Journal: bioRxiv

Article Title: Combinations of approved oral nucleoside analogues confer potent suppression of alphaviruses in vitro and in vivo

doi: 10.1101/2025.01.24.633564

Figure Lengend Snippet: Dose-response activity of (A,D) EIDD-1931 (MPV), (B,E) SOF and (C,F) FAV against cytopathic effect induced by CHIKV, SFV, VEEV, RRV or SINV (AR86 and HRsp strain), quantified in (A-C) skin fibroblasts or (D-F) Huh7 cells by the MTS or ATP method at 72 hours post infection. Data represent percentage of virus inhibition or cell viability compared to virus or cell control samples, respectively, and is shown as mean values ± standard deviation from at least three independent experiments.

Article Snippet: SFV Vietnam strain (GenBank EU350586.1 ) and SINV HRsp strain (GenBank J02363.1 ) belong to the collection of the Rega Institute of Medical Research (KU Leuven, Belgium).

Techniques: Activity Assay, Infection, Virus, Inhibition, Control, Standard Deviation

Journal: bioRxiv

Article Title: Combinations of approved oral nucleoside analogues confer potent suppression of alphaviruses in vitro and in vivo

doi: 10.1101/2025.01.24.633564

Figure Lengend Snippet: Antiviral efficacy of combinations of EIDD-1931 (MPV) and FAV, EIDD-1931 (MPV) and SOF or FAV and SOF against (A) VEEV TC83, (B) SINV AR86, (C) SINV HRsp in Huh7 cells. Inhibition of virus-induced CPE was quantified by the ATP method at 72 hours post infection. Data were analyzed and visualized using SynergyFinder 3.0 based on the Bliss independence model. Left panels: overall Bliss Synergy score averaged over all dose combination measurements of the matrix, representing the percentage excess response compared to the expected responses. Bliss Synergy scores lower than –10, between –10 and 10 or larger than 10 indicate combinations which are likely to be antagonistic, additive or synergistic, respectively. Right panels: maximum Bliss synergy score representing the most synergistic 3-by-3 window in the full dose-response matrix (MSA). Data in panels A-C indicate the mean of 2-4 independent experiments (with each experiment denoted by a dot). MSA; most synergistic area.

Article Snippet: SFV Vietnam strain (GenBank EU350586.1 ) and SINV HRsp strain (GenBank J02363.1 ) belong to the collection of the Rega Institute of Medical Research (KU Leuven, Belgium).

Techniques: Inhibition, Virus, Infection

Journal: bioRxiv

Article Title: DEAD box RNA helicases 5 and 17 are new host factors for Sindbis virus infection

doi: 10.1101/2023.09.21.558232

Figure Lengend Snippet: A ) Confocal microscopy analysis of SINV (+) RNA and DDX5 protein localization in mock and infected HCT116 cells at 24 hpi by RNA fluorescence in situ hybridization (FISH) (in magenta) combined with protein immunostaining (in green). DAPI staining (in blue) and merge of the different channels are shown. Magnification 40X, scale bar, 10µm. B ) RT-qPCR on SINV genomic (g) RNA upon DDX5 RIP or IgG RIP. Results are expressed as percentage of Input (total RNA) and represent the mean ± standard deviation (SD) of three biological replicates (n = 3). C ) Anti-dsRNA dot blot assay on serial dilutions of the total RNA (INPUT) and the undiluted RNA samples from DDX5-RIP or IgG-RIP, in mock and SINV infected conditions. J2 antibody was used to detect dsRNAs.

Article Snippet: Cells were fixed again with 3.7 % formaldehyde (Biosearch technologies) diluted in PBS 1X for10 min at room temperature and incubated over night at room temperature with the SINV genome specific LGC Biosearch Technologies’ Stellaris® RNA FISH Probe diluted in RNA FISH hybridization buffer (Stellaris, Biosearch technologies).

