Journal: Science Advances

Article Title: Protein modification with ISG15 blocks coxsackievirus pathology by antiviral and metabolic reprogramming

doi: 10.1126/sciadv.aay1109

Figure Lengend Snippet: Wild-type (wt), ISG15 −/− , and Ube1L −/− mice were infected with 1 × 10 5 pfu of CV Nancy and sacrificed at the indicated points in time post infection (p.i.). Tissue from ( A ) liver and ( B ) heart of wild-type mice was subjected to Western blot analysis using an ISG15-specific antibody. Each lane represents tissue homogenates obtained from a different animal. ( C and D ) Infectious viral particles were quantified in the respective organs obtained from wild-type, ISG15 −/− , and Ube1L −/− mice by plaque assay during CV infection. Each dot represents a different animal; data are summarized as median values. Student’s t tests were conducted. P values of <0.05 are indicated in the graph. ( E to J ) USP18C61A and wild-type littermate controls were infected with 1 × 10 5 pfu of CV and sacrificed at the indicated points in time. (E) Heart tissue was homogenized and subjected to Western blot analysis for detection of ISG15. Each lane represents tissue obtained from a different animal, and the shown example for each group and point in time is representative for n = 3 mice. (F) At day 6 after infection, infectious viral particles were determined in heart by TaqMan qPCR and plaque assay. Each dot represents a different animal. Data are summarized as means ± SEM; t tests were performed, and P values of <0.05 are depicted. (G) Cardiomyocytes derived from USP18C61A ISG15 −/− and ISG15 −/− embryos were transduced with Ad5 vectors encoding murine ISG15 and stimulated with poly(I:C). Cellular lysates were subjected to Western blot analysis. (H to J) ISG15-rescued cardiomyocytes from USP18C61A ISG15 −/− and ISG15 −/− embryos were infected with CV at an MOI of 0.1 for 24 hours. (H) Total RNA was isolated to determine CV genome copy numbers by TaqMan qPCR; ΔΔ C t values obtained from duplicates are shown for two independent experiments. Data are summarized as means ± SEM. (I) Viral load was determined in cellular lysates by Western blot analysis of CV VP1. Densitometric analysis of VP1 and the GAPDH loading control was performed to calculate the mean (± SEM) relative VP1 expression in three independent experiments. (J) Release of infectious viral particles was assessed by plaque assay. One representative out of three independent experiments yielding the same result is depicted. Unpaired t test (plaque assay) and one-sample t tests (VP1 and CV RNA) were performed, and P values are depicted.

Article Snippet: The following primary antibodies were used: β-actin (C4; Merck), FLAG (M2; Sigma-Aldrich), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (FL-335; Santa Cruz Biotechnology or 6C5; Invitrogen), ISG15 (lab stocks), human IFIT1 (polyclonal; Abcam), human IFIT3 (polyclonal; Abcam), murine IFIT1 (polyclonal; Abcam), murine IFIT3 (polyclonal; Antibodies Online), α-tubulin (GT114; GeneTex), and VP1 (31A2; Mediagnost).

Techniques: Infection, Western Blot, Plaque Assay, Derivative Assay, Transduction, Isolation, Expressing

Journal: Science Advances

Article Title: Protein modification with ISG15 blocks coxsackievirus pathology by antiviral and metabolic reprogramming

