BIOCHEMICAL CHARACTERIZATION OF ENTEROVIRAL 2C PROTEINS

Enteroviruses represent an existing and emerging threat to public health, especially children. Because of the high rate of mutation of RNA viruses and their capacity to recombine, there is a high risk of virulent strain evolution. It is impossible to predict the next dangerous serotype of these viruses, leading to the need of development of pan-viral or pan-enteroviral therapeutics. All enteroviruses encode an enzyme called 2C that is essential for both enteroviral replication and assembly. In this dissertation, we develop an experimental framework to define mechanism of action of enteroviral 2C inhibitors. In Chapter 1, we summarize what is known about the viral protein 2C and structurally related viral helicases. We discuss compounds that have been previously known to inhibit 2C protein with unknown mechanisms of action. Chapter 1 ends with the admission that historically there has been very little learned about RNA-specific superfamily 3 helicases, to which 2C belongs. The dearth of mechanistic studies of the enteroviral 2C protein was because of the lack of an established biochemical study system. In Chapter 2, we detail our success at purifying a full-length, RNA-stimulated 2C ATPase protein; we also develop a robust, truncated 2C study system that does not require detergent that will be essential for future biophysical characterization. In Chapter 3, we define how enteroviral 2C protein has RNA-stimulated ATPase activity that relies on a two-step mechanism for both binding RNA and ATP. Additionally, 2C protein is stimulated only by RNA and not DNA, nor other forms of RNA lacking an accessible 2’-OH. In Chapter 4, we begin to characterize the effects of archetypical 2C inhibitors like guanidine hydrochloride and 5-(3,4-dichlorophenyl)-methylhydantoin (hydantoin) on enteroviral 2C ATPase activity and RNA-binding capabilities in vitro. In Chapter 5, we conclude with the remaining critical questions pertaining to 2C and the many ways that we can leverage the 2C study system developed herein to answer them.