Techniques: Confocal Microscopy, Infection, Fluorescence, In Situ Hybridization, Immunostaining, Staining, Quantitative RT-PCR, Standard Deviation, Dot Blot

Journal: Pathogens

Article Title: High-Sensitivity RT-LAMP for Molecular Detection of O’nyong-nyong ( Alphavirus onyong )

doi: 10.3390/pathogens13100892

Figure Lengend Snippet: Multiple alignment overview of the design of primers. ( a ) Multiple alignment scheme of ONNV, SINV, and CHIKV genomes, showing each position of the genomes, whether they are a match, mismatch, or gap relative to the reference genome ONNV Gulu strain. The region of interest for LAMP primer design is shown in red. ( b ) Detailed view from Bioedit of the region of interest. Bars with arrows represent each primer design for RT-LAMP ONNV . The gap between genomes relative to the reference genome ONNV Gulu strain is shown in red. Each subclade of ONNV (reference ) is represented by subclade 1 (●) and subclade 2 (●●).

Article Snippet: Additionally, three other alphaviruses—namely, CHIKV (strain S-27; laboratory stock), SINV (strain EfAr339; laboratory stock), and MAYV (strain TRVL-4675, laboratory stock recently prepared after obtaining the virus through BEI Resources-NIAID-NIH)—all belonging to the Semliki Forest serological complex (all stored at −80 °C after replication on VeroE6 cells) were used for viral RNA extraction and included in the assay’s specificity evaluation.

Techniques:

Journal: Pathogens

Article Title: High-Sensitivity RT-LAMP for Molecular Detection of O’nyong-nyong ( Alphavirus onyong )

doi: 10.3390/pathogens13100892

Figure Lengend Snippet: Comparison of the limit of detection of RT-LAMP ONNV between two different reaction times and specificity of RT-LAMP ONNV . ( a ) Limit of detection for a 20 min reaction time. ( b ) Limit of detection for a 30 min reaction time. ( c ) Agarose gel electrophoresis analysis of the specificity of the assay to detect ONNV, when performed with RNA of CHIKV, MAYV, and SINV. M: Molecular marker—GeneRuler 1 kb Plus DNA Ladder (Invitrogen). NTC: Negative control. RNA: ONNV RNA extract.

Article Snippet: Additionally, three other alphaviruses—namely, CHIKV (strain S-27; laboratory stock), SINV (strain EfAr339; laboratory stock), and MAYV (strain TRVL-4675, laboratory stock recently prepared after obtaining the virus through BEI Resources-NIAID-NIH)—all belonging to the Semliki Forest serological complex (all stored at −80 °C after replication on VeroE6 cells) were used for viral RNA extraction and included in the assay’s specificity evaluation.

Techniques: Comparison, Agarose Gel Electrophoresis, Marker, Negative Control

Journal: bioRxiv

Article Title: XRN1 supplies free nucleotides to feed alphavirus replication

doi: 10.1101/2024.12.09.625895

Figure Lengend Snippet: (A) Fluorescence microscopy analysis of A549 WT and XRN1-KO cells infected with SINV for 18 h (MOI 2). n = 3. Cellular mRNA was detected by smFISH with an Oligo(dT) 25 probe (polyA RNA), nuclei were stained with DAPI, and SINV capsid was visualized by immunofluorescence. Yellow arrows indicate the accumulation of polyA RNA in the viral factories marked with SINV capsid antibody. (B) MA plot comparing the read coverage and the log2 fold change between SINV infected (8 hpi top; or 18 hpi bottom) and uninfected condition of each transcript detected in the RNA sequencing experiment for HEK293 WT, XRN1 partial, and full KO cells. Blue dots represent RNAs enriched with p.adj < 0.05 while grey dots represent non-significant changes. Dashed lines: black indicates the zero log2 fold change while grey represents the median fold changes across all transcripts. (C) Proportion of normalized human and SINV reads from RNAseq experiment at 18 hpi. (D) Boxplot of the Synonymous dinucleotide usage (SDU) of the three most important features in upregulated (pos) and downregulated (neg) transcripts upon SINV infection. (E) Comparison of the SDU of the indicated dinucleotides between SINV genome and cellular transcripts upregulated and downregulated upon SINV infection. (F) Luciferase activity of Rluc-WT or reporters with altered dinucleotide codon usage (Rluc-up and Rluc-down) measured at 6 hours post transfection (hpt) relative to the luciferase levels at 4 hpt in SINV infected conditions. n = 4; error bars: standard error; * p < 0.05.

