Journal: Journal of Virology

Article Title: ISG15 Modulates Type I Interferon Signaling and the Antiviral Response during Hepatitis E Virus Replication

doi: 10.1128/JVI.00621-17

Figure Lengend Snippet: HEV induces ISG15 both in vitro and in vivo. (A) Replication kinetics of viral RNAs from the HEV P6 infectious cDNA clone and HEV P6GLuc replicon in Huh7-S10-3 liver cells. The cells were transfected with capped RNA transcripts from the genotype 3 HEV P6 infectious cDNA clone (HEV P6) or with the HEV P6GLuc replicon (HEV P6GLuc; P6 encoding Gaussia luciferase clone). The culture supernatants were collected at various time points and used to measure viral RNA replication levels by HEV real-time quantitative RT-PCR or by the Gaussia luciferase assay (GLuc assay). RLU, relative light units. (B) ISG15 mRNA expression levels in Huh7-S10-3 liver cells transfected with capped RNA transcripts from the HEV P6 infectious cDNA clone or the HEV P6GLuc replicon at 1 and 5 days posttransfection (dpt) were determined by Sybr green-based quantitative PCR (qPCR). The ISG15-mRNA fold change was calculated using the 2−ΔΔCT method for comparisons to untransfected control cells, and the RPS18 gene was used as the housekeeping gene. (C and D) ISG15 protein expression levels in Huh7-S10-3 cells transfected with capped RNA from the HEV P6 infectious cDNA clone or the HEV P6GLuc replicon (C and D) or the HEV P6 replication-deficient infectious cDNA clone (P6GAD) (D). CC, untransfected cell control. The ISG15 protein expression, at 5 days posttransfection, in cell lysates (40 μg/lane) was analyzed by Western blotting using anti-ISG15 antibody (1:1,000 dilution). Fold change in band density, as calculated via normalization of ISG15 with β-actin (loading control), was measured using ImageJ software (NIH, Bethesda, MD). (E) ISG15 and USP18 mRNA expression levels in swine liver tissue samples obtained from animals experimentally infected with HEV (n = 3) at 3 weeks postinfection. The fold changes in swine ISG15 and USP18 mRNA levels were calculated using the 2−ΔΔCT method compared to the uninfected control animals (n = 2), and the RPS18 gene was used as the housekeeping gene. The data represent means ± standard errors of the means (SEM) of results from three independent transfection experiments (A, B, and C), one experiment representative of two independent transfection experiments (D), and HEV-infected animals (n = 3) compared to uninfected animals (n = 2) (E). **, P ≤ 0.01.

Article Snippet: Recombinant human interferon alpha (IFN-α; catalog no. 11100-1) and antibodies specific for ISG15 (catalog no. 21900-1; 1:1,000 dilution) were purchased from PBL Assay Science (Piscataway, NJ).

Techniques: In Vitro, In Vivo, Transfection, Luciferase, Quantitative RT-PCR, Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Control, Western Blot, Software, Infection

Journal: Journal of Virology

Article Title: ISG15 Modulates Type I Interferon Signaling and the Antiviral Response during Hepatitis E Virus Replication

doi: 10.1128/JVI.00621-17

Figure Lengend Snippet: ISG15 is not required for HEV replication. Huh7-S10-3 liver cells were cotransfected with 20 nM ISG15-targeted siRNA (siISG15) or control siRNA (siCnt) along with HEV P6GLuc replicon RNA (A and C) or HEV P6 infectious clone viral genomic RNA (B and D). The GLuc activity levels in culture supernatant (A) and the intracellular viral RNA levels (B) were determined by GLuc assay and qPCR, respectively, at 5 dpt. The efficiency of knockdown of ISG15 by siISG15 was determined by measuring ISG15 protein levels in siRNA and HEV P6GLuc replicon RNA cotransfected samples at 5 dpt (C) and ISG15 mRNA levels in siRNA and HEV P6 viral RNA cotransfected samples at 5 dpt (D); fold change compared to untransfected control cells was calculated using the 2−ΔΔCT method, and the RPS18 gene was used as the housekeeping gene. The data represent means ± SEM of results from two independent transfection experiments.

Article Snippet: Recombinant human interferon alpha (IFN-α; catalog no. 11100-1) and antibodies specific for ISG15 (catalog no. 21900-1; 1:1,000 dilution) were purchased from PBL Assay Science (Piscataway, NJ).

Techniques: Control, Activity Assay, Knockdown, Transfection

Journal: Journal of Virology

Article Title: ISG15 Modulates Type I Interferon Signaling and the Antiviral Response during Hepatitis E Virus Replication

doi: 10.1128/JVI.00621-17

Figure Lengend Snippet: ISG15 silencing enhances the type I IFN-mediated antiviral effect against HEV. Huh7-S10-3 liver cells were cotransfected with 20 nM ISG15-targeted siRNA (siISG15) or with control siRNA (siCnt) along with HEV P6GLuc replicon RNA (A) or HEV P6 infectious cDNA clone viral genomic RNA (B) at 24 hpt. The cells were either left untreated (w/o IFN-α) or treated with 100 IU/ml IFN-α (w IFN-α 100IU), and the amounts of HEV RNA were monitored at 5 dpt. The GLuc activity levels in culture supernatants (A) and the intracellular HEV P6 viral RNA levels (B) were normalized to the IFN-α-untreated control levels. The data represent means ± SEM of results of n = 5 (A) and n = 2 (B) independent transfection experiments. ***, P ≤ 0.001 (compared to siCnt w/o IFN-α); aaa, P ≤ 0.001 (compared to siCnt w IFN-α100IU).

Article Snippet: Recombinant human interferon alpha (IFN-α; catalog no. 11100-1) and antibodies specific for ISG15 (catalog no. 21900-1; 1:1,000 dilution) were purchased from PBL Assay Science (Piscataway, NJ).

Techniques: Control, Activity Assay, Transfection

Journal: Journal of Virology

Article Title: ISG15 Modulates Type I Interferon Signaling and the Antiviral Response during Hepatitis E Virus Replication

doi: 10.1128/JVI.00621-17

Figure Lengend Snippet: Loss of UBE1L and UBE2L6 did not result in an enhanced type I IFN-mediated antiviral effect against HEV. (A) ISG15 protein levels as measured by Western blot analysis in IFN-α-treated cells during ISG15 knockdown (siISG15) or during UBE1L (UBE1L) and UBE2L6 (UBE2L6) double knockdown (siE1E2). Huh7-S10-3 liver cells were transfected with 20 nM siCnt or siISG15 or siE1E2. At 24 hpt, cells were treated with 100 IU/ml IFN-α. Cell lysates were collected at 24 h post-IFN-α treatment to measure conjugated as well as free-form ISG15. The ISG15 protein levels in cell lysates (40 μg/lane) were analyzed by Western blotting using anti-ISG15 antibody (1:750 dilution). The data are representative of the results of one of three independent experiments. (B) GLuc activity levels in culture supernatant at 5 dpt. Cells were cotransfected with siCnt/siISG15/siE1E2 along with HEV P6GLuc replicon RNA; at 24 hpt, the cells were treated with 100 IU/ml IFN-α. The relative GLuc activity levels were determined compared to the IFN-α-untreated control levels. The data represent means ± SEM of n = 4 independent transfection experiments. ***, P ≤ 0.001 (compared to siCnt plus HEV P6GLuc); aaa, P ≤ 0.001; aa, P ≤ 0.01 (compared to siCnt plus P6GLuc plus IFN-α); ##, P ≤ 0.01 (compared to siE1E2 plus HEV P6GLuc plus IFN-α). Statistical analysis was done using ANOVA and a post hoc Tukey test.

Article Snippet: Recombinant human interferon alpha (IFN-α; catalog no. 11100-1) and antibodies specific for ISG15 (catalog no. 21900-1; 1:1,000 dilution) were purchased from PBL Assay Science (Piscataway, NJ).

