Journal: Antiviral research

Article Title: Identification of 2′-deoxy-2′-fluorocytidine as a potent inhibitor of Crimean-Congo hemorrhagic fever virus replication using a recombinant fluorescent reporter virus

doi: 10.1016/j.antiviral.2017.10.008

Figure Lengend Snippet: a. Schematic representation of the large (L), medium (M), and small (S) genome segments (antigenomic sense) of Crimean-Congo hemorrhagic fever virus (CCHFV). Arrows represent the viral protein coding sequences for RNA-dependent RNA polymerase (RdRp), glycoprotein precursor (GPC), and nucleoprotein (NP). CCHFV strain IbAr10200 S segment was modified by inserting the ZsGreen1 (ZsG) protein coding sequence fused to the P2A sequence (amino acid sequence shown) upstream of the NP coding region (rS). b. Western blot analysis of viral protein expression in Huh7 cells infected with CCHFV/ZsG at MOI 1. Cell lysates were harvested 72 h post infection (hpi) to determine cellular expression levels of NP and ZsG, using tubulin as a loading control. c. Cellular localization of virally expressed proteins. Huh7 cells were infected with either CCHFV or CCHFV/ZsG at MOI 1, and imaged 48 hpi. CCHFV NP was detected using immunofluorescence, and ZsG fluorescence was imaged using standard fluorescent microscopy. d. Viral growth curves in Huh7, SW13, and HAP1 cells infected with either CCHFV or CCHFV/ZsG at MOI 0.1. Titers (TCID50) were determined at 24, 48, and 72 hpi, with ZsG fluorescence determined concurrently at the same time points (RLU, relative light units). Representative fluorescent microscopy images of CCHFV/ZsG-infected cell monolayers at 72 hpi are presented.

Article Snippet: Primary antibodies used were: α-CCHFV NP (IBT Bioservices, #04-0011), α-ZsGreen1 (Clonetech, #632598), and anti-α-tubulin (Sigma-Aldrich, #T5168).

Techniques: Virus, Modification, Sequencing, Western Blot, Expressing, Infection, Control, Immunofluorescence, Fluorescence, Microscopy

Journal: Antiviral research

Article Title: Identification of 2′-deoxy-2′-fluorocytidine as a potent inhibitor of Crimean-Congo hemorrhagic fever virus replication using a recombinant fluorescent reporter virus

doi: 10.1016/j.antiviral.2017.10.008

Figure Lengend Snippet: a. Dose-response curves in Huh7 or SW13 cells treated with a 2-fold serial dilution of ribavirin before infection with CCHFV/ZsG at MOI 0.1. At 72 h post treatment, the reduction in ZsG fluorescence (green) was determined, and all values were normalized to those in mock-treated (DMSO only) cells. Cell viability was determined concurrently using the same serial-fold dilution of compound, with ATP content (blue) normalized to content in mock-treated cells. Each point represents the mean of quadruplicate wells, with error bars indicating standard deviation. Graphs are representative of 3 independent experiments. b. Representative concentration-response curves in Huh7 cells infected with CCHFV/ZsG and treated with T-705, 2′-deoxy-2′-fluorocytidine (2′-dFC), mycophenolic acid, or mycophenolate mofetil. Each point represents the mean of quadruplicate wells, with error bars indicating standard deviation. Graphs are representative of 3 independent experiments. c. Confirmatory counter-screen titer reduction assays were performed using wild-type CCHFV. Huh7 cells were treated with 4 dilutions of each compound centered around their calculated EC50 value 1 h prior to infection with CCHFV at MOI 0.1. At 48 hpi, virus-containing supernatants were harvested, and viral titers were determined by TCID50. Each point represents the mean of triplicate wells, with error bars indicating standard deviation. Dotted line represents the limit of detection for this assay (1.89 × 101 TCID50/mL). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used were: α-CCHFV NP (IBT Bioservices, #04-0011), α-ZsGreen1 (Clonetech, #632598), and anti-α-tubulin (Sigma-Aldrich, #T5168).