Article Snippet: The pcDNA5-Rluc-WT, up, or down constructs containing the wild-type DNA Renilla luciferase sequence from pRL-CMV (Promega AF025843) or the di-nucleotide optimized Rluc sequence optimised to resemble SINV (Upregulated transcripts, up) or Mock (downregulated transcripts, down) conditions were purchased from GeneScript and inserted into pcDNA™/FRT/TO (ThermoFisher # V652020) via BamHI and NotI sites.

Techniques: Fluorescence, Microscopy, Infection, Staining, Immunofluorescence, RNA Sequencing Assay, Comparison, Luciferase, Activity Assay, Transfection

Journal: bioRxiv

Article Title: XRN1 supplies free nucleotides to feed alphavirus replication

doi: 10.1101/2024.12.09.625895

Figure Lengend Snippet: (A) Western blot analysis of samples from (MOI = 1. n = 3). (B) Correlation of the RNAseq and RT-qPCR data from for randomly selected transcripts. Error bars represent standard error of three independent experiments. (C) GO term enrichment for upregulated transcripts in SINV infected WT (left) and XRN1-KO (right) cells. (D) MA plot comparing the read coverage and the log2 fold change between WT and XRN1-KO uninfected cells of each gene detected in the RNAseq experiment from . Red, blue, and grey dots represent significantly upregulated, downregulated, and unchanged transcripts levels, respectively. Purple dots represent ISGs in HEK293 cells, as defined in . Density plots displaying distribution of fold changes among each colour group are shown at the right side of the figure. (E) GO term enrichment of transcripts upregulated in XRN1-KO cells in non-infected conditions (XRN1 WT versus KO cells in non-infected conditions, left) and transcripts upregulated in XRN1-KO cells 18 hpi (XRN1 WT versus KO cells in infected conditions, right). (F) Western blot analysis of antiviral response proteins OAS1, OAS3, and IFIT1 in WT and XRN1/5’-3’ RNA decay components KO cells in non-infected conditions. n = 3 (G) Top 20 most important features and their corresponding importance scores identified by Synonymous Dinucleotide Usage (SDU) and Codon Usage comparing up versus down-regulated transcripts upon SINV infection (RNAseq experiment fig 3B). (H) Comparison of the SDU between SINV genome and cellular transcripts upregulated and downregulated upon SINV infection. (I) Luciferase activity of Rluc-WT or reporters with altered dinucleotide codon usage (Rluc-up and Rluc-down) measured at 6 hours post transfection (hpt) relative to the luciferase levels at 4 hours post transfection (hpt) in mock conditions. n = 4; error bars: standard error. (J-K) RNA fold change of RLuc-WT or reporters with altered dinucleotide codon usage (RLuc-up and RLuc-down) measured at 6 hours post transfection (hpt) relative to RNA levels at 4 hours post transfection (hpt) in mock (J) and infected conditions (K). n = 3; error bars: standard error.

Article Snippet: The pcDNA5-Rluc-WT, up, or down constructs containing the wild-type DNA Renilla luciferase sequence from pRL-CMV (Promega AF025843) or the di-nucleotide optimized Rluc sequence optimised to resemble SINV (Upregulated transcripts, up) or Mock (downregulated transcripts, down) conditions were purchased from GeneScript and inserted into pcDNA™/FRT/TO (ThermoFisher # V652020) via BamHI and NotI sites.

Techniques: Western Blot, Quantitative RT-PCR, Infection, Comparison, Luciferase, Activity Assay, Transfection

Journal: bioRxiv

Article Title: XRN1 supplies free nucleotides to feed alphavirus replication

doi: 10.1101/2024.12.09.625895

Figure Lengend Snippet: (A) Principal component analysis of data from immunoprecipitated (IP) and size match input (SMI) samples from iCLIP2 experiments in uninfected and SINV-infected cells at 4 or 18 hpi. (B) Visualisation of IR adaptor of size match input (SMI) and immunoprecipitated (IP) samples from iCLIP2 experiment, in mock and infected conditions. (C) MA plot comparing the read coverage and the log2 fold change between the differentially regulated transcripts in XRN1-KO cells pre- and post-infection with the XRN1 targets (left) and not targets (right) identified by iCLIP2. Blue and red dots represent significantly downregulated and upregulated RNAs, respectively. (D) Bar plots reflecting the same data shown in . Instead of absolute counts, the percentage of transcripts is plotted to better visualize the effect seen in KO cells. (E) Comparison of binding profile of TRIM25 and XRN1on SINV genome at 9 and 18hpi, respectively. (F) SINV genome coverage from RNAseq (WT and XRN1-KO samples) and iCLIP2 data with inset showing a ‘zoomed in’ view of the 3’ end of the viral genome. (G) Silver staining analysis of samples from anti-GFP-beads for mass spectrometry analysis from . (H-I) Volcano plots showing XRN1 interactor proteins enriched in XRN1-IP compared to WCL in mock (H) and infected (I) conditions.

