RUI: Ubiquitin-Mediated Degradation of Picornavirus Proteins

Bates College, Lewiston ME

Investigators

Abstract

The ubiquitin/26S proteasome system is the major process by which proteins are selectively degraded in eukaryotic cells. Little is currently understood about how the ubiquitin system selects substrates, and the polyubiquitinating mechanism is not understood clearly. A small number of viral proteins have been shown to be substrates for ubiquitin-mediated destruction. These include the 3C proteases of the encephalomyocarditis virus (EMCV) and hepatitis A virus (HAV) and the 3D RNA polymerase of HAV. These viral substrates for ubiquitin/26S proteasome system offer the opportunity to study the mechanistic details by which proteins are selected and ubiquitinated by the ubiquitin-conjugating machinery, as well as to examine the potential role of selective protein destruction in picornavirus replication. A major goal of this project is to employ the EMCV and HAV 3C proteases as substrates for measurements of binding affinities to ubiquitin system enzymes and for addressing the fundamental questions of whether polyubiquitin chain synthesis occurs via a processive mechanism and whether it can involve more than one ubiquitinating enzyme complex. This research will also initiate a characterization of the ubiquitin-conjugating system that targets the HAV 3D polymerase for degradation, in preparation for future studies of how this substrate is selected and what function its destruction serves in HAV replication. This project includes an endeavor to develop an inducible expression system for 3C protease genes cloned into mammalian cells, and this system will be used to evaluate the cytotoxicity of the EMCV 3C protease and to determine whether a failure to ubiquitinate the 3C protease effects the efficiency with which EMCV is replicated. These studies will involve undergraduate students in research on the mechanism of some of the crucial steps in protein degradation.