doi: 10.1126/sciadv.aay1109

Figure Lengend Snippet: ( A ) ISG15 expression was deleted in HeLa cells using CRISPR-Cas9 gene editing as described in . ISG15-ko cells and wild-type cells were infected with CV (MOI 0.1) for 16 hours. Expression of CV VP1 was determined by Western blot analysis in four independent experiments, and the obtained signal normalized to GAPDH was compared to wild-type samples. Infectious virus particles were quantified in five independent experiments by plaque assay. Data are summarized as means ± SEM. ( B ) HeLa cells were transfected with siRNA targeting human ISG15 or a nontargeting control siRNA. Cells were subsequently infected with CV (MOI 0.01) for 16 hours. VP1 protein expression was determined and normalized to the control sample in four independent experiments. Plaque assays were performed in two independent experiments. Data are summarized as described in (A). ( C ) HeLa cells stably expressing FLAG-tagged human ISG15 and respective control cells were infected with CV (MOI 0.1) for 16 hours. VP1 expression and virus titer were determined as described in (A) in three independent experiments. ( D ) Primary embryonic cardiomyocytes obtained from ISG15 −/− mice were transduced with Ad5 vectors expressing human ISG15 (hISG15) or control for 48 hours at MOI 25 before CV infection (MOI 0.1) for 24 hours. VP1 levels were determined by Western blot analysis, and infectious viral particles were quantified by plaque assay in three independent experiments. ( E ) Cardiomyocytes derived from USP18C61A ISG15 −/− and ISG15 −/− embryos were transduced with Ad5 vectors encoding hISG15 and infected with CV in three independent experiments for detection of the viral load by Western blot analysis of VP1 as well as plaque assay. One-sample t tests were performed for all summarized VP1 data. Unpaired t tests were conducted for all plaque assay data. ( F and G ) CVB isolates were obtained from patients presenting with neurological symptoms that may have been of infectious origin. (F) ISG15-ko HeLa cells and control cells were infected with the indicated CV serotypes (MOI 0.1), and infectious virus particles were determined after 16 hours by plaque assay. The relative increase of the viral titer in ISG15-ko HeLa cells as compared to control cells is depicted for a representative experiment. Three independent experiments demonstrated similar results. (G) The ISG15 system was induced in USP18C61A ISG15 −/− and ISG15 −/− cardiomyocytes by transduction of Ad5 vectors encoding hISG15. Cells were infected with CV serotypes as follows: CVB1 425, MOI 1 (2 days); CVB1 506, MOI 1 (1 day); CVB3 1072, MOI 10 (2 days); CVB3 180, MOI 10 (1 day); CVB4 686, MOI 10 (2 days); CVB4 120, MOI1 (2 days); CVB5 679, MOI 1 (1 days); CVB5 800, MOI 10 (1 day). Infectious viral particles were quantified by plaque assay. Relative reduction of the viral load in USP18C61A ISG15 −/− cells as compared to ISG15 −/− cardiomyocytes with restored ISG15 expression is depicted for a representative of at least three independent experiments. Data are means ± SEM; one-sample t tests were performed, and P values are depicted.

Article Snippet: The following primary antibodies were used: β-actin (C4; Merck), FLAG (M2; Sigma-Aldrich), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (FL-335; Santa Cruz Biotechnology or 6C5; Invitrogen), ISG15 (lab stocks), human IFIT1 (polyclonal; Abcam), human IFIT3 (polyclonal; Abcam), murine IFIT1 (polyclonal; Abcam), murine IFIT3 (polyclonal; Antibodies Online), α-tubulin (GT114; GeneTex), and VP1 (31A2; Mediagnost).

Techniques: Expressing, CRISPR, Infection, Western Blot, Plaque Assay, Transfection, Stable Transfection, Transduction, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: Inhibitor of κB Kinase ϵ (IKKϵ), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection*

doi: 10.1074/jbc.m111.285205

Figure Lengend Snippet: FIGURE 1. IKK and IFIT2 impose restriction on WNV infection. WT or IKK/ MEFs were mock-infected or infected with WNV-MAD at an m.o.i. of 1 (A) and mock-stimulated or stimulated with 100 IU/ml IFN- (B). Protein lysate was collected at the indicated times and immunoblotted using IFIT2, IFIT3, IFIT1, PKR, and IKK antibodies. Tubulin was used as loading control. C, HEK293 cells were cotransfected with pCMV-Renilla and either pIFN--Luciferase (top left panel), pISG15-Luciferase (top right panel), pADAR1-Luciferase (bottom left panel), or pIFIT2-Luciferase (bottom right panel). 16 h later, cells were either mock-stimu- lated (vector cotransfection); stimulated by transfection with 25 ng, 50 ng, or 100 ng of an IKK expression plasmid; or treated with 100 IU/ml IFN-. Cells were harvested 48 h post-transfection, and luciferase expression was measured and normalized to Renilla. The relative luciferase value was calculated as fold induction over induction of the vector that was set to 1. Statistical analysis was performed with Student’s t test. D, WT or IFIT2/ MEFs were infected with WNV-MAD at an m.o.i. of 5. At the indicated time points post-infection, culture supernatants were collected, and virus titers were determined by plaque assay on BHK cells. C and D, data are mean S.D. of three independent experiments performed in triplicate. p values were calculated using Student’s t test to determine statistical significance.

Article Snippet: The following primary antibodies were used for immunoblot analyses: -ADAR1 (Abnova); -IRF-3 (M. David, UCSD); -IFIT1, -murine IFIT2 and -murine IFIT3 (G. Sen, Cleveland Clinic); -ISG15 (A. Haas, Louisiana State University); -WNV (Centers for Disease Control and Prevention); -pSTAT1 Tyr-701, -p-STAT1 Ser-727, -STAT1, -p-IRF-3 (Cell Signaling Technology, Inc.); -murine IRF-3 (Invitrogen); -IKK (Imgenex); -PKR (Santa Cruz Biotechnology, Inc.); -SenV (Biodesign International); -FLAG (M2), and -Tubulin (Sigma).

Techniques: Infection, Control, Luciferase, Plasmid Preparation, Cotransfection, Transfection, Expressing, Virus, Plaque Assay