Article Snippet: The pcDNA5-Rluc-WT, up, or down constructs containing the wild-type DNA Renilla luciferase sequence from pRL-CMV (Promega AF025843) or the di-nucleotide optimized Rluc sequence optimised to resemble SINV (Upregulated transcripts, up) or Mock (downregulated transcripts, down) conditions were purchased from GeneScript and inserted into pcDNA™/FRT/TO (ThermoFisher # V652020) via BamHI and NotI sites.

Techniques: Immunoprecipitation, Infection, Comparison, Binding Assay, Silver Staining, Mass Spectrometry

Journal: bioRxiv

Article Title: XRN1 supplies free nucleotides to feed alphavirus replication

doi: 10.1101/2024.12.09.625895

Figure Lengend Snippet: (A) XRN1 binding site and target gene counts from iCLIP2 analysis of HEK293-Flp-In T-REx-XRN1-eGFP cells. Cells were either uninfected or infected with SINV for 4 or 18 h (MOI 10). n = 3. (B) Venn diagram representing the overlap of target genes identified in iCLIP2 in mock and SINV infected cells (4 h and 18 h). (C) Density plot of distribution of XRN1 binding sites on mature target mRNAs. (D) MA plots from (of WT 18 hpi sample) showing XRN1 targets (left, 4hpi) and non-targets (right) identified by iCLIP2. Blue and red dots represent significantly downregulated and upregulated RNAs, respectively. (E) Bar plots showing the percentage of XRN1 target genes (based on iCLIP2) that are downregulated, unchanged, and upregulated (based on RNAseq) in WT, XRN1 partial (PKO), and full KO (KO). (F) Bar plots showing the number of XRN1 target genes and non-target genes (based on iCLIP2) that are upregulated and downregulated (based on RNAseq) in WT, XRN1 partial (PKO), and full KO (KO). (G) Top: Binding profile of XRN1 on SINV genome at 4 hpi and 18 hpi. Bottom: schematic representation of SINV genome features. (H) Volcano plot showing proteins enriched in HEK293-Flp-In T-REx-XRN1-eGFP IP in mock versus SINV-infected cells (18 hpi, MOI 10). n = 3. (I) Volcano plot showing proteins enriched in HEK293-Flp-In T-REx-XRN1-eGFP IP compared to WCL in SINV-infected cells (18 hpi, MOI 10). n = 3. (J) Western blot analysis of input and anti-GFP-beads IP (pull down of GFP-CTRL and XRN1-GFP) in mock and infected conditions. n = 3.

Article Snippet: The pcDNA5-Rluc-WT, up, or down constructs containing the wild-type DNA Renilla luciferase sequence from pRL-CMV (Promega AF025843) or the di-nucleotide optimized Rluc sequence optimised to resemble SINV (Upregulated transcripts, up) or Mock (downregulated transcripts, down) conditions were purchased from GeneScript and inserted into pcDNA™/FRT/TO (ThermoFisher # V652020) via BamHI and NotI sites.

