, a group which includes numerous viruses ranging from the common cold to hepatitis A. Elucidating the mechanisms by which polio enters and navigates cells will enhance our basic understanding of viral pathogenesis and will lead to possible targets for therapies based on the inhibition of viral entry or transport.