Techniques: Western Blot, Knockdown, Transfection, Activity Assay, Control

Journal: Journal of Virology

Article Title: ISG15 Modulates Type I Interferon Signaling and the Antiviral Response during Hepatitis E Virus Replication

doi: 10.1128/JVI.00621-17

Figure Lengend Snippet: ISG15 regulates type I IFN signaling. (A) Relative pSTAT1 levels. Huh7-S10-3 liver cells were transfected with 20 nM control siRNA (siCnt) or ISG15-siRNA (siISG15) or UBE1L-siRNA plus UBE2L6-siRNA (siE1E2). At 24 hpt, cells were treated with IFN-α (100 IU/ml) for 30 min to 7 h. Cell lysate (20 μg/lane) was analyzed by Western blotting with the indicated antibody, anti-pSTAT1 (1:1,000 dilution), anti-STAT1 (1:1,000 dilution), and anti-β-actin (1:1,000 dilution). Fold change in band intensity was determined using ImageJ software (NIH, Bethesda, MD). The data represent means ± SEM of results from five independent experiments. a, P ≤ 0.05; aa, P ≤ 0.01 (compared with siCnt plus IFN-α at the given time point). (B) IFN-stimulated response element (ISRE) promoter activity levels. Huh7-S10-3 liver cells were cotransfected with siCnt/siISG15/siE1E2 along with pGL4.45[luc2P/ISRE/Hygro] (firefly luciferase) and pGL4.74[hRluc/TK] (renilla luciferase). At 24 hpt, cells were treated with various concentrations of IFN-α. Relative levels of fold induction of the cell-associated firefly luciferase activity, compared to the corresponding untreated cell control levels, at 18 h post-IFN-α treatment were estimated using a dual-luciferase assay kit and were normalized with renilla luciferase expression levels. The data represent means ± SEM of results from triplicate sample experiments. aa, P ≤ 0.01 (compared to siCnt plus IFN-α). (C to F) ISG mRNA levels in siRNA-transfected and IFN-α-treated samples were measured for Mx1 (C), OAS1 (D), PKR (E), and ISG15 (F) using Sybr green qPCR. Fold change in mRNA levels compared to the untransfected control was calculated using the 2−ΔΔCT method, and the RPS18 gene was used as the housekeeping gene. The data represent means ± SEM of results from three independent experiments. a, P ≤ 0.05; aa, P ≤ 0.01; aaa, P ≤ 0.001 (compared to siCnt plus IFN-α).

Article Snippet: Recombinant human interferon alpha (IFN-α; catalog no. 11100-1) and antibodies specific for ISG15 (catalog no. 21900-1; 1:1,000 dilution) were purchased from PBL Assay Science (Piscataway, NJ).

Techniques: Transfection, Control, Western Blot, Software, Activity Assay, Luciferase, Expressing, SYBR Green Assay

Journal: Journal of Virology

Article Title: ISG15 Modulates Type I Interferon Signaling and the Antiviral Response during Hepatitis E Virus Replication

doi: 10.1128/JVI.00621-17

Figure Lengend Snippet: Loss of ISG15 increases type I IFN-induced Mx1 levels in HEV RNA-transfected Huh7-S10-3 liver cells. (A and B) Mx1 (A) and ISG15 (B) mRNA levels in Huh7-S10-3 liver cells cotransfected with capped RNA from the HEV P6 infectious cDNA clone and siRNA. At 24 hpt, cells were either left untreated (w/o IFN-α 100IU) or treated with 100 IU/ml IFN-α (w IFN-α 100IU). Fold change in mRNA levels compared to the untransfected control levels was calculated using the 2−ΔΔCT method, and the RPS18 gene was used as the housekeeping gene The data represent means ± SEM of results from three independent experiments. **, P ≤ 0.01 (compared to siCnt plus P6); aa, P ≤ 0.01 (compared to siCnt plus P6 plus IFN-α using the Student t test).

Article Snippet: Recombinant human interferon alpha (IFN-α; catalog no. 11100-1) and antibodies specific for ISG15 (catalog no. 21900-1; 1:1,000 dilution) were purchased from PBL Assay Science (Piscataway, NJ).

Techniques: Transfection, Control

Journal: Journal of Virology

Article Title: ISG15 Modulates Type I Interferon Signaling and the Antiviral Response during Hepatitis E Virus Replication

doi: 10.1128/JVI.00621-17

Figure Lengend Snippet: ISG15 silencing mediates an enhanced antiviral effect of IFN-α against HEV via PKR and OAS1. Huh7-S10-3 liver cells were cotransfected with HEV RNA and control siRNA (siCnt)/ISG15-siRNA (siISG15), along with siRNA targeted against Mx1 (siMx1) or OAS1 (siOAS1) or PKR (siPKR). At 24 hpt, cells were either left untreated or treated with 100 IU/ml IFN-α. (A) GLuc activity levels in cell culture supernatant at 5 dpt. (B) Intracellular HEV P6 viral RNA levels. The relative changes in GLuc/viral RNA levels were calculated with respect to the corresponding IFN-α-untreated sample. (C to E) Representative Western blot analyses to show the siRNA knockdown efficiency. The Huh7-S10-3 liver cells were cotransfected with control siRNA (siCnt)/ISG15-siRNA (siISG15), along with siRNA targeted against Mx1 (siMx1) (C) or OAS1 (siOAS1) (D) or PKR (siPKR) (E). At 24 hpt, cells were either left untreated or treated with 100 IU/ml IFN-α. After 24 h post-IFN-α treatment, Mx1/PKR/OAS1 protein levels were estimated using Western blot analyses to determine the siRNA knockdown efficiency. The data represent means ± SEM of results from three independent experiments (A) and duplicate cultures (B). a, P ≤ 0.05; aaa, P ≤ 0.001 (compared to siCnt plus IFN-α); #, P ≤ 0.05; ##, P ≤ 0.01 (compared to siISG15 plus IFN-α). N.S, not significant.

Article Snippet: Recombinant human interferon alpha (IFN-α; catalog no. 11100-1) and antibodies specific for ISG15 (catalog no. 21900-1; 1:1,000 dilution) were purchased from PBL Assay Science (Piscataway, NJ).

Techniques: Control, Activity Assay, Cell Culture, Western Blot, Knockdown

Journal: Journal of Virology

Article Title: ISG15 Modulates Type I Interferon Signaling and the Antiviral Response during Hepatitis E Virus Replication

doi: 10.1128/JVI.00621-17

Figure Lengend Snippet: Oligonucleotide primer sequences used for qRT-PCR in the study a

Article Snippet: Recombinant human interferon alpha (IFN-α; catalog no. 11100-1) and antibodies specific for ISG15 (catalog no. 21900-1; 1:1,000 dilution) were purchased from PBL Assay Science (Piscataway, NJ).

Techniques: Sequencing

Journal: Cell Reports Medicine

Article Title: RSAD2 : A pathogenic interferon-stimulated gene at the maternal-fetal interface of patients with systemic lupus erythematosus

doi: 10.1016/j.xcrm.2025.101974

Figure Lengend Snippet:

Article Snippet: The membrane was blocked with 5% skimmed milk at 37°C for 1 h, and then stained using the primary antibodies anti-RSAD2 (D5T2X) (rabbit, CST, 13996) and anti-ISG15 (22D2) (rabbit, CST, 2758T) (for human samples) and the primary antibodies anti-RSAD2 (rabbit, Proteintech, 28089-1-AP) and anti-ISG15 (F-9) (mouse, Santa Cruz, sc-166755) (for murine samples) overnight at 4°C.

Techniques: Recombinant, SYBR Green Assay, Multiplex Assay, Immunohistochemical staining, Staining, Reverse Transcription, Modification, Plasmid Preparation, Software, Microscopy

Journal: Mucosal Immunology

Article Title: Human rhinovirus-induced ISG15 selectively modulates epithelial antiviral immunity

doi: 10.1038/mi.2013.128

Figure Lengend Snippet: Human rhinovirus (HRV)-16 infection induces the expression of interferon-stimulated gene 15 kDa (ISG15) mRNA and protein in human bronchial epithelial (HBE) cells. ( a ) Induction of ISG15 mRNA expression after HRV-16 infection in HBE cells assessed by quantitative reverse transcriptase-PCR (femtograms±s.e.m.; n =6). ( b ) Immunoblot detection of intracellular ISG15 protein production following HRV-16 infection of HBE cells (representative of n =3). ( c ) Levels of released ISG15 into the supernatants of HRV-16-infected HBE (μg ml −1 ±s.e.m.; n =6). M, medium control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HRV, HRV-16. GAPDH was used as an internal control. Asterisk indicates significant difference between medium and HRV-16-infected cells at the respective time point ( P <0.05).

Article Snippet: The following reagents were purchased: Eagle's minimal essential medium, Hank's balanced salt solution, penicillin–streptomycin–amphotericin B, and L -glutamine, TRIzol reagent, sodium pyruvate, nonessential amino acids, gentamicin, and fetal bovine serum (Invitrogen, Burlington, ON, Canada); bronchial epithelial cell basal medium, and additives to create bronchial epithelial cell growth medium (BEGM; Lonza, Walkersville, MD); TaqMan Master Mix, 20 × glyceraldehyde-3-phosphate dehydrogenase, RNase inhibitor, and reverse transcriptase (Applied Biosystems, Streetsville, ON, Canada); TransIT LTI (Mirius, Madison, WI); firefly luciferase reporter plasmid pGL4.10[luc2], and 5 × passive lysis buffer (Promega, Madison, WI); firefly luciferase assay kit (Biotium, Hayward, CA); protease inhibitor tablets (Roche, Mississauga, ON, Canada); horseradish peroxidase-conjugated anti-rabbit Ig Ab and enhanced chemoluminescent substrate reagent (GE Healthcare Bio-Sciences, Piscataway, NJ); specific antibodies: ISG15 and RIG-I (Cell Signaling Technology, Danvers, MA), IRF-1 (Santa Cruz Biotechnology, Santa Cruz, CA), MDA5 (Enzo Life Sciences, Plymouth Meeting, PA); FLAG (Sigma-Aldrich, Oakville, ON, Canada); recombinant active ISG15 and pro-ISG15 (Boston Biochem, Boston, MA).