Techniques: Binding Assay, Infection, Western Blot

Journal: bioRxiv

Article Title: XRN1 supplies free nucleotides to feed alphavirus replication

doi: 10.1101/2024.12.09.625895

Figure Lengend Snippet: (A) Fluorescence microscopy analysis of mock and SINV-infected cells at the indicated time-points post infection (MOI 1). N = 3. NME3, HPRT1, and APRT were detected by immunofluorescence, SINV-nsP3 was visualized by tagging with mScarlet, and nuclei were stained with DAPI. Yellow arrows indicate colocalization of nsP3 with NME3 (top) HPRT1 (middle), and APRT (bottom). (B) Western blot analysis of A549-WT cells (CTRL siRNA) and cells depleted of components of the nucleotide salvage pathway with two different siRNAs mix (Pool-1: HPRT1, CTPS1/2, APRT, UPRT, CDA, and NME3; and Pool-2: HPRT1, CTPS1, APRT, CDA, and UPRT), infected with SINV for 18 h (MOI 0.1). n = 3. (C) Western blot analysis of HEK293-Flp-In T-REx WT and XRN1 KO cells complemented with the indicated XRN1 mutants, in presence or absence of externally supplemented nucleosides and infected with SINV for 18 h (MOI 0.1). n = 3. Below the blots the average normalized capsid level of three independent experiments, in bracket standard error. * p < 0.05. Expression of XRN1 constructs was induced by doxycycline treatment (Dox) for at least 96 hours prior infection. (D) Western blot analysis of HEK293T KO cells of the indicated member of the RNA decay machinery, supplemented with nucleosides (2X) and infected with SINV for 18 h (MOI 0.01). n = 3.

Article Snippet: The pcDNA5-Rluc-WT, up, or down constructs containing the wild-type DNA Renilla luciferase sequence from pRL-CMV (Promega AF025843) or the di-nucleotide optimized Rluc sequence optimised to resemble SINV (Upregulated transcripts, up) or Mock (downregulated transcripts, down) conditions were purchased from GeneScript and inserted into pcDNA™/FRT/TO (ThermoFisher # V652020) via BamHI and NotI sites.

Techniques: Fluorescence, Microscopy, Infection, Immunofluorescence, Staining, Western Blot, Expressing, Construct

Journal: bioRxiv

Article Title: XRN1 supplies free nucleotides to feed alphavirus replication

doi: 10.1101/2024.12.09.625895

Figure Lengend Snippet: (A) Fluorescence microscopy analysis of mock and SINV-infected cells at the indicated time-points post infection (MOI 1). n = 3. PPAT or NME1 were detected by immunofluorescence, SINV-nsP3 was visualized by tagging with mScarlet, and nuclei were stained with DAPI. Yellow arrows indicate colocalization of nsP3 with PPAT (top), or NME1 (bottom). (B) Cell count of WT cells and cells depleted of components of the nucleotide salvage pathway with two different siRNA mixes (Pool-1: HPRT1, CTPS1/2, APRT, UPRT, CDA, and NME3; and Pool-2: HPRT1, CTPS1, APRT, CDA, and UPRT), infected with SINV for 18 h (MOI 0.1). n = 3. Error bars: standard error. (C) Western blot analysis of HEK293 WT and XRN1 partial KO (XRN1-PKO) cells supplemented with nucleosides and infected with SINV for 18 h. (MOI 0.1). n = 5. Below the blots the average normalized capsid level of four independent experiments, in bracket standard error. * p < 0.05. (D) Western blot analysis of DDX6-KO cells, supplemented with nucleosides and infected with SINV for 18 h. (MOI 0.01). n = 3. (E) Schematic model representing the role of the RNA decay machinery and nucleotide salvage pathway in regulating alphavirus infection. XRN1 and the 5-3DM localize to SINV viral factories and degrade cellular transcripts in proximity to the replication centre to provide high local concentration of nucleotides to sustain viral replication.

Article Snippet: The pcDNA5-Rluc-WT, up, or down constructs containing the wild-type DNA Renilla luciferase sequence from pRL-CMV (Promega AF025843) or the di-nucleotide optimized Rluc sequence optimised to resemble SINV (Upregulated transcripts, up) or Mock (downregulated transcripts, down) conditions were purchased from GeneScript and inserted into pcDNA™/FRT/TO (ThermoFisher # V652020) via BamHI and NotI sites.

Techniques: Fluorescence, Microscopy, Infection, Immunofluorescence, Staining, Cell Counting, Western Blot, Concentration Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response

doi: 10.1073/pnas.1621508114

Figure Lengend Snippet: TIPARP limits replication of viruses belonging to the family Togaviridae. (A) Domain architecture of ZAP, PARP12, and TIPARP. (B) U373-CD14 cells were treated with the indicated siRNAs and then infected with SINV (multiplicity of infection [MOI] = 1) for 24 h. The levels of viral protein and β-actin in the cell lysates were determined by immunoblotting analysis. (C and D) U373-CD14 cells were treated with control or TIPARP siRNA. (C) The cells were infected with rubella virus (MOI = 0.1) for 96 h. The levels of rubella virus RNA in the culture supernatants were measured by quantitative reverse transcription-PCR. (D) The cells were infected with VSV (MOI = 1), CVB3 (MOI = 1), JEV (MOI = 1), IAV (PR8, 100 hemagglutinin unit), or HSV-1 (MOI = 1) for 24 h. The levels of viral proteins and β-actin in the cell lysates were determined by immunoblotting analysis. Experiments were performed three times, and representative data are shown (means ± SD of three independent samples). *P < 0.05.

