Enteroviral 2C protein as a therapeutic target

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Project Abstract Non-polio human enteroviruses (NPEVs) represent an existing and emerging threat to public health, especially to infants and children. With more than 100 NPEVs known and the high rate of mutation and recombination of these viruses, the risk for evolution of virulent strains is high. Because we cannot predict the serotype of any newly emerging strain, addressing this problem effectively will require pan-enterovirus solutions. All NPEVs encode an enzyme, which has been termed 2C. This enzyme is as well conserved as the enterovirus RNA- dependent RNA polymerase (RdRp). Indeed, inhibitors of 2C with activity against multiple enteroviruses have been known for decades, with even more reported over the past few years alone. A major obstacle to further development of these compounds is the absence of an established experimental framework to define mechanism of action and to guide design of pan-enterovirus activity. Two of the earliest inhibitors of 2C: guanidine hydrochloride (GuHCl); and 5-(3,4-dichlorophenyl)- methylhydantoin (hydantoin), revealed roles for 2C both in genome replication and virion assembly. Because 2C protein is a member of helicase superfamily 3, the prevailing view has been that 2C is a hexameric helicase that cooperates with RdRp by unwinding RNA structure during genome replication and by facilitating genome encapsidation during virion assembly. Unfortunately, only indirect evidence exists to support these views. Nearly five years ago, our laboratory initiated an effort to connect the biochemical and biophysical properties of 2C to its biological functions using poliovirus (PV) as our model. The advances made to date, all unpublished, have changed the way we think about structure-dynamics-function relationships of 2C, the subcellular locations in which 2C manifests its virion-assembly function, and the physical properties of the virus-induced membranes with which 2C must interact during infection. In this application, we propose to add Enterovirus A71 (EV-A71), Coxsackievirus B3 (CVB3), and Enterovirus D68 (EV-D68) to our studies of PV to establish unifying models for the biological functions of 2C that will provide a framework to establish 2C as a pan-enterovirus therapeutic target. We will do so by pursuing the following specific aims: Elucidate the quaternary structure and kinetic mechanism of 2C ATPase (Aim 1); Elucidate the impact of drug resistance on 2C ATPase activity and viral fitness (Aim 2), and Characterize sites contributing to genome encapsidation (Aim 3).