Techniques: Infection, Expressing, Reverse Transcription, Western Blot, Control

Journal: Mucosal Immunology

Article Title: Human rhinovirus-induced ISG15 selectively modulates epithelial antiviral immunity

doi: 10.1038/mi.2013.128

Figure Lengend Snippet: Healthy volunteers with naturally acquired human rhinovirus (HRV) colds have increased levels of interferon-stimulated gene of 15 kDa (ISG15) protein in nasal lavages. ISG15 protein (ng ml −1 ) and corresponding viral titers (log TCID 50 U ml −1 ) in samples from HRV-infected individuals at the onset of cold symptoms (visit 1), 2 subsequent days (visits 2 and 3), and 4 weeks after onset of symptoms and clearance of viral infection (visit 4). Data represent mean±s.e.m. ( n =10). Asterisks indicate significant ( P <0.05) difference between visit 1 and visit 4 (within each respective group).

Article Snippet: The following reagents were purchased: Eagle's minimal essential medium, Hank's balanced salt solution, penicillin–streptomycin–amphotericin B, and L -glutamine, TRIzol reagent, sodium pyruvate, nonessential amino acids, gentamicin, and fetal bovine serum (Invitrogen, Burlington, ON, Canada); bronchial epithelial cell basal medium, and additives to create bronchial epithelial cell growth medium (BEGM; Lonza, Walkersville, MD); TaqMan Master Mix, 20 × glyceraldehyde-3-phosphate dehydrogenase, RNase inhibitor, and reverse transcriptase (Applied Biosystems, Streetsville, ON, Canada); TransIT LTI (Mirius, Madison, WI); firefly luciferase reporter plasmid pGL4.10[luc2], and 5 × passive lysis buffer (Promega, Madison, WI); firefly luciferase assay kit (Biotium, Hayward, CA); protease inhibitor tablets (Roche, Mississauga, ON, Canada); horseradish peroxidase-conjugated anti-rabbit Ig Ab and enhanced chemoluminescent substrate reagent (GE Healthcare Bio-Sciences, Piscataway, NJ); specific antibodies: ISG15 and RIG-I (Cell Signaling Technology, Danvers, MA), IRF-1 (Santa Cruz Biotechnology, Santa Cruz, CA), MDA5 (Enzo Life Sciences, Plymouth Meeting, PA); FLAG (Sigma-Aldrich, Oakville, ON, Canada); recombinant active ISG15 and pro-ISG15 (Boston Biochem, Boston, MA).

Techniques: Infection

Journal: Mucosal Immunology

Article Title: Human rhinovirus-induced ISG15 selectively modulates epithelial antiviral immunity

doi: 10.1038/mi.2013.128

Figure Lengend Snippet: Human rhinovirus (HRV)-16-induced interferon-stimulated gene of 15 kDa (ISG15) expression is controlled at the level of transcription. A 634-bp ISG15 promoter sequence and various truncated forms were cloned upstream into a pGL4.10 firefly luciferase plasmid and transiently transfected into BEAS-2B cells using lipid-mediated techniques. Cells were exposed to medium (M) or infected with HRV-16 (V) for 24 h and luciferase activity was measured. Data are presented as ( a ) fold increase over medium control and ( b ) relative light units (RLUs), and represent mean±s.e.m. ( n =6). Asterisk indicates significant difference between 634-bp vs. truncated forms in HRV-16-infected cells ( P <0.05). Transcription factor binding sites in the ISG15 promoter: CRE, cAMP response element; IRF, interferon regulatory factor; κB, nuclear factor-κB; STAT, signal transducer and activator of transcription.

Article Snippet: The following reagents were purchased: Eagle's minimal essential medium, Hank's balanced salt solution, penicillin–streptomycin–amphotericin B, and L -glutamine, TRIzol reagent, sodium pyruvate, nonessential amino acids, gentamicin, and fetal bovine serum (Invitrogen, Burlington, ON, Canada); bronchial epithelial cell basal medium, and additives to create bronchial epithelial cell growth medium (BEGM; Lonza, Walkersville, MD); TaqMan Master Mix, 20 × glyceraldehyde-3-phosphate dehydrogenase, RNase inhibitor, and reverse transcriptase (Applied Biosystems, Streetsville, ON, Canada); TransIT LTI (Mirius, Madison, WI); firefly luciferase reporter plasmid pGL4.10[luc2], and 5 × passive lysis buffer (Promega, Madison, WI); firefly luciferase assay kit (Biotium, Hayward, CA); protease inhibitor tablets (Roche, Mississauga, ON, Canada); horseradish peroxidase-conjugated anti-rabbit Ig Ab and enhanced chemoluminescent substrate reagent (GE Healthcare Bio-Sciences, Piscataway, NJ); specific antibodies: ISG15 and RIG-I (Cell Signaling Technology, Danvers, MA), IRF-1 (Santa Cruz Biotechnology, Santa Cruz, CA), MDA5 (Enzo Life Sciences, Plymouth Meeting, PA); FLAG (Sigma-Aldrich, Oakville, ON, Canada); recombinant active ISG15 and pro-ISG15 (Boston Biochem, Boston, MA).

Techniques: Expressing, Sequencing, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Infection, Activity Assay, Control, Binding Assay

Journal: Mucosal Immunology

Article Title: Human rhinovirus-induced ISG15 selectively modulates epithelial antiviral immunity

doi: 10.1038/mi.2013.128

Figure Lengend Snippet: Human rhinovirus (HRV)-16-induced interferon-stimulated gene of 15 kDa (ISG15) transcriptional upregulation is mediated specifically through the interferon regulatory factor (IRF)-B DNA-binding site. Specific point mutations were introduced into either individual ( a ) or multiple ( b ) DNA-binding sites in the 634-bp ISG15 promoter sequence using site-directed mutagenesis techniques. BEAS-2B cells were transiently transfected using lipid-mediated techniques. Cells were infected with HRV-16 for 24 h and luciferase activity was measured in cell lysates. Data are presented as ( a , c ) fold increase over medium control and ( b , d ) as relative light units (RLUs), and represent mean±s.e.m. ( n =6). Asterisk indicates significant difference between wild type and mutated forms in HRV-16-infected cells ( P <0.05). Transcription factor binding sites in the ISG15 promoter: CRE, cAMP response element; κB, nuclear factor-κB; STAT, signal transducer and activator of transcription; M, medium control; V, infection with HRV-16.

Article Snippet: The following reagents were purchased: Eagle's minimal essential medium, Hank's balanced salt solution, penicillin–streptomycin–amphotericin B, and L -glutamine, TRIzol reagent, sodium pyruvate, nonessential amino acids, gentamicin, and fetal bovine serum (Invitrogen, Burlington, ON, Canada); bronchial epithelial cell basal medium, and additives to create bronchial epithelial cell growth medium (BEGM; Lonza, Walkersville, MD); TaqMan Master Mix, 20 × glyceraldehyde-3-phosphate dehydrogenase, RNase inhibitor, and reverse transcriptase (Applied Biosystems, Streetsville, ON, Canada); TransIT LTI (Mirius, Madison, WI); firefly luciferase reporter plasmid pGL4.10[luc2], and 5 × passive lysis buffer (Promega, Madison, WI); firefly luciferase assay kit (Biotium, Hayward, CA); protease inhibitor tablets (Roche, Mississauga, ON, Canada); horseradish peroxidase-conjugated anti-rabbit Ig Ab and enhanced chemoluminescent substrate reagent (GE Healthcare Bio-Sciences, Piscataway, NJ); specific antibodies: ISG15 and RIG-I (Cell Signaling Technology, Danvers, MA), IRF-1 (Santa Cruz Biotechnology, Santa Cruz, CA), MDA5 (Enzo Life Sciences, Plymouth Meeting, PA); FLAG (Sigma-Aldrich, Oakville, ON, Canada); recombinant active ISG15 and pro-ISG15 (Boston Biochem, Boston, MA).

Techniques: Binding Assay, Sequencing, Mutagenesis, Transfection, Infection, Luciferase, Activity Assay, Control

Journal: Mucosal Immunology

Article Title: Human rhinovirus-induced ISG15 selectively modulates epithelial antiviral immunity

doi: 10.1038/mi.2013.128

Figure Lengend Snippet: Interferon (IFN) regulatory factor (IRF)-1 knockdown reduces human rhinovirus (HRV)-16-induced IFN-stimulated gene of 15 kDa (ISG15) protein production in human bronchial epithelial (HBE) cells. HRV-16-induced ISG15 protein (48 h after infection) was assessed by immunoblotting following lipid-mediated transfection (24 h) of 10 n M IRF-1 short-interfering RNA (siRNA) duplexes A or B compared with control, nontargeting siRNA. ISG15 blot is representative of three separate experiments. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. M, medium control; HRV, HRV-16.

Article Snippet: The following reagents were purchased: Eagle's minimal essential medium, Hank's balanced salt solution, penicillin–streptomycin–amphotericin B, and L -glutamine, TRIzol reagent, sodium pyruvate, nonessential amino acids, gentamicin, and fetal bovine serum (Invitrogen, Burlington, ON, Canada); bronchial epithelial cell basal medium, and additives to create bronchial epithelial cell growth medium (BEGM; Lonza, Walkersville, MD); TaqMan Master Mix, 20 × glyceraldehyde-3-phosphate dehydrogenase, RNase inhibitor, and reverse transcriptase (Applied Biosystems, Streetsville, ON, Canada); TransIT LTI (Mirius, Madison, WI); firefly luciferase reporter plasmid pGL4.10[luc2], and 5 × passive lysis buffer (Promega, Madison, WI); firefly luciferase assay kit (Biotium, Hayward, CA); protease inhibitor tablets (Roche, Mississauga, ON, Canada); horseradish peroxidase-conjugated anti-rabbit Ig Ab and enhanced chemoluminescent substrate reagent (GE Healthcare Bio-Sciences, Piscataway, NJ); specific antibodies: ISG15 and RIG-I (Cell Signaling Technology, Danvers, MA), IRF-1 (Santa Cruz Biotechnology, Santa Cruz, CA), MDA5 (Enzo Life Sciences, Plymouth Meeting, PA); FLAG (Sigma-Aldrich, Oakville, ON, Canada); recombinant active ISG15 and pro-ISG15 (Boston Biochem, Boston, MA).

Techniques: Knockdown, Infection, Western Blot, Transfection, Small Interfering RNA, Control

Journal: Mucosal Immunology

Article Title: Human rhinovirus-induced ISG15 selectively modulates epithelial antiviral immunity

doi: 10.1038/mi.2013.128

Figure Lengend Snippet: Interferon-stimulated gene of 15 kDa (ISG15) knockdown markedly inhibits human rhinovirus (HRV)-16-induced ISG15 protein expression, but does not alter HRV-16 replication in human bronchial epithelial (HBE) cells. ( a ) HRV-16-induced ISG15 protein expression (48 h after infection) is reduced after transfection of 10 n M ISG15 short-interfering RNA (siRNA) duplexes A or B compared with control, nontargeting siRNA ( n =3). HBE cells were initially transfected with either ISG15-specific siRNA duplex or control siRNA followed by HRV-16 infection for 3 h, washed with Hank's balanced salt solution (HBSS) three times, and fresh medium was added and collected every 24 h (72 h total). ( b ) HRV-16 RNA levels were measured (72 h after infection) in HBE lysates (white bars) and supernatants (black bars) using 5′-untranslated region (UTR)-specific primers and probe (cycle threshold counts±s.e.m.; n =3). ( c ) HRV-16 titers from HBE were measured (72 h after infection) from supernatants using WI-38 lung fibroblast viral titer assay (log TCID 50 U ml −1 ±s.e.m.; n =3).

Article Snippet: The following reagents were purchased: Eagle's minimal essential medium, Hank's balanced salt solution, penicillin–streptomycin–amphotericin B, and L -glutamine, TRIzol reagent, sodium pyruvate, nonessential amino acids, gentamicin, and fetal bovine serum (Invitrogen, Burlington, ON, Canada); bronchial epithelial cell basal medium, and additives to create bronchial epithelial cell growth medium (BEGM; Lonza, Walkersville, MD); TaqMan Master Mix, 20 × glyceraldehyde-3-phosphate dehydrogenase, RNase inhibitor, and reverse transcriptase (Applied Biosystems, Streetsville, ON, Canada); TransIT LTI (Mirius, Madison, WI); firefly luciferase reporter plasmid pGL4.10[luc2], and 5 × passive lysis buffer (Promega, Madison, WI); firefly luciferase assay kit (Biotium, Hayward, CA); protease inhibitor tablets (Roche, Mississauga, ON, Canada); horseradish peroxidase-conjugated anti-rabbit Ig Ab and enhanced chemoluminescent substrate reagent (GE Healthcare Bio-Sciences, Piscataway, NJ); specific antibodies: ISG15 and RIG-I (Cell Signaling Technology, Danvers, MA), IRF-1 (Santa Cruz Biotechnology, Santa Cruz, CA), MDA5 (Enzo Life Sciences, Plymouth Meeting, PA); FLAG (Sigma-Aldrich, Oakville, ON, Canada); recombinant active ISG15 and pro-ISG15 (Boston Biochem, Boston, MA).

Techniques: Knockdown, Expressing, Infection, Transfection, Small Interfering RNA, Control, Titer Assay

Journal: Mucosal Immunology

Article Title: Human rhinovirus-induced ISG15 selectively modulates epithelial antiviral immunity

doi: 10.1038/mi.2013.128

Figure Lengend Snippet: Interferon-stimulated gene of 15 kDa (ISG15) does not alter human rhinovirus (HRV)-16-induced RIG-I protein expression, but ISG15 binding to RIG-I is increased following HRV infection. ( a ) HRV-16-induced RIG-I and MDA-5 protein levels in human bronchial epithelial (HBE) cell lysates (48 h after infection) were assessed by western blotting after lipid-mediated transfection (24 h) of 10 n M ISG15 short-interfering RNA (siRNA) duplexes A or B, compared with control, nontargeting siRNA. ( b ) HRV-16 infection induces ISG15 binding to RIG-I in BEAS-2B cells. A pTIS plasmid encoding FLAG-tagged RIG-I was transiently transfected using lipid-mediated techniques. Cells were infected with HRV-16 for 48 h and cells were lysed. ( c ) HRV-16-induced ISG15 and RIG-I protein levels (24 h after infection) in HBE cells were reduced after lipid-mediated transfection (24 h) of 10 n M RIG-I siRNA duplexes A or B compared with control, nontargeting siRNA. Blots are representative of three separate experiments. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. M, medium control; HRV, HRV-16.

Article Snippet: The following reagents were purchased: Eagle's minimal essential medium, Hank's balanced salt solution, penicillin–streptomycin–amphotericin B, and L -glutamine, TRIzol reagent, sodium pyruvate, nonessential amino acids, gentamicin, and fetal bovine serum (Invitrogen, Burlington, ON, Canada); bronchial epithelial cell basal medium, and additives to create bronchial epithelial cell growth medium (BEGM; Lonza, Walkersville, MD); TaqMan Master Mix, 20 × glyceraldehyde-3-phosphate dehydrogenase, RNase inhibitor, and reverse transcriptase (Applied Biosystems, Streetsville, ON, Canada); TransIT LTI (Mirius, Madison, WI); firefly luciferase reporter plasmid pGL4.10[luc2], and 5 × passive lysis buffer (Promega, Madison, WI); firefly luciferase assay kit (Biotium, Hayward, CA); protease inhibitor tablets (Roche, Mississauga, ON, Canada); horseradish peroxidase-conjugated anti-rabbit Ig Ab and enhanced chemoluminescent substrate reagent (GE Healthcare Bio-Sciences, Piscataway, NJ); specific antibodies: ISG15 and RIG-I (Cell Signaling Technology, Danvers, MA), IRF-1 (Santa Cruz Biotechnology, Santa Cruz, CA), MDA5 (Enzo Life Sciences, Plymouth Meeting, PA); FLAG (Sigma-Aldrich, Oakville, ON, Canada); recombinant active ISG15 and pro-ISG15 (Boston Biochem, Boston, MA).

