Enteroviral 2C protein as a therapeutic target

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 The advances made to date, all unpublished,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).

Grant Number: 5R01AI169462-04
NIH Institute/Center: NIH
Principal Investigator: CRAIG CAMERON