HERC6 is the main E3 ligase for global ISG15 conjugation in mouse cells

• Published in: PloS one 2012-01, Vol.7 (1), p.e29870
• Main Authors: Oudshoorn, Diede, van Boheemen, Sander, Sánchez-Aparicio, Maria Teresa, Rajsbaum, Ricardo, García-Sastre, Adolfo, Versteeg, Gijs A
• Format: Article
• Language: English
• Subjects: Analysis; Animals; Antiviral activity; Binding Sites; Biological response modifiers; Biology; Blotting, Western; Cancer; Cell Line; Cell Line, Tumor; Cells (Biology); Conjugation; Cytokines - genetics; Cytokines - metabolism; Cytoplasm - drug effects; Cytoplasm - metabolism; Enzymes; Feasibility studies; Gene expression; Gene Expression - drug effects; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HEK293 Cells; HeLa Cells; Homology; Human behavior; Humans; Infections; Influenza; Interferon; Interferon-beta - genetics; Interferon-beta - metabolism; Interferon-beta - pharmacology; Ligases; Localization; Medicine; Mice; Mice, Knockout; Microscopy, Confocal; Newcastle disease; Newcastle disease virus - genetics; Newcastle disease virus - metabolism; NIH 3T3 Cells; Phosphorylation; Plasmids; Promoter Regions, Genetic - genetics; Protein Binding; Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Stomatitis; Transcription factors; Ubiquitin; Ubiquitin-protein ligase; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Ubiquitins - genetics; Ubiquitins - metabolism; Virology; Viruses
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0029870

Type I interferon (IFN) stimulates expression and conjugation of the ubiquitin-like modifier IFN-stimulated gene 15 (ISG15), thereby restricting replication of a wide variety of viruses. Conjugation of ISG15 is critical for its antiviral activity in mice. HECT domain and RCC1-like domain containing protein 5 (HerC5) mediates global ISGylation in human cells, whereas its closest relative, HerC6, does not. So far, the requirement of HerC5 for ISG15-mediated antiviral activity has remained unclear. One of the main obstacles to address this issue has been that no HerC5 homologue exists in mice, hampering the generation of a good knock-out model. However, mice do express a homologue of HerC6 that, in contrast to human HerC6, can mediate ISGylation.Here we report that the mouse HerC6 N-terminal RCC1-like domain (RLD) allows ISG15 conjugation when replacing the corresponding domain in the human HerC6 homologue. In addition, sequences in the C-terminal HECT domain of mouse HerC6 also appear to facilitate efficient ISGylation. Mouse HerC6 paralleled human HerC5 in localization and IFN-inducibility. Moreover, HerC6 knock-down in mouse cells abolished global ISGylation, whereas its over expression enhanced the IFNβ promoter and conferred antiviral activity against vesicular stomatitis virus and Newcastle disease virus. Together these data indicate that HerC6 is likely the functional counterpart of human HerC5 in mouse cells, suggesting that HerC6(-/-) mice may provide a feasible model to study the role of human HerC5 in antiviral responses.