Fig 2: ISG15 covalently modifies RTN4 and MAGT1 following infection.ISG15-overexpression does not lead to changes in SEC61 or KDEL distribution or to changes in ER mass. Listeria infection increases IL-8 and IL-6 secretion and Isg15−/− MEFs secrete less IL-6 than Isg15+/+ mefs. (A) Cells were infected with L. innocua expressing InlB or Listeria for 24 hr (MOI 10) and harvested for immunoprecipitation (input is 1.5% of IP). Higher molecular weight species corresponding to ISGylated RTN4 or MAGT1 are indicated. Asterisks denote background bands from antibody heavy and light chain. (B) Control or ISG15-overexpressing cells transfected with GFP-SEC61 and BFP-KDEL (scale bar is 10 μm). (C) Mean-fluorescence intensity of ER-tracker staining of control or ISG15-overexpressing cells. (D) IL-8 and IL-6 production in HeLa cells following infection with Listeria or L. innocua expressing InlB. The indicated cells were infected in a 96-well format, supernatants were collected, and 100 μl of supernatant was used for the ELISAs. (E) IL-6 secretion in Isg15+/+ or Isg15−/− MEFs (SV40 transformed) infected with Listeria (MOI 25) for 4 hr; CFUs/well in Isg15+/+ or Isg15−/− MEFs following Listeria infection. Statistical significance determined using a Mann–Whitney test. Statistical significance is indicated as follows: NS, nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001.DOI: http://dx.doi.org/10.7554/eLife.06848.014

Fig 3: Type I IFN response is blunted in virus‐infected IRGM knockdown cells A–DControl and si‐IRGM transfected THP‐1 IFN reporter cells were kept uninfected (Mock) or infected with (A) JEV (MOI 5) or (B) CHIKV (MOI 5) or (C) HSV‐1 (MOI 2.5) or (D) VSV‐eGFP (MOI 1) and the supernatant collected 8 hpi were subjected to luciferase assay. The graphs depict fold change in interferon response. (n = 3, mean ± SD, *P < 0.05, **P < 0.005, ***P < 0.0005, Student’s unpaired t‐test).E–JControl and si‐IRGM transfected HT‐29 cells uninfected or infected with CHIKV and qRT–PCR analysis were performed with several ISG’s (E) SAMHD1 (F) HERC5 (G) ISG15 (H) viperin/RSAD2 (I) MX1 (J) APOBEC3G. (n = 3, mean ± SE, **P < 0.005, ***P < 0.0005, ****P < 0.00005, Student’s unpaired t‐test).KWestern blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and si‐IRGM transfected THP‐1 cells and probed with the indicated antibodies. S.E, short exposure; L.E, long exposure.LWestern blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and IRGM knockdown HT‐29 cells and probed with the indicated antibodies.MWestern blot analysis with cell lysates of mock and JEV (MOI 5, 24 h) infected control and si‐IRGM transfected HT‐29 cells and probed with the indicated antibodies.NWestern blot analysis with cell lysates of THP‐1 control or IRGM knockdown cells, untransfected or transfected with, heat‐killed whole CHIKV or CHIKV viral RNA and probed with the indicated antibodies.OqRT–PCR analysis to determine the knockdown efficiencies of PRR’s and adaptor proteins as indicated (n = 3, mean ± SE, ***P < 0.0005, Student’s unpaired t‐test).PqRT–PCR analysis with total RNA isolated from control and IRGM knockdown HT‐29 cells transfected with siRNA combinations as indicated that were infected with CHIKV (MOI 5, 24 h). (n = 3, mean ± SE, ***P < 0.0005, ****P < 0.00005, Student’s unpaired t‐test).QWestern blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and IRGM knockdown HT‐29 cells transfected with indicated siRNA and probed with the indicated antibodies. Source data are available online for this figure.

Fig 4: ISG15 is not secreted as a cytokine from HeLa cells following TNFα treatment or Listeria infection.(A) Control and ISG15-overexpressing cells were treated with 20 ng/ml TNFα for 24 hr. Supernatants were collected and assayed for secreted ISG15. (B) Control and ISG15-overexpressing cells were infected with Listeria for 24 hr, supernantants were collected and assayed for secreted ISG15.DOI: http://dx.doi.org/10.7554/eLife.06848.016

Fig 5: siRNA-mediated knockdown effectively depletes ISG15 during Listeria infection.(A) Cells were treated with siControl or siISG15 for 24 hr and subsequently infected for the indicated times with Listeria (MOI 25), lysed and immunoblotted with α-ISG15 and α-ACTIN antibodies. Extremely efficient knockdown was achieved.DOI: http://dx.doi.org/10.7554/eLife.06848.010