title: Functional characterization of residues required for the herpes simplex virus 1 E3 ubiquitin ligase ICP0 to interact with the cellular E2 ubiquitin-conjugating enzyme UBE2D1 (UbcH5a).
abstract: The viral ubiquitin ligase ICP0 is required for efficient initiation of herpes simplex virus 1 (HSV-1) lytic infection and productive reactivation of viral genomes from latency. ICP0 has been shown to target a number of specific cellular proteins for proteasome-dependent degradation during lytic infection, including the promyelocytic leukemia protein (PML) and its small ubiquitin-like modified (SUMO) isoforms. We have shown previously that ICP0 can catalyze the formation of unanchored polyubiquitin chains and mediate the ubiquitination of specific substrate proteins in vitro in the presence of two E2 ubiquitin-conjugating enzymes, namely, UBE2D1 (UbcH5a) and UBE2E1 (UbcH6), in a RING finger-dependent manner. Using homology modeling in conjunction with site-directed mutagenesis, we identify specific residues required for the interaction between the RING finger domain of ICP0 and UBE2D1, and we report that point mutations at these residues compromise the ability of ICP0 to induce the colocalization of conjugated ubiquitin and the degradation of PML and its SUMO-modified isoforms. Furthermore, we show that RING finger mutants that are unable to interact with UBE2D1 fail not only to complement the plaque-forming defect of an ICP0-null mutant virus but also to mediate the derepression of quiescent HSV-1 genomes in cell culture. These data demonstrate that the ability of ICP0 to interact with cellular E2 ubiquitin-conjugating enzymes is fundamentally important for its biological functions during HSV-1 infection.
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom.
authors.forename: Emilia
authors.initials: E
authors.lastname: Vanni
authors.forename: Derek
authors.initials: D
authors.lastname: Gatherer
authors.forename: Lily
authors.initials: L
authors.lastname: Tong
authors.forename: Roger D
authors.initials: RD
authors.lastname: Everett
authors.forename: Chris
authors.initials: C
authors.lastname: Boutell
mesh_terms: D019943:Amino Acid Substitution; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000472:pathogenicity; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: D017874:Immediate-Early Proteins; D050505:Mutant Proteins; C097308:UBE2D1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; C051804:Vmw110 protein, Human herpesvirus 1