Techniques: Expressing, Binding Assay, Infection, Western Blot, Transfection, Small Interfering RNA, Control, Plasmid Preparation

Journal: Mucosal Immunology

Article Title: Human rhinovirus-induced ISG15 selectively modulates epithelial antiviral immunity

doi: 10.1038/mi.2013.128

Figure Lengend Snippet: Human rhinovirus (HRV)-16-induced CXCL10 protein production from human bronchial epithelial (HBE) cells is modulated in the presence of interferon-stimulated gene of 15 kDa (ISG15) short-interfering RNA (siRNA) and recombinant, active ISG15 protein. ( a ) HRV-16-induced CXCL10 protein release in the presence of ISG15-specific or control, nontargeting siRNA was assessed by enzyme-linked immunosorbent assay (ELISA) at 48 h after infection. Asterisk indicates significant difference between HRV-16-induced CXCL10 production in ISG15 siRNA vs. control siRNA-treated cells ( P <0.05). HRV-16-induced ( b ) CXCL10 or ( d ) CXCL8 protein levels in supernatants were assessed using ELISA in the presence of various concentrations of either active or inactive ISG15 protein after 24 h incubation. ( c ) BEAS-2B cells were transiently transfected with a full-length 972-bp CXCL10 promoter-luciferase construct and incubated with either active or inactive ISG15 (30 μg ml −1 ) in the presence or absence of HRV-16 for 24 h. Luciferase activity was measured in cell lysates. Data are presented as fold increase over medium control and represent mean±s.e.m. ( n =6). CXCL10 and CXCL8 protein data are means±s.e.m. ( n =3–5). Asterisk indicates significant difference between HRV-16 alone and HRV-16+active ISG15 ( P <0.05).

Article Snippet: The following reagents were purchased: Eagle's minimal essential medium, Hank's balanced salt solution, penicillin–streptomycin–amphotericin B, and L -glutamine, TRIzol reagent, sodium pyruvate, nonessential amino acids, gentamicin, and fetal bovine serum (Invitrogen, Burlington, ON, Canada); bronchial epithelial cell basal medium, and additives to create bronchial epithelial cell growth medium (BEGM; Lonza, Walkersville, MD); TaqMan Master Mix, 20 × glyceraldehyde-3-phosphate dehydrogenase, RNase inhibitor, and reverse transcriptase (Applied Biosystems, Streetsville, ON, Canada); TransIT LTI (Mirius, Madison, WI); firefly luciferase reporter plasmid pGL4.10[luc2], and 5 × passive lysis buffer (Promega, Madison, WI); firefly luciferase assay kit (Biotium, Hayward, CA); protease inhibitor tablets (Roche, Mississauga, ON, Canada); horseradish peroxidase-conjugated anti-rabbit Ig Ab and enhanced chemoluminescent substrate reagent (GE Healthcare Bio-Sciences, Piscataway, NJ); specific antibodies: ISG15 and RIG-I (Cell Signaling Technology, Danvers, MA), IRF-1 (Santa Cruz Biotechnology, Santa Cruz, CA), MDA5 (Enzo Life Sciences, Plymouth Meeting, PA); FLAG (Sigma-Aldrich, Oakville, ON, Canada); recombinant active ISG15 and pro-ISG15 (Boston Biochem, Boston, MA).

Techniques: Small Interfering RNA, Recombinant, Control, Enzyme-linked Immunosorbent Assay, Infection, Incubation, Transfection, Luciferase, Construct, Activity Assay

Journal: Mucosal Immunology

Article Title: Human rhinovirus-induced ISG15 selectively modulates epithelial antiviral immunity

doi: 10.1038/mi.2013.128

Figure Lengend Snippet: Human rhinovirus (HRV)-16-induced interferon (IFN)-stimulated gene of 15 kDa (ISG15) expression and CXCL10 production is not dependent on interleukin (IL)-29 (type III IFN). BEAS-2B cells were infected with HRV-16 (HRV) or exposed to medium (M) or exogenous IL-29 (1 ng ml −1 ). ( a ) Western blotting for ISG15 expression (representative of n =3). ( b ) Enzyme-linked immunosorbent assay (ELISA) to determine levels of IL-29 in supernatants at 24 and 48 h post stimulation (data are mean±s.e.m. from three experiments). ( c ) Levels of CXCL10 secretion in supernatants at 24 and 48 h post stimulation (data are mean±s.e.m. from three experiments).

Article Snippet: The following reagents were purchased: Eagle's minimal essential medium, Hank's balanced salt solution, penicillin–streptomycin–amphotericin B, and L -glutamine, TRIzol reagent, sodium pyruvate, nonessential amino acids, gentamicin, and fetal bovine serum (Invitrogen, Burlington, ON, Canada); bronchial epithelial cell basal medium, and additives to create bronchial epithelial cell growth medium (BEGM; Lonza, Walkersville, MD); TaqMan Master Mix, 20 × glyceraldehyde-3-phosphate dehydrogenase, RNase inhibitor, and reverse transcriptase (Applied Biosystems, Streetsville, ON, Canada); TransIT LTI (Mirius, Madison, WI); firefly luciferase reporter plasmid pGL4.10[luc2], and 5 × passive lysis buffer (Promega, Madison, WI); firefly luciferase assay kit (Biotium, Hayward, CA); protease inhibitor tablets (Roche, Mississauga, ON, Canada); horseradish peroxidase-conjugated anti-rabbit Ig Ab and enhanced chemoluminescent substrate reagent (GE Healthcare Bio-Sciences, Piscataway, NJ); specific antibodies: ISG15 and RIG-I (Cell Signaling Technology, Danvers, MA), IRF-1 (Santa Cruz Biotechnology, Santa Cruz, CA), MDA5 (Enzo Life Sciences, Plymouth Meeting, PA); FLAG (Sigma-Aldrich, Oakville, ON, Canada); recombinant active ISG15 and pro-ISG15 (Boston Biochem, Boston, MA).

Techniques: Expressing, Infection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Chinese Herbal Medicines

Article Title: Anemoside B4 inhibits SARS-CoV-2 replication in vitro and in vivo

doi: 10.1016/j.chmed.2023.09.005

Figure Lengend Snippet: AB4 inhibited viral replication through IFN signaling pathway and modulated protein expression in cells infected with SARS-CoV-2. HEK293T cell lysates were analyzed by Western blotting for IFNβ signal pathway (A − B) and ISG15 (C). (D) Vero E6 cells were pretreated with AB4 (0–200 μmol/L) for 4 h before viral infection. Cells were infected with SARS-CoV-2 at an MOI of 0.1. At 3 dpi, the cytopathic effect caused by SARS-CoV-2 infection was quantitatively analyzed using CCK-8 according to the manufacturer’s protocol. n = 2 biological replicates. (E) IHC staining of lung tissues for IFNβ. Representative images are shown; Scale bar: 100 µm. Data were expressed as mean ± SD. # P < 0.05, ## P < 0.01, ### P < 0.001 vs vehicle group; * P < 0.05, ** P < 0.01, *** P < 0.001 vs SARS-CoV-2-infected group.

Article Snippet: SARS-CoV-2 N Protein antibody (Cat# A20142; 1:1 000), SARS-CoV-2 3CLpro antibody (Cat# A20198; 1:1 000), SARS-CoV-2 Spike antibody (Cat# A20497; 1:1 500), Hsp90α/β antibody (Cat# A5027; 1:1 000), TBK1 antibody (Cat# A3458; 1:1 000), phospho-IRF3-S386 antibody (Cat# AP0995; 1:1 000) and ISG15 antibody (Cat# A2416; 1:1 000) were provided by ABclonal (Wuhan, China).

Techniques: Expressing, Infection, Western Blot, CCK-8 Assay, Immunohistochemistry

Journal: Nature immunology

Article Title: Mapping Systemic Lupus Erythematosus heterogeneity at the single-cell level

doi: 10.1038/s41590-020-0743-0

Figure Lengend Snippet: (a). Individual UMAP plots from 11 cHD (left), 33 cSLE (right) based on monocyte SCs (Mono-SCs, n=8). Each color represents a distinct SC. (b). Flow cytometry detection of ISG15 in PBMCs gated on CD14+ monocytes from 17 cSLE patients with different SLEDAI scores, as well as 14 cHD. (c). Percentage of ISG15+ CD14+ monocytes in cHD and cSLE as categorized based on SLEDAI (cHD cells ‘none’ in gray, SLEDAI <=4, in yellow; n=9, and SLEDAI >4, in red; n=8). T-test was used for statistical analysis. P-values are shown for the respective comparisons. (d). Individual UMAP plots from 11 cHD (left), 33 cSLE (right) based on B cells SCs (B-SCs, n=7). Each color represents a distinct SC.

Article Snippet: Subsequent to surface staining and staining with live/dead fixable dye (Aqua, 1:1000, Thermo-Fisher), cells were fixed and permeabilized according to the manufacturer’s instructions (Cytofix/Cytoperm and Perm/Wash Buffer; BD Biosciences), and stained for 30 min on ice for Granzyme A (GB9, 1:50, BioLegend), Granzyme B (GB11, 1:50, BioLegend), Perforin (B-D48, 1:50, BD Biosciences), and ISG15 (IC8044P, 1:50, R&D Systems).

