title: A quantitative assay to monitor HSV-1 ICP0 ubiquitin ligase activity in vitro.
abstract: The ubiquitin-proteasome system is an essential cellular process that plays a fundamental role in the regulation of protein stability. This pathway is tightly controlled by a sequential cascade of enzymatic steps that culminates in the formation of a poly-ubiquitin chain onto the substrate protein targeted for 26S proteasome degradation. Through a process of co-evolution viruses have evolved mechanisms to utilize or suppress this pathway in order to enhance their replication and spread. One of the first proteins to be expressed during herpes simplex virus 1 (HSV-1) infection is ICP0, a viral RING-finger E3 ubiquitin ligase that targets a variety of cellular proteins for ubiquitination and proteasome-dependent degradation. This activity is required in order for ICP0 to efficiently stimulate the onset of HSV-1 lytic infection and viral reactivation from latency. While it is clear that the RING-finger domain of ICP0 plays an important role in the biology of HSV-1, methods for accurately quantifying its biochemical activity are currently lacking. Here we describe a protocol that enables the quantitative measurement of the ubiquitin ligase activity of ICP0 using near-infrared (IR) western blot imaging. The use of such imaging technology provides an accurate means to examine the biochemical and kinetic parameters of RING-finger ubiquitin ligases in solution, and may provide significant application for inhibitor studies.
authors: MRC-University of Glasgow Centre for Virus Research, 464 Bearsden Road, Glasgow G61 1QH, UK. Electronic address: chris.boutell@glasgow.ac.uk.
authors: Chris
authors: C
authors: Boutell
authors: Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66049, USA.
authors: David J
authors: DJ
authors: Davido
mesh_terms: D015153:Blotting, Western / Q000379:methods*; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000472:pathogenicity; D017874:Immediate-Early Proteins / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D017874:Immediate-Early Proteins; D044767:Ubiquitin-Protein Ligases; C051804:Vmw110 protein, Human herpesvirus 1
keywords: HSV-1; ICP0; Infrared; LI-COR; RING; Ubiquitin