Article Snippet: Anti-SINV E2 antibody was originally prepared by D. E. Griffin (Johns Hopkins University School of Medicine) and was obtained from Y. Yoshinaka (Tokyo Medical and Dental University).

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

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response

doi: 10.1073/pnas.1621508114

Figure Lengend Snippet: Loss of TIPARP enhances SINV replication. (A) Primary Tiparp+/+ and Tiparp−/− MEFs were infected with SINV (MOI = 1) for 12 h. Fixed samples were subjected to an RNA fluorescence in situ hybridization analysis of SINV RNA and to Hoechst 33342 staining of genomic DNA. (B and C) Primary Tiparp+/+ and Tiparp−/− MEFs were infected with SINV (MOI = 0.1) (B) or VSV (MOI = 0.1) (C) for 24 h. The viral titers in culture supernatants were determined by 50% tissue culture infectious dose assay. (D and E) ELISA of IFN-β and CXCL10 in culture supernatants of primary Tiparp+/+ and Tiparp−/− MEFs. (D) Cells were infected with SINV (MOI = 1) or VSV (MOI = 1) for 24 h. (E) Cells were stimulated with 5′PPP dsRNA (1 μg/mL), poly rI:rC (1 μg/mL), and IFN stimulatory DNA (ISD) (1 μg/mL), together with Lipofectamine 2000, for 24 h. (Scale bar, 20 μm.) Experiments were performed at least three times, and representative data are shown (means ± SD of three independent samples). *P < 0.05.

Article Snippet: Anti-SINV E2 antibody was originally prepared by D. E. Griffin (Johns Hopkins University School of Medicine) and was obtained from Y. Yoshinaka (Tokyo Medical and Dental University).

Techniques: Infection, Fluorescence, In Situ Hybridization, Staining, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response

doi: 10.1073/pnas.1621508114

Figure Lengend Snippet: TIPARP deficiency renders mice susceptible to SINV infection. (A and B) Ten-day-old Tiparp+/+ and Tiparp−/− mice (n = 5 each) were s.c. inoculated with SINV (100 plaque-forming units per mouse). At day 5 postinfection, the brains of SINV-infected Tiparp+/+ and Tiparp−/− mice were isolated. (A) The viral titers (plaque-forming units per tissue weight) in brains were determined by 50% tissue culture infectious dose. (B) The levels of Ifnb1, Cxcl10, Il1b, and Il6 mRNA in brains were measured by quantitative reverse transcription-PCR. Experiments were performed five times, and representative data are shown (means ± SD of five independent samples). *P < 0.05.

Article Snippet: Anti-SINV E2 antibody was originally prepared by D. E. Griffin (Johns Hopkins University School of Medicine) and was obtained from Y. Yoshinaka (Tokyo Medical and Dental University).

Techniques: Infection, Isolation, Reverse Transcription

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response

doi: 10.1073/pnas.1621508114

Figure Lengend Snippet: TIPARP binds to SINV RNA and induces SINV RNA degradation. (A) Wild-type MEFs stably expressing the indicated FLAG-tagged proteins were infected with SINV (MOI = 0.1) for 24 h. The levels of viral E2 protein, FLAG-tagged protein, and β-actin in cell lysates were determined by immunoblotting analysis. (B) Wild-type MEFs stably expressing the indicated expression plasmids were infected with SINV for 24 h. The levels of SINV RNA binding to the indicated FLAG-tagged proteins were measured by RNA immunoprecipitation, coupled with quantitative reverse transcription-PCR. (C) U373-CD14 cells were transiently transfected with the indicated expression plasmids. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody and to immunoblot analysis with anti-FLAG and anti-Myc antibodies. The experiments were performed three times, and representative data are shown (means ± SD of three independent samples). *P < 0.05.

Article Snippet: Anti-SINV E2 antibody was originally prepared by D. E. Griffin (Johns Hopkins University School of Medicine) and was obtained from Y. Yoshinaka (Tokyo Medical and Dental University).

Techniques: Stable Transfection, Expressing, Infection, Western Blot, RNA Binding Assay, RNA Immunoprecipitation, Reverse Transcription, Transfection, Immunoprecipitation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response

doi: 10.1073/pnas.1621508114

Figure Lengend Snippet: TIPARP recruits EXOSC5 to degrade Sindbis virus (SINV) RNA. (A) Wild-type MEFs were stably expressed with control shRNA or Exosc5 shRNA. The levels of Exosc5 mRNA expression were measured by quantitative reverse transcription-PCR. (B and C) Wild-type MEFs stably expressing control shRNA or Exosc5 shRNA were infected with SINV (MOI = 0.05) (B) or VSV (MOI = 0.05) (C) for 24 h. Levels of viral protein and β-actin in cell lysates were measured by immunoblotting. Experiments were performed three times, and representative data are shown (means ± SD of three independent samples). *P < 0.05.

Article Snippet: Anti-SINV E2 antibody was originally prepared by D. E. Griffin (Johns Hopkins University School of Medicine) and was obtained from Y. Yoshinaka (Tokyo Medical and Dental University).

Techniques: Virus, Stable Transfection, Control, shRNA, Expressing, Reverse Transcription, Infection, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response

doi: 10.1073/pnas.1621508114

Figure Lengend Snippet: Neither SINV nor IFN-β induces expression of Tiparp mRNA. (A) Wild-type MEFs were stimulated with IFN-β (10 ng/mL) or TNF (10 ng/mL) for the indicated durations. Levels of Tiparp, Zc3hav1, and Parp12 mRNA expression were measured by quantitative reverse transcription-PCR. (B) Wild-type MEFs were infected with SINV (MOI = 1) for the indicated durations. Levels of Tiparp mRNA expression were measured by quantitative reverse transcription-PCR. Experiments were performed three times, and representative data are shown (means ± SD of three independent samples).

Article Snippet: Anti-SINV E2 antibody was originally prepared by D. E. Griffin (Johns Hopkins University School of Medicine) and was obtained from Y. Yoshinaka (Tokyo Medical and Dental University).

Techniques: Expressing, Reverse Transcription, Infection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response

doi: 10.1073/pnas.1621508114

Figure Lengend Snippet: Cytoplasmic accumulation of TIPARP promotes elimination of SINV. (A) Wild-type MEFs stably expressing TIPARP-FLAG were infected with SINV (MOI = 5) for 12 h. Fixed samples were subjected to RNA fluorescence in situ hybridization analysis of SINV RNA, immunocytochemistry analysis of TIPARP-FLAG, and Hoechst 33342 staining of genomic DNA. (B and C) Wild-type MEFs stably expressing TIPARP-FLAG were treated with tunicamycin (5 μg/mL), etoposide (10 μM), Leu-Leu methyl ester hydrobromide (LLOMe) (100 μM), or CCCP (10 μM) for 6 h (B). Wild-type MEFs stably expressing TIPARP-FLAG were infected with SINV (MOI = 5), together with Z-VAD (10 μM), cyclosporin A (5 μM), or BHA (5 μM) for 12 h (C). Fixed samples were subjected to immunocytochemistry analysis of FLAG-tagged protein and Hoechst 33342 staining of genomic DNA. Frequencies of MEFs with cytoplasmic accumulation of TIPARP were determined. (D) Wild-type MEFs were infected with SINV (MOI = 5) or stimulated with CCCP (10 μM), with or without BHA (5 μM), for 12 h, and then stained with MitoSOX Red. Samples were subjected to flow cytometric analysis to measure the level of mitochondrial reactive oxygen species. SSC-A, side scatter area. (E) U373-CD14 cells were infected with SINV (MOI = 5) or stimulated with CCCP (50 μM) in the presence or absence of BHA (5 μM) for 24 h. Cellular extracts were treated with or without 100 mM DTT. Samples were suspended in 2-mercaptoethanol-free loading buffer and were subjected to immunoblot analysis of Nup62 and lamin A/C. (Scale bars, 20 μm.) The experiments were performed three times, and representative data are shown (means ± SD of three independent samples). *P < 0.05.

Article Snippet: Anti-SINV E2 antibody was originally prepared by D. E. Griffin (Johns Hopkins University School of Medicine) and was obtained from Y. Yoshinaka (Tokyo Medical and Dental University).

Techniques: Stable Transfection, Expressing, Infection, Fluorescence, In Situ Hybridization, Immunocytochemistry, Staining, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response

doi: 10.1073/pnas.1621508114

Figure Lengend Snippet: Cytoplasmic accumulation enhances anti-SINV activity of TIPARP. (A) Wild-type MEFs stably expressing TIPARP-FLAG and TIPARPNES-FLAG tagged protein were infected with SINV (MOI = 5) for 12 h. Fixed samples were subjected to immunocytochemistry analysis of FLAG-tagged protein and Hoechst 33342 staining of genomic DNA. (B and C) Wild-type MEFs stably expressing TIPARP-FLAG and TIPARPNES-FLAG-tagged protein were infected with SINV (MOI = 0.1) for the indicated durations (B) or VSV (MOI = 0.1) for 24 h (C). Viral titers in culture supernatants were determined by 50% tissue culture infectious dose assay. (D) Primary Mavs+/− or Mavs−/− MEFs stably expressing TIPARP-FLAG were infected with SINV (MOI = 5) for 12 h. Fixed samples were subjected to immunocytochemistry analysis of FLAG-tagged protein and Hoechst 33342 staining of genomic DNA. (E) Wild-type MEFs stably expressing TIPARP-FLAG were stimulated with IFN-β (10 ng/mL) or poly rI:rC (1 μg/mL) for 12 h. Fixed samples were subjected to immunocytochemistry analysis of FLAG-tagged protein and Hoechst 33342 staining of genomic DNA. (F) Wild-type MEFs were stably expressed with Nup62-specific and control shRNA. The levels of Nup62 mRNA were measured by quantitative reverse transcription-PCR. (G) Wild-type MEFs stably expressing TIPARP-FLAG with Nup62-specific shRNA or control shRNA. Fixed samples were subjected to immunocytochemistry analysis of TIPARP-FLAG and Hoechst 33342 staining of genomic DNA. (Scale bars, 20 μm.) Experiments were performed three times, and representative data are shown (means ± SD of three independent samples). *P < 0.05.

Article Snippet: Anti-SINV E2 antibody was originally prepared by D. E. Griffin (Johns Hopkins University School of Medicine) and was obtained from Y. Yoshinaka (Tokyo Medical and Dental University).

Techniques: Activity Assay, Stable Transfection, Expressing, Infection, Immunocytochemistry, Staining, Control, shRNA, Reverse Transcription

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response

doi: 10.1073/pnas.1621508114

Figure Lengend Snippet: BAX- and BAK1-dependent generation of mitochondrial reactive oxygen species causes cytoplasmic accumulation of TIPARP to limit SINV replication. (A) Bax+/+/Bak1+/+ and Bax−/−/Bak1−/− MEFs stably expressing TIPARP-FLAG were infected with SINV (MOI = 5) or stimulated with CCCP (10 μM) for 12 h. Fixed samples were subjected to immunocytochemistry analysis of TIPARP-FLAG and Hoechst 33342 staining of genomic DNA. (B) Bax+/+/Bak1+/+ and Bax−/−/Bak1−/− MEFs were infected with SINV (MOI = 5) or stimulated with CCCP (10 μM) for 12 h, and then stained with MitoSOX Red. Samples were subjected to flow cytometric analysis to measure levels of mitochondrial reactive oxygen species. (C and D) Bax+/+/Bak1+/+ and Bax−/−/Bak1−/− MEFs (C) or primary Mavs+/− and Mavs−/− MEFs (D) stably expressing TIPARP-FLAG were then infected with SINV (MOI = 5) for 24 h. Viral titers in culture supernatants were determined by 50% tissue culture infectious dose assay. (Scale bar, 20 μm.) Experiments were performed three times, and representative data are shown (means ± SD of three independent samples). *P < 0.05.

Article Snippet: Anti-SINV E2 antibody was originally prepared by D. E. Griffin (Johns Hopkins University School of Medicine) and was obtained from Y. Yoshinaka (Tokyo Medical and Dental University).

Techniques: Stable Transfