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

Abstract

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.

Figure 1. The mouse HerC6 RLD confers efficient ISG15 conjugation.

A. Schematic overview of the HerC6 chimeras with the RLD and HECT domains. B. HerC expression levels were determined in HEK-293T cells transfected with plasmids expressing wild-type HA-tagged HerC proteins and human-mouse chimeric HerC6 proteins. C/D. HEK-293T cells transfected with plasmids expressing human E1 and E2 enzymes, indicated HerC proteins and either V5-tagged human or mouse wild-type ISG15 (C) or human-mouse ISG15 chimeras (D) were analyzed for global ISGylation by V5-specific immunoblot.

Figure 2. mHerC6 is induced by type I interferon and localizes exclusively in the cytoplasm.

A. Indicated murine and human cell lines were stimulated with recombinant type I IFN and subsequently analyzed for HerC mRNA regulation by RT-qPCR. Values are relative fold-change over mock-induced samples. Experiments were reproduced at least twice; a representative experiment is shown. Error bars represent standard deviation of qPCR replicates. B. HeLa cells transfected with an empty control plasmid and subsequently infected with SeV were immuno-stained with IRF-3 specific antibodies. C. Localization of overexpressed HA-tagged HerC proteins in the presence of subsequent SeV infection was determined in HeLa cells by immuno-fluorescence assay using HA- and SeV-specific antibodies.

Figure 3. mHerC6 is essential for global cellular ISG15 conjugation in mouse cells.

(A) mRNA knock-down of HerC6 was analyzed by RT-qPCR in mouse L929 cells transfected with siRNAs specifically targeting human or mouse HerC6 and subsequently treated with recombinant type I IFN. mRNA levels in mHerC6 knock-down cells are plotted relative to mRNA levels in cells with non-targeting hHerC6 siRNA. Experiments were reproduced at least twice; a representative experiment is shown. Error bars represent standard deviation of qPCR replicates. B/C. L-929 cells were transfected with (B) indicated siRNAs, stimulated with IFN for 48 h and probed for endogenous ISG15 on a Western blot or (C) simultaneously transfected with a V5-tagged mouse ISG15 plasmid and indicated siRNAs, stimulated with IFN for 48 h and subsequently analyzed for global ISG15 conjugation by V5-specific immunoblot.

Figure 4. mHerC6 stimulates the IFNβ promoter and confers antiviral activity against VSV and NDV.

(A) HEK-293T cells were transfected with an IFNβ reporter construct controlling firefly luciferase, a constitutively active renilla luciferase internal control plasmid, a limiting amount of a plasmid expressing a constitutively active form of RIG-I (RIG-I(2CARD)) and the indicated HerC5/6 proteins. After 24 h, cells were lysed and luciferase measured. Values were normalized to the internal control and plotted relative to the GST control. (B) L-929 cells were transfected with the indicated plasmids. After 48 h, the cells were infected with VSV-GFP or NDV-GFP at an m.o.i. of 5. At 7 h p.i. supernatant was harvested from the VSV-GFP infected cells and at 16 h p.i. from the NDV-GFP infected cells. The supernatants were tittered by TCID50 on HEK-293T cells.