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

<p>(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.</p

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

<p>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.</p

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

<p>(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.</p