Techniques: Flow Cytometry

Journal: Nature immunology

Article Title: Mapping Systemic Lupus Erythematosus heterogeneity at the single-cell level

doi: 10.1038/s41590-020-0743-0

Figure Lengend Snippet: (a). Individual UMAP plots from 11 cHD (left), 33 cSLE (right) based on T cells SCs (T-SCs, n=6). Each color represents a distinct SC. (b). Percentage of GzB+, or Perforin+ CD8+ T cells within cHD and cSLE as categorized based on SLEDAI categories (cHD cells ‘none’ in gray, SLEDAI <=4 in yellow; n=9 and SLEDAI >4, in red; n=8). (c). Flow cytometry detection of GzB and perforin proteins in PBMCs, gated on CD8+ T cells, from cSLE (n=17) with different SLEDAI scores and cHD (n=14). T-test was used for statistical analysis. P-values are shown for the respective comparisons. (d). Individual UMAP plots from 11 cHD (left), 33 cSLE (right) based on NK SCs (NK-SCs, n=4). Each color represents a distinct SC. (e). Flow staining of ISG15 on PBMCs gated on CD57+ NK cells from six cSLE patients (in purple) with different SLEDAI scores and nine matched cHD (in green). MFI values are represented. (f). Boxplots representing the ESR (left panel), C4 (middle panel) and C3 (right panel) levels across the six cSLE subcluster groups (SCGs) depicted in . (g). Dotplot representing the correlation between hemoglobin (HGB) levels (g/dL) and proportion of cells from the Erythrocyte cluster across the cSLE samples.

Article Snippet: Subsequent to surface staining and staining with live/dead fixable dye (Aqua, 1:1000, Thermo-Fisher), cells were fixed and permeabilized according to the manufacturer’s instructions (Cytofix/Cytoperm and Perm/Wash Buffer; BD Biosciences), and stained for 30 min on ice for Granzyme A (GB9, 1:50, BioLegend), Granzyme B (GB11, 1:50, BioLegend), Perforin (B-D48, 1:50, BD Biosciences), and ISG15 (IC8044P, 1:50, R&D Systems).

Techniques: Flow Cytometry, Staining

Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: a , Biological processes involved in immune responses triggered by viral infections. b-f , Schematic showing how ISG15 and genes selected in bats are involved in viral entry into cells and detecting viral patterns (b), regulating antiviral and inflammatory responses (c-d), B cell signaling (e), and activation of the complement system (f). Genes positively-selected in the ancestral branches of Chiroptera (white font), Rhinolophidae–Hipposideridae (yellow font), or Rhinolophidae (orange font) have a blue background. Genes highlighted in panels b-d are described in the main text. e , Inflammation triggers the release of chemokines that direct the migration of leukocytes to sites of infection. GO ‘leukocyte migration’ ( GO:0050900 , P cor = 0.0016) is only enriched for genes selected in bats. We identified selection in two CC chemokine receptors, CCR2 (repeatedly selected in Chiroptera, Rhinolophidae–Hipposideridae and Rhinolophidae) and CCR5 (selected in Rhinolophidae–Hipposideridae) that promote the infiltration of pro-inflammatory cells , . Importantly, CCR2 and CCR5 are located in a major risk locus for severe COVID-19 that contains SNPs linked to increased CCR2/5 expression . Consistent with CCR2/5 mediated hyperinflammation, CCR2/CCR5 receptor antagonists can reduce cytokine storms in patients with severe COVID-19 (ref. ). The GO term ‘lymphocyte activation’ ( GO:0046649 , P cor = 2.46 × 10 − 5 ) has the most significant enrichment for genes under selection in bats, which includes two key factors for B cell signaling, CD79A (selected in Rhinolophidae–Hipposideridae) and BTK (described in the main text). CD79A is the signal transduction subunit of the B cell antigen receptor that upon phosphorylation mediates BTK phosphorylation , . Activated BTK promotes B cell receptor mediated survival of B cells. f , The complement system helps to phagocytose or lyse pathogens, stimulates adaptive immunity and inflammation, but excessive complement activation can lead to hyperinflammation and thrombosis during COVID-19 . GO ‘Regulation of immune effector process’ ( GO:0002697 , P cor = 1.55 × 10 − 8 ; Fig. ), ‘Complement system’ (WikiPathways WP2806, P cor = 8.92 × 10 − 8 ) and ‘Complement and coagulation cascades’ (KEGG:04610, P cor = 0.0003; Supplementary Table ) have the most significant enrichment for genes selected in bats. We detected two complement components, C7 and C1S (both under selection in Rhinolophidae–Hipposideridae). C1S encodes a serine protease involved in early activation of the classical pathway . C7 is a component of the membrane attack complex that forms membrane-disrupting pores and, when endocytosed, activates noncanonical NF-κB signaling and inflammasome assembly . Pictograms of cellular elements were custom-drawn.

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques: Activation Assay, Migration, Infection, Selection, Expressing, Transduction, Phospho-proteomics, Coagulation, Membrane

Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: a , Alignment of functionally important ISG15 residues. The sequence logos show the protein conservation of mammalian orders. b , Representative western blot of HEK293-subclone-ΔISG15 cells overexpressing Myc-tagged wild-type or mutated ISG15 . Lysates were treated with 1,6-bis(maleimido)hexane (BMH) to cross-link disulfide bonds. c , Quantification of ISG15 western blots. Two-tailed t -tests test for differences in dimer formation; n = 4. d – g , Viral output, measured by TCID 50 ( f and g ) or a TCID 50 surrogate (Crystal Violet absorbance at 450 nm; d and e ) in a HEK293-subclone ( d ), Vero-E6 ( e ), stable ANPEP-expressing HEK293 cells ( f ) and stable ACE2-expressing A549 cells ( g ) that express ISG15 constructs. d , VSV–GFP infection (multiplicity of infection (MOI) of 0.1 for 24 h); n = 3. e , H1N1 PR8 IAV infection (MOI of 0.1 for 48 h); n = 4. f , HCoV-229E infection (MOI of 0.1 for 72 h); n = 3. g , SARS-CoV-2 infection (MOI of 0.1 for 48 h); n ≥ 3 for ISG15, n = 7 for SARS-CoV-2 control. h , Quantification of western blots (Extended Data Fig. ) of ISG15-transfected HEK293-subclone-ΔISG15 cells, co-transfected with SARS-CoV-2 PLpro (NSPC3C/L); n = 4. i , Quantification of ISG15 conjugated in cells after PLpro cleavage (matched to h ). Decrease in conjugation with PLpro indicates de-ISGylation; n = 4. j , ISG15 released into the supernatant after PLpro cleavage (matched to h ). In supernatants, ISG15 is not detected before infection; n = 4. k , Quantification of western blots for ISG15 cleavage and release into the supernatant (example in j ), on a logarithmic scale; n = 4. Data are presented as mean (solid oval) and standard error of the mean (s.e.m.) (bars), showing individual data points of biological replicates as grey circles ( c – g , h , i and k ). All quantifications are from independent experiments. Significant differences to vector control (blue asterisks) or wild-type ISG15 (black asterisks) were determined by two-tailed t -tests (* P < 0.05, ** P < 0.01, *** P < 0.001). Uncropped images, P values and all data are in and Supplementary Tables , , , and – . WT, wild type; C78A, Cys78 to Ala mutation; ΔC78, Cys78 deletion; S77C, Ser77 to Cys mutation. All animal silhouettes apart from the bat were obtained from PhyloPic ( phylopic.org ). Mus musculus , by Daniel Jaron under a CC0 1.0 Universal Public Domain licence; Gorilla gorilla gorilla by T. Michael Keesey (after Colin M. L. Burnett) under a CC0 1.0 Universal Public Domain licence; Leporidae , by Sarah Werning under a CC BY 3.0 licence; Erinaceus europaeus by Roberto Díaz Sibaja under a CC BY 3.0 licence; Equus ferus przewalskii , by Mercedes Yrayzoz (vectorized by T. Michael Keesey) under a CC BY 3.0 licence; Camelus dromedarius , by Steven Traver under a CC0 1.0 Universal Public Domain licence; Panthera pardus , by Margot Michaud under a CC0 1.0 Universal Public Domain licence; Manis culionensis , by Steven Traver under a CC0 1.0 Universal Public Domain licence. The bat silhouette was vectorized by A.E.M., the Afrotheria (Thai Elephant) silhouette was obtained from OpenClipArt ( https://openclipart.org/ ) under a CC0 1.0 Universal Public Domain licence.

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques: Sequencing, Western Blot, Two Tailed Test, Expressing, Construct, Infection, Control, Transfection, Conjugation Assay, Plasmid Preparation, Mutagenesis

Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: a , HEK293-CD13-myc ISG15 stable cells were infected with HCoV-229E at an MOI of 0.01 for 72 h prior to collection of cell lysates (lysates) and supernatant (S/N) prior to western blot with anti-myc Ab (as above) to detect transfected ISG15. The level of ISG15 in the supernatant was normalized to cell lysates and loading control (GAPDH), and was expressed relative to the most abundant protein in the supernatant ( Mops condylurus ). Only ISG15 of Mops condylurus was significantly secreted into the supernatant and secretion further increased during HCoV-229E infection. Mean and standard error of the mean are displayed, including individual points (black) for n = 3 independent experiments. Significance was tested with a two-tailed t-test comparing infection to ISG15 alone. Raw data are provided in Supplementary Table . b , An example western blot image (as per panel a) showing high and low exposure of ISG15 supernatant (S/N), HCoV-229E N protein, GAPDH and ISG15 bands in the HEK293-subclone-ΔISG15 cell lysate with protein ladders. Lanes are as indicated. c , Example western blot image with low and high contrast showing ISG15 conjugation (α-myc) after transfection with ISG15’s and HCoV-229E infection (−/+). Arrows indicate bands not seen in the empty vector control (conjugated proteins). d , Western blot of E1 ligase (UBE1L), E2 ligase (UBE2L6) and E3 ligase (HERC5) expression, together with GAPDH in the same samples shown in panel c, indicating sufficient expression of ISGylation machinery in the HEK293-subclone-ΔISG15 cell line. e , HEK293-subclone-ΔISG15 cells were transfected with ISG15 constructs as indicated and with/without NSP3C/L (SARS-CoV-2 PLpro). As per panel c, arrows indicate ISGylation bands, or ISG15 dimer/monomer. The amount of ISG15 present was calculated, normalized to GAPDH and quantified from three western blots. One of the three western blots is shown as an example. f , Representative western blot for the amount of ISG15 present in the supernatant (matched to e). g , ISGylation machinery expression, as per panel d, for NSP3C samples used in panel e and f and Fig. . Western blots in panels b-g are matched to the quantification in Fig. .

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques: Infection, Western Blot, Transfection, Control, Two Tailed Test, Conjugation Assay, Plasmid Preparation, Expressing, Construct

Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: We used structural modeling to explore whether the Cys78 deletion affects homodimer formation of bat ISG15. We hypothesize two alternative outcomes. A stable homodimer will not be formed if we delete Cys78 due to the removal of the disulfide bond. Alternatively, stable homodimers might still form if the dimeric interface is maintained by compensating sets of non-covalent interactions (e.g., hydrogen bonds, hydrophobic interactions). To test these hypotheses, we initially performed 3D structural modeling using AlphaFold2 and compared the putative ISG15 homodimers of human and four bats lacking Cys78 (Supplementary Fig. ). However, the AlphaFold models showed discrepancies between the dimeric interface confidence scores generated and previous experimental studies for human ISG15 , , as well as predicted variations in stability between the bat species (Supplementary Fig. ); therefore we evaluated the stability of predicted dimers by molecular dynamics simulations shown in this figure. We conducted three replicate simulations of 1 μs for human ISG15 and for ISG15 of one representative rhinolophid and hipposiderid bat (total duration 3 μs, about 550,000 CPU hours for each species) (Supplementary Figs. - show the simulation boxes and equilibration phase). a-c , Conformations of putative dimers of human ( a ), Doryrhina cyclops ( b ), and Rhinolophus sinicus ( c ) ISG15. The dimer that was estimated with AlphaFold is circled, whereas the other dimers are major conformations, observed during molecular dynamics simulations (see ). Helices are shown in cyan, whereas β-strands in red. The percentages correspond to the proportion of each conformation across the simulations. The figure was rendered with UCSF ChimeraX v.1.2 . a , Both major conformations are very similar to the AlphaFold predicted conformation. Importantly, while the monomers slightly rotate around the disulfide bond, the Cys78-mediated disulfide bond is sufficient to maintain a dimeric interface with a highly similar conformation, indicative of a stable dimer (see also Supplementary Videos – ). The molecular dynamics simulation results of human ISG15 are consistent with experimental studies , and serve as a positive control as the disulfide bond cannot be broken in the simulation. b , If a stable dimer would exist, we would expect to observe a single dominant structural conformation, or several conformations that are highly similar, as observed for human ISG15. In contrast, for D. cyclops ISG15, the lack of the disulfide bond appears to result in an unstable dimer that adopts a range of conformations, showing remarkable differences in the spatial arrangement of the monomers (see also Supplementary Videos – ). c , As for D. cyclops ISG15, the lack of the Cys78-mediated disulfide bond appears to consistently result in an unstable dimer that adopts two major conformations that differ considerably in the spatial arrangement of the monomers, both from each other and from the starting AlphaFold structure (see also Supplementary Videos – ). This result was observed both at physiological (top) and at salt concentrations as low as required to neutralize the system (middle) for a comparable simulation time of up to 0.5 μs, indicating that different salt concentrations consistently result in unstable dimers. Comparing low salt conditions for a simulation time of 0.5 vs. 1 μs (middle vs. bottom), we found even larger structural differences between the major conformations, which is consistent with the expectation that as the simulation time increases, unstable dimers would increasingly deviate from the initial structure. Overall, these tests also indicate that our results are robust to variation in the salt concentration used in the simulations.

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques: Generated, Positive Control, Concentration Assay

Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: a , Rhinolophus sinicus ISG15; b , Doryrhina cyclops ISG15. While the three simulations initially exhibit highly similar dimeric interfaces, because the starting point is the same AlphaFold-predicted dimer after energy minimization and equilibration, the interfaces increasingly deviate from each other as simulation time increases, indicative of an unstable dimer. See also Supplementary Videos – . The figure was rendered with PyMOL v. 2.5.0 .

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques:

Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: To examine how human and various Cys78-lacking bat ISG15 constructs affect VSV entry and replication and cell viability, we synthesized in addition to Rhinolophus affinis ISG15 from five additional rhinolophid ( R. perniger lanosus, R. yonghoiseni, R. sinicus, R. trifoliatus, R. ferrumequinum ) and three hipposiderid bats ( Aselliscus stoliczkanus , H. larvatus , Doryrhina cyclops ). a , Viral entry, quantified as the percentage of VSV-GFP positive cells. HEK293 cells were transiently transfected with ISG15 constructs and infected with VSV-GFP. GFP-positive cells after 16 h infection with an MOI of 0.01 were measured by FACS, examining mCherry (or transfected) cells only. The percentage of GFP-positive cells is shown for three separate experiments (n > 10,000 cells each). Data is presented as mean (solid oval) and standard deviation (bars) with individual data points for the three biological replicates displayed as grey circles. Grey and light yellow backgrounds indicate empty vector and human, respectively. All ISG15 proteins reduce viral entry, with the exception of R. trifoliatus where the effect is not significant compared to the empty vector. Notably, viral entry differs substantially between ISG15 of different rhinolophid and hipposiderid bats. For example, ISG15 of R. yonghoiseni / R. trifoliatus / R. perniger lanosus (comprising a phylogenetic clade) had a significantly reduced ability to block viral entry compared to human ISG15. Because ISG15 of all rhinolophid and hipposiderid bats lacks Cys78 (Fig. ), this indicates that other amino acid mutations apart from the shared Cys78 deletion alter ISG15 function. P-values and raw data are in Supplementary Table . b , Viral replication, measured by GFP intensity during early infection stages. Viral load for the data shown in Fig. , measured by mean fluorescence intensity (VSV-GFP) in HEK293 cells transiently transfected with ISG15 (IRES-mcherry) constructs, relative to the control vector (no ISG15). Data are presented as mean (solid oval) and standard deviation (bars) with individual data points of three biological replicates shown as grey circles. P-values and raw data are in Supplementary Table . c , ISG15 of some bats affects growth of uninfected HEK293 cells. Since we observed large differences between initial infection, intracellular viral load and final viral release into the supernatant, we additionally examined cell viability in the presence of ISG15. The panel shows FACS measurements of Ki-67, a cellular marker for proliferation, in HEK293 cells that were stably transfected with the various ISG15 constructs. Measurements were taken at 16 h post-infection and are normalized to the 0 h timepoint and the vector control. Thus, values > 1 indicate an increase in cell growth compared to the vector control. Three biological replicates are indicated by grey dots and black bars show standard error per treatment. P-values and raw data are in Supplementary Table . For uninfected cells (light blue bars), expression of human ISG15 and ISG15 of A. stoliczkanus , R. ferrumequinum and R. sinicus leads to a significant decrease in cell growth, compared to the vector control. In contrast, expression of ISG15 of the six other bats ( D. cyclops, H. larvatus, R. affinis, R. yonghoiseni, R. trifoliatus , and R. perniger lanosus ) results in a significantly increased growth of uninfected cells, showing that ISG15 of some bats positively affects cell growth in the absence of viral infection. Furthermore, ISG15 of six bats induces a significantly higher cell growth compared to human ISG15, while A. stoliczkanus ISG15 induces a significantly lower cell growth. For cells infected with GFP-VSV (red bars), none of the differences are significant when comparing bat to human ISG15. Human ISG15 was, however, still significantly different between infected and uninfected (p = 0.0017). Unlike human, several bats show large decreases in proliferation between uninfected and infected cells, indicating different dynamics inside the cell during virus replication. Furthermore, the basal increase in proliferation may have a substantial consequence on later virus production by allowing cells to continue producing higher amounts of virus prior to cell death , or by altering the basal immune state, possibly explaining a reduced effect of human or bat wildtype ISG15 in blocking VSV production. a-c , Significant differences to the vector control or to human ISG15 were determined with a two-tailed t-test and is indicated with * P < 0.05, ** P < 0.01, *** P < 0.001. New Bat1K genomes are indicated in bold font.

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques: Construct, Synthesized, Transfection, Infection, Standard Deviation, Plasmid Preparation, Blocking Assay, Fluorescence, Control, Marker, Stable Transfection, Expressing, Virus, Two Tailed Test

Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: a , ISG15 Cys mutants do not alter ISG expression during H1N1 PR8 Influenza A virus (IAV) infection or ISG15 supernatant treatment in epithelial cells. To investigate why mutating or deleting Cys78 from human ISG15 and restoring Cys78 in R. affinis ISG15 significantly increased IAV production, we tested whether the ISG15 Cys mutants but not wildtype ISG15 impacted IRF3 activation (known to be modulated by ISG15 ), IFN production, and induction of key antiviral interferon-stimulated genes (MX1 and IFIT1). IAV was infected at an MOI of 0.1 for 48 h after transfection with wildtype ISG15 constructs and ISG15 Cys78 mutants (indicated on right). The representative western blot shows that IAV infection causes some IRF3 activation and only minimal MX1 or IFIT1 protein induction in Vero-E6 cells when transfected with an empty vector or most ISG15 constructs. While R. affinis S77C induced more MX1 and IFIT1, this did not alter IAV production (Fig. ), indicating minimal effect on the final production of infectious IAV particles. This is consistent with VeroE6 cells having limited IFN signal amplification, and shows that while ISG15 modulates IFN signaling and some ISG-induction in Vero-E6 cells, other effects of ISG15 on viral production are more relevant. Importantly, there is no impact of Cys-removal from human ISG15 on MX1 or IFIT1 in infected cells, indicating that the observed pro-viral outcome of ISG15 Cys mutants is likely not due to downregulating interferon-stimulated gene expression. A representative western blot image is shown from three independent experiments. b , Extracellular ISG15 does not enhance NFκB and IFN signaling in A549 cells. Wildtype and Cys-mutant ISG15 showed limited anti-IAV activity. To rule out a reliance on extracellular cytokine enhancement from ISG15, we measured the effect of extracellular wildtype or mutant ISG15 on NFκB and IFN signaling. As this process requires the ISG15 receptor, LFA-1 , which is not expressed in VeroE6 cells, we used A549 cells that are IFNɣ-competent and express a low-level of LFA-1. ISG15-containing supernatants from IAV-infected VeroE6 cells were UV-treated with 10000 gy of UV-C to deactivate IAV. A549 cells were then treated with these supernatants overnight. We then measured A549-cell lysates for TNFα and IL-6 to assess NFκB-signaling, and IFIT1, IFITM3, and endogenous ISG15 to assess IFN signaling. We observed no obvious induction of these pathways in these cells between wildtype and Cys-mutant ISG15, indicating the pro-viral effect of ISG15 mutants is likely not caused by impairing ISG15’s cytokine enhancement function in these cells. A representative western blot image is shown from three independent experiments.

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques: Expressing, Virus, Infection, Activation Assay, Transfection, Construct, Western Blot, Plasmid Preparation, Amplification, Gene Expression, Mutagenesis, Activity Assay

Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: a – c , Viral output, expressed relative to the empty vector (IRES–mCherry) control, in A549 ( a ), stable ANPEP-expressing HEK293 ( b ) and stable ACE2-expressing A549 ( c ) cells that express ISG15 constructs. a , H1N1 PR8 IAV infection (as per Fig. ). A549 cell supernatants titrated on Vero-E6 cells by plaque assays; n = 4. b , HCoV-229E infection (as per Fig. ). Viral output was measured by TCID 50 in Huh7 cells; n = 3. c , SARS-CoV-2 infection (as per Fig. ). Viral output from A549 cells was measured by TCID 50 in Vero76 cells; n ≥ 4 for ISG15, n = 7 for SARS-CoV-2 control. d , As c except using wild-type (WT) and LRGG>LRAA ISG15 mutants that remove the ISG15 conjugation motif; n ≥ 5 for LRGG mutations, n = 7 for SARS-CoV-2 control. Graphs represent mean and s.e.m. with individual data points in grey; n refers to independent biological replicates. Significant differences to vector control (black asterisks) or human ISG15 (orange asterisks) were determined by two-tailed t -tests (* P < 0.05, ** P < 0.01, *** P < 0.001). All data are given in Supplementary Tables – .

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques: Plasmid Preparation, Control, Expressing, Construct, Infection, Conjugation Assay, Two Tailed Test

Journal: Nature

Article Title: Bat genomes illuminate adaptations to viral tolerance and disease resistance

doi: 10.1038/s41586-024-08471-0

Figure Lengend Snippet: a , Predicted Aligned Error (PAE) plots obtained with Alphafold2-Multimer in ColabFold . Alphafold2 modeled SARS-CoV-2 (wildtype, Wuhan-1) PLpro chain (amino acids 819–2763 of ORF1ab) monomer in complex with a monomer of ISG15 of the five indicated species. The PAE plots represent the error in the position of each amino acid. The crystal structure of PLpro in complex with ISG15 revealed it exists as a pentamer of five units of PLpro with five monomeric ISG15 units in a large complex . This AlphaFold2 model represents the native monomer:monomer structure as folded in Alphafold2-Multimer (Mmseqs2 v 1.5.5) via ColabFold, due to computational limits. See also Supplementary Videos – . b , Snapshots from Supplementary Videos – showing PLpro in a complex with ISG15 of bats and human. Images on the left show a whole protein view of the complex (as per panel a). The middle images show a zoom view of the active site and the right images display the flexible C-terminal tail of ISG15 adjacent to the PLpro binding groove with contacts visualized in green (Supplementary Videos – show the details). pLDDT scores and number of contacts for each of the five species are 64.84: 422, 65.77: 156, 66.04: 761, 60.44: 354, 65.79: 126, respectively. ISG15 is colored by pLDDT values. PLpro in white. The linear C-terminal tail of human ISG15 resembles that of R. trifoliatus ISG15, loading directly into the PLpro catalytic site, with additional contact sites in R. trifoliatus that seem to stabilize the interaction.

Article Snippet: For ISGylation detection, protein samples were mixed with 4× NuPAG LDS sample buffer (Invitrogen, NP0007), separated by NuPAGE 4–12% Bis-Tris gels in running buffer (Invitrogen, NP0001) for 70 min under 120 V, and transferred (Invitrogen, NP000061) for 90 min under 100 V. The following antibodies were used for detection: rabbit anti-MX1 polyclonal antibody (clone N2C2, Genetex, GTX110256, dilution 1:1,000); rabbit anti-ISG15 polyclonal antibody (middle region, Aviva Systems Biology, ARP59386_P050, dilution 1:1,000); rabbit anti-GAPDH monoclonal antibody (clone 14C10, Cell Signaling, 2118, dilution 1:2,000); rabbit anti-CD13 polyclonal antibody (Sino Biological, 10051-T60, dilution 1:2,000); rabbit HCoV-229E nucleocapsid polyclonal antibody (Sino Biological, 40640-T62, dilution 1:2,000); mouse anti-MYC monoclonal antibody (Sino Biological, 100029-MM08, dilution 1:2,000 for cell lysate and 1:1,000 for cell supernatants); rabbit anti-UBE1L monoclonal antibody (Huabio, HA721228, dilution:1:500); rabbit polyclonal anti-UBE2L6 antibody (Abclonal, A13670 ); rabbit polyclonal anti-HERC5 antibody (Abclonal, A14889 ); and HRP-conjugated goat anti-rabbit IgG (Transgen Biotech, HS101-01, dilution 1:5,000).

Techniques: Binding Assay