Techniques: Serial Dilution, Infection, Fluorescence, Standard Deviation, Concentration Assay, Virus

Journal: Antiviral research

Article Title: Identification of 2′-deoxy-2′-fluorocytidine as a potent inhibitor of Crimean-Congo hemorrhagic fever virus replication using a recombinant fluorescent reporter virus

doi: 10.1016/j.antiviral.2017.10.008

Figure Lengend Snippet: Compounds evaluated in the antiviral screening assay, showing the 50% effective concentrations (EC 50 ) and 50% cytotoxicity concentrations (CC 50 ) of each compound in Huh7 cells. All values are in μM; selectivity indices (SI) shown in parentheses. RdRp, RNA-dependent RNA polymerase; IMPDH, inosine monophosphate dehydrogenase.

Article Snippet: Primary antibodies used were: α-CCHFV NP (IBT Bioservices, #04-0011), α-ZsGreen1 (Clonetech, #632598), and anti-α-tubulin (Sigma-Aldrich, #T5168).

Techniques: Screening Assay

Journal: Antiviral research

Article Title: Identification of 2′-deoxy-2′-fluorocytidine as a potent inhibitor of Crimean-Congo hemorrhagic fever virus replication using a recombinant fluorescent reporter virus

doi: 10.1016/j.antiviral.2017.10.008

Figure Lengend Snippet: Dose-response curve in SW13 cells treated with a 2-fold serial dilution of 2′-dFC before infection with CCHFV at MOI 0.1. At 48 hpi, cells were fixed and stained with anti-CCHFV polyclonal antibodies, CellMask Red, and Nuc-Blue to visualize viral proteins, cell cytoplasm, and cell nuclei, respectively. a. The percentage of infected cells at each concentration of compound was calculated by determining the proportion of cells expressing CCHFV proteins compared to total cell number (based on CellMask Red). b. Cell viability assessed by determining the number of Nuc-Blue-stained nuclei. c. Representative images at indicated 2′-dFC concentrations show the reduction in CCHFV protein production (green) in the absence of any cytotoxicity (Nuc-Blue-stained nuclei). CellMask Red staining was omitted from these images for clarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used were: α-CCHFV NP (IBT Bioservices, #04-0011), α-ZsGreen1 (Clonetech, #632598), and anti-α-tubulin (Sigma-Aldrich, #T5168).

Techniques: Serial Dilution, Infection, Staining, Concentration Assay, Expressing

Journal: Antiviral research

Article Title: Identification of 2′-deoxy-2′-fluorocytidine as a potent inhibitor of Crimean-Congo hemorrhagic fever virus replication using a recombinant fluorescent reporter virus

doi: 10.1016/j.antiviral.2017.10.008

Figure Lengend Snippet: Huh7 cells were treated with a 6 × 6 compound combination matrix prior to infection with CCHFV/ZsG at MOI 0.1. At 72 h post treatment, the reduction in ZsG fluorescence was determined, and all values normalized to mock-treated (DMSO only) cells. The compound combinations evaluated were: a. T-705 with ribavirin; b. T-705 with 2′-dFC; and c. 2′-dFC with ribavirin. Dose-response surface curves were created for each combination (Bliss model), from which Bliss synergy and antagonism surface isograms were generated. Matrix tables indicate the maximum synergy value for each combination (% synergy effect observed over expected), with standard deviation indicated. Significance of synergy effect over expected results is indicated following a one-sample t-test (*p < 0.05; **p < 0.01, ***p < 0.001).

Article Snippet: Primary antibodies used were: α-CCHFV NP (IBT Bioservices, #04-0011), α-ZsGreen1 (Clonetech, #632598), and anti-α-tubulin (Sigma-Aldrich, #T5168).

Techniques: Infection, Fluorescence, Generated, Standard Deviation

Journal: Antiviral research

Article Title: Identification of 2′-deoxy-2′-fluorocytidine as a potent inhibitor of Crimean-Congo hemorrhagic fever virus replication using a recombinant fluorescent reporter virus

doi: 10.1016/j.antiviral.2017.10.008

Figure Lengend Snippet: a. Huh7 cells were treated with the indicated compounds either individually or in combination at the following concentrations: T-705, 2 μM; 2′-dFC, 200 nM; ribavirin, 10 μM. Cell were infected with wild-type CCHFV at MOI 0.1, and viral titers were determined at 48 hpi. Each point represents the mean of triplicate wells, with error bars indicating standard deviation. Dotted line represents the limit of detection for this assay (1.89 × 101 TCID50/mL). b. Cell viability (ATP content) was determined concurrently and normalized to ATP content in mock-treated (DMSO only) Huh7 cells.

Article Snippet: Primary antibodies used were: α-CCHFV NP (IBT Bioservices, #04-0011), α-ZsGreen1 (Clonetech, #632598), and anti-α-tubulin (Sigma-Aldrich, #T5168).

Techniques: Infection, Standard Deviation

Journal: Cell reports

Article Title: Crimean-Congo Hemorrhagic Fever Virus suppresses Innate Immune Responses via a Ubiquitin and ISG15 Specific Protease

doi: 10.1016/j.celrep.2017.08.040

Figure Lengend Snippet: (A) CCHF virus-like particles (VLPs) were generated by transfecting Huh7 cells with plasmids encoding CCHFV polymerase (L), nucleoprotein (NP), and glycoprotein (GPC), as well as a minigenome encoding luciferase and T7 polymerase. After 3 days, supernatants containing the VLPs were harvested and transferred to new Huh7 cells, and the luciferase signal was determined as a measure of VLP activity. Data are presented as mean ± SD of 3 biological replicates and * indicate p < 0.05 (B) A model of the CCHFV OTU in complex with ubiquitin (PDB entry 3PRP) was used to assist with the design of mutations that disrupt the OTU-Ub and OTU-ISG15 binding. The protease surface that forms the binding interface with ubiquitin is shaded in brown, with the residues targeted for mutation in this study indicated. The residues forming the shared consensus sequence of ubiquitin and ISG15, LRLRGG, are shown in purple. (C) OTU mutant activity profiles on 7-amino-4-methylcoumarin (AMC) fluorogenic substrates of monoubiquitin (Ub-AMC) and ISG15 (ISG-AMC) was assessed in vitro. Data are presented as mean of two biological replicates ± SD and * indicate p < 0.05 relative to WT (D) Overview of selected OTU mutations and their predicted effect on deubiquitinase and deISGylase activities. between the wild-type OTU or L protein and the individual mutants.

Article Snippet: CCHFV N was detected with hyperimmune mouse ascetic fluid (in house) or CCHFV anti-N antibody (IBT Bioservices).

Techniques: Virus, Generated, Luciferase, Activity Assay, Ubiquitin Proteomics, Binding Assay, Mutagenesis, Sequencing, In Vitro

Journal: Cell reports

Article Title: Crimean-Congo Hemorrhagic Fever Virus suppresses Innate Immune Responses via a Ubiquitin and ISG15 Specific Protease

doi: 10.1016/j.celrep.2017.08.040

Figure Lengend Snippet: To assess the DUB and deISGylase activity of the OTU mutants, Huh7 cells were co-transfected with plasmids expressing HA-Ub or V5-ISG15, Ube1L, UbcH8 and HERC5. Cell lysates were harvested 2 days post transfection, and proteins analyzed for Ub- or ISG15-conjugates. The OTU activities of both the single OTU domain (A) and the OTU in the context of the complete L protein were assessed by western blot (B). (C) The cellular levels of CCHFV L protein, co-expressed with HA-Ub or ISG15, were analyzed in Huh7 cells by western blot.

Article Snippet: CCHFV N was detected with hyperimmune mouse ascetic fluid (in house) or CCHFV anti-N antibody (IBT Bioservices).

Techniques: Activity Assay, Transfection, Expressing, Western Blot

Journal: Cell reports

Article Title: Crimean-Congo Hemorrhagic Fever Virus suppresses Innate Immune Responses via a Ubiquitin and ISG15 Specific Protease

doi: 10.1016/j.celrep.2017.08.040

Figure Lengend Snippet: To assess the effect of OTU on the RIG-I-mediated IFN response, HEK293T cells were transfected with plasmids expressing the CCHFV OTU domain, the constitutively active RIG-I CARDs to induce IFN-β expression and a firefly luciferase reporter gene under control of the promoter of IFN-β (A), or containing binding sites for IRF3 (B) or NF-κB (C). Data are presented as mean ± SD of 3 biological replicates and * indicate statistical significance of p<0.05 between the wild-type OTU -and the individual mutants. OTU expression and activation of the RIG-I mediated immune response was confirmed by western blot analysis (D).

Article Snippet: CCHFV N was detected with hyperimmune mouse ascetic fluid (in house) or CCHFV anti-N antibody (IBT Bioservices).

Techniques: Transfection, Expressing, Luciferase, Control, Binding Assay, Activation Assay, Western Blot

Journal: Cell reports

Article Title: Crimean-Congo Hemorrhagic Fever Virus suppresses Innate Immune Responses via a Ubiquitin and ISG15 Specific Protease

doi: 10.1016/j.celrep.2017.08.040

Figure Lengend Snippet: Wild-type and mutant CCHFV growth kinetics were measured by TCID50. (A) A549, BSR-T7/5, Huh7, SW13, and HAP1 cells were infected with CCHFV at an MOI of 0.01. Monocyte-derived macrophages were infected with CCHFV (MOI 1). Data are mean ± SD of two independent experiments. * represent p < 0.05 between WT and OTU mutants. (B) HUVEC and primary skin fibroblast cells were infected at an MOI of 0.5. Data are mean ± SD of three biological replicates.

Article Snippet: CCHFV N was detected with hyperimmune mouse ascetic fluid (in house) or CCHFV anti-N antibody (IBT Bioservices).

Techniques: Mutagenesis, Infection, Derivative Assay

Journal: Cell reports

Article Title: Crimean-Congo Hemorrhagic Fever Virus suppresses Innate Immune Responses via a Ubiquitin and ISG15 Specific Protease

doi: 10.1016/j.celrep.2017.08.040

Figure Lengend Snippet: (A) Western blot of ubiquitinated and ISGylated proteins in CCHFV-infected cells. Huh7 cells were infected with CCHFV at an MOI of 5. Cell lysates were harvested 24h post infection, separated by SDS-PAGE and probed for total Ub or K48- and K63-linked poly-Ub chains. (B) To assess protein ISGylation in infected cells, IFN β (1000 IU/ml) was added to the cell culture media 4 h post infection. Conjugation of ISG15 was visualized using western blotting.

Article Snippet: CCHFV N was detected with hyperimmune mouse ascetic fluid (in house) or CCHFV anti-N antibody (IBT Bioservices).

Techniques: Western Blot, Infection, SDS Page, Cell Culture, Conjugation Assay

Journal: Cell reports

Article Title: Crimean-Congo Hemorrhagic Fever Virus suppresses Innate Immune Responses via a Ubiquitin and ISG15 Specific Protease

doi: 10.1016/j.celrep.2017.08.040

Figure Lengend Snippet: (A) A549 cells were infected with CCHFV or RVFV vaccine strain at an MOI of 3.3. RNA was isolated from samples harvested 24 h post infection, and transcripts of the indicated genes were quantified using RT-PCR. Data are mean ± SD of a representative experiment performed using 3 biological replicates, *p < 0.05. (B) CCHFV NP levels were determined by western blotting.

Article Snippet: CCHFV N was detected with hyperimmune mouse ascetic fluid (in house) or CCHFV anti-N antibody (IBT Bioservices).

Techniques: Infection, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot