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

Chimeric mouse TRIM25 containing the CCD of human TRIM25 recovers NS1 binding.

<p>(<b>A</b>) Schematic representations of human TRIM25 (hT25), mouse TRIM25 (mT25) and the mouse/human TRIM25 chimera (mChimT25_h191–379). Numbers indicate amino acid. (<b>B</b>) WCLs of HEK293T cells, that had been transfected with empty vector, V5-hTRIM25, V5-mTRIM25 or V5-mChimT25_h191–379 together with NS1-PR8, were subjected to IP with anti-V5 antibody, followed by IB with anti-NS1. (<b>C</b>) Localization of NS1-PR8 in HeLa cells determined by confocal microscopy. At 30 h posttransfection with NS1-PR8 alone, NS1-PR8 together with V5-hTRIM25, V5-mTRIM25, or V5-mChimT25_h191–379, HeLa cells were stained with anti-NS1 (green), anti-V5 (red) and DAPI (nucleus, blue). (<b>D</b>) Quantification of cytoplasmic NS1 localization from (C). From three independent experiments, 50 cells with expression of NS1 alone or NS1 together with hTRIM25, mTRIM25 or mChimT25_h191–379, respectively, were counted and the percentage of cells with cytoplasmic NS1 is shown, ***p<0.001 by Fisher's exact test.</p

Influenza A Virus NS1 interacts with mouse Riplet.

<p>(<b>A</b>) At 30 h posttransfection with Myc-tagged mouse Riplet (Myc-mRiplet) together with NS1-PR8, HEK293T cells were mock-treated or infected with SeV (10 HA units/ml) for 10 h. V5-tagged hTRIM25 was co-transfected as positive control. WCLs were subjected to IP with anti-Myc (Riplet) or anti-V5 (TRIM25), followed by IB with anti-NS1 antibody. (<b>B</b>) Hepa1.6 cells were transfected with Flag-tagged mouse Riplet. At 30 h posttransfection, cells were either mock-treated or infected with recombinant A/PR/8/34 virus at an MOI of 2. 18 h later, WCLs were subjected to IP with anti-NS1 antibody, followed by immunoblotting using the indicated antibodies. (<b>C</b>) Localization of NS1-PR8 and mouse Riplet in HeLa cells determined by confocal microscopy. At 30 h posttransfection with Myc-mRiplet alone, NS1-PR8 alone, or NS1 together with Myc-mRiplet, HeLa cells were stained with anti-Myc (green), anti-NS1 (red), and DAPI (nucleus, blue). Fifty cells from three independent experiments were counted and the percentage of cells with cytoplasmic NS1 is shown, ***p<0.001 by Fisher's exact test. (<b>D and E</b>) Hepa1.6 cells were transfected with Flag-tagged mouse Riplet. At 30 h posttransfection, cells were either mock-treated or infected with recombinant A/PR/8/34 virus expressing NS1 PR8, Cal04, HK156, Tx91, SwTx98, or Pan99 at an MOI of 2 (<b>D</b>), or R38A/K41A or E96A/E97A NS1 mutant at an MOI of 4 (<b>E</b>). 18 h later, WCLs were subjected to IP with anti-NS1 antibody, followed by immunoblotting using the indicated antibodies.</p

NS1 proteins from human influenza strains bind and inhibit human Riplet.

<p>(<b>A</b>) HEK293T cells were transfected with empty vector or HA-tagged human Riplet (HA-hRiplet). At 30 h posttransfection, cells were either mock-treated, or infected with the indicated recombinant A/PR/8/34 viruses at an MOI of 2. 18 h later, WCLs were subjected to IP with anti-HA antibody, followed by immunoblotting using the indicated antibodies. (<b>B</b>) Tx91 recombinant virus suppresses the endogenous RIG-I ubiquitination more potently than PR8 virus. HEK293T cells, that had been transfected with HA-tagged ubiquitin, were either mock-treated, or infected with ΔNS1 PR8, PR8 WT, or Tx91-NS1 recombinant virus at an MOI of 2 for 18 h. WCLs were subjected to IP with anti-RIG-I antibody, followed by IB with anti-HA or anti-RIG-I antibody. Expression of HA-ubiquitin, viral NS1, and Actin was further determined in the WCLs. (<b>C</b>) A549 cells were infected with PR8-NS1 or Tx91-NS1 recombinant virus at an MOI of 0.1. Cells were collected at the indicated time points and IFN-β mRNA was measured by qPCR. (<b>D and E</b>) A549 cells were transiently transfected with non-silencing control siRNA (si.C), or with siRNA specific for TRIM25 (si.TRIM25), Riplet (si.Riplet), or both. At 40 h posttransfection, cells were infected with PR8 WT or Tx91 recombinant virus at an MOI of 2 for 30 h. The mRNA levels of TRIM25 and Riplet were measured by qPCR for analyzing their knockdown efficiency (<b>D</b>). Furthermore, IFN-β mRNA levels were assessed by qPCR (<b>E</b>). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003059#s2" target="_blank">Results</a> are triplicates from 3 independent experiments. NS; statistically non-significant.</p

NS1-mediated suppression of RIG-I ubiquitination and IFN induction in mouse is TRIM25-independent.

<p>(<b>A</b>) Ubiquitination of the RIG-I CARDs in WT and <i>TRIM25 −/−</i> MEF cells. WT and <i>TRIM25 −/−</i> MEFs were transfected with human GST-RIG-I 2CARD (GST-h2CARD) or mouse GST-RIG-I 2CARD (GST-m2CARD). At 40 h posttransfection, WCLs were subjected to GST-pulldown (GST-PD), followed by IB with anti-GST antibody. Arrow heads indicate ubiquitinated bands. Arrow indicates non-ubiquitinated 2CARD. (<b>B</b>) Ubiquitination of the RIG-I CARDs in <i>TRIM25 −/−</i> cells reconstituted with mouse or human TRIM25. At 40 h posttransfection with human or mouse GST-RIG-I 2CARD together with empty vector, V5-tagged hTRIM25 or mTRIM25, WCLs of <i>TRIM25 −/−</i> MEFs were subjected to GST-PD, followed by IB with anti-GST or anti-ubiquitin (Ub) antibody. Expression of TRIM25 proteins was determined by IB with anti-V5 antibody. Arrow heads indicate ubiquitinated bands. Arrow indicates non-ubiquitinated 2CARD. (<b>C</b>) NS1 inhibits the RIG-I CARD ubiquitination induced by human TRIM25 but not by mouse TRIM25. <i>TRIM25 −/−</i> MEFs were transfected with GST, human GST-RIG-I 2CARD (GST-h2CARD) or mouse GST-RIG-I 2CARD (GST-m2CARD) together with vector, hTRIM25-V5 or mTRIM25-V5 with or without NS1. At 40 h posttransfection, WCLs were subjected to GST-PD, followed by IB with anti-GST for detecting RIG-I CARD ubiquitination, anti-V5 for detecting TRIM25 binding, or anti-NS1 antibody for detecting NS1 binding. Arrow heads indicate ubiquitinated bands. Arrow indicates non-ubiquitinated 2CARD. (<b>D</b>) NS1 interacts with mouse RIG-I in a TRIM25-independent manner. <i>TRIM25 −/−</i> MEFs were transfected with empty vector, V5-hTRIM25, or V5-mTRIM25 together with NS1-PR8. WCLs were subjected to IP with anti-RIG-I antibody, followed by IB with anti-NS1, anti-V5, or anti-RIG-I antibody. Expression of NS1 and TRIM25 proteins in the WCLs was determined by anti-NS1 and anti-V5 antibody, respectively. (<b>E</b>) NS1 inhibits endogenous mouse RIG-I ubiquitination in a TRIM25-independent manner. <i>TRIM25 −/−</i> MEFs were transfected with increasing amounts of NS1-PR8. At 32 h posttransfection, cells were infected with SeV (50 HA units/ml) for 10 h. WCLs were subjected to IP with anti-RIG-I antibody, followed by IB with anti-Ub or anti-RIG-I antibody. (<b>F</b>) NS1 inhibits IFN-β induction in murine cells in a TRIM25-independent manner. WT and <i>TRIM25 −/−</i> MEFs were transfected with an IFN-β luciferase construct together with empty vector or NS1. At 24 h posttransfection, cells were either mock-treated, or infected with SeV (10 HA units/ml) for 16 h. Samples were then subjected to a dual luciferase assay. Data represent the mean ± SD (n = 3).</p

Proposed model of the species-specific inhibition of RIG-I by influenza A virus NS1 protein.

<p>The NS1 proteins of avian influenza viruses bind to chicken TRIM25, thereby suppressing IFN induction. The substrate of TRIM25 in avian cells has yet to be determined. The NS1 proteins from human influenza viruses bind to and inhibit both human TRIM25 and human Riplet, thereby suppressing the ubiquitination of the RIG-I CARD and CTD. The NS1 proteins from avian, human and mouse-adapted influenza viruses block Riplet but not TRIM25 for the inhibition of RIG-I signaling in mouse cells.</p

NS1 inhibits the Riplet-dependent RIG-I ubiquitination and IFN induction in murine cells.

<p>(<b>A</b>) Mouse Hepa 1.6 cells were transfected with vector or Flag-tagged mRiplet with or without NS1-PR8. WCLs were subjected to IP with RIG-I antibody, followed by IB with anti-Ub or anti-RIG-I antibody. Expression of mRiplet, NS1, and ubiquitin (Ub) was determined in the WCLs by IB with anti-Flag, anti-NS1 or anti-Ub antibody. (<b>B</b>) Mouse Hepa1.6 cells were transfected with IFN-β luciferase reporter plasmid together with empty vector or mRIG-I together with or without mRiplet and NS1-PR8. At 24 h posttransfection, cells were lysed and subjected to luciferase assay. Data shown is representative of 3 independent experiments and depicted is the mean ± SD (n = 3). (<b>C</b>) Influenza NS1 protein specifically inhibits the Riplet-dependent ubiquitination of mouse RIG-I. Hepa1.6 cells were transiently transfected with non-silencing control siRNA (si.C), or with a siRNA specific for mouse Riplet (si.Riplet) together with empty vector or NS1-PR8. At 24 h posttransfection, cells were infected with SeV (50 HA units/ml) for 22 h. WCLs were used for IP with anti-RIG-I antibody, followed by IB with anti-Ub or anti-RIG-I antibody. (<b>D–F</b>) Knockdown of endogenous Riplet in mouse embryonic fibroblasts enhances influenza A virus replication. WT or <i>TRIM25 −/−</i> MEFs were transfected with non-silencing control siRNA (si.C), or with a siRNA specific for mouse Riplet (si.Riplet). At 30 h posttransfection, cells were infected with recombinant A/PR/8/34 WT virus (MOI 0.1). Knockdown of endogenous Riplet was confirmed by RT-PCR (<b>D</b>). Supernatants were assayed for progeny virus yields 24 h postinfection in standard plaque titrations (<b>E</b>). Virus yields are depicted in Pfu/ml. The results of three independent experiments are shown. Furthermore, viral NS1 protein expression was determined in the WCLs of infected cells (<b>F</b>).</p

Influenza A Virus NS1 does not interact with mouse TRIM25.

<p>(<b>A</b>) WCLs of human (HEK293T) or mouse (Hepa1.6) cells, that had been mock-treated or infected with PR8 virus at MOI 2 for 18 h, were subjected to IP with or without anti-NS1 antibody, followed by IB with anti-TRIM25. Expression of NS1 and endogenous TRIM25 was determined by IB with anti-NS1 or anti-TRIM25 antibody. (<b>B</b>) Mouse Hepa1.6 or human HEK293T cells were transfected with empty vector, V5-tagged mouse TRIM25 (V5-mTRIM25), or V5-tagged human TRIM25 (V5-hTRIM25), together with NS1-PR8. At 30 h posttransfection, WCLs were prepared and subjected to IP with anti-V5 antibody, followed by immunoblotting with anti-NS1 or anti-V5. (<b>C</b>) <i>In vitro</i> binding of NS1 with TRIM25. Maltose-binding protein (MBP), GST, and the recombinant fusion proteins MBP-human TRIM25-Flag (MBP-hTRIM25-Flag), MBP-mouse TRIM25-Flag (MBP-mTRIM25-Flag), and GST-NS1 (PR8) were purified from bacteria. Purified MBP-hTRIM25-Flag or MBP-mTRIM25-Flag was incubated with GST-NS1. As controls for binding, purified MBP was incubated with GST-NS1, and MBP-hTRIM25-Flag was incubated with GST. Protein complexes were then precipitated with Amylose agarose gel, and precipitates resolved by SDS-PAGE, followed by Coomassie staining. Asterisk indicates the main degradation product of MBP-hTRIM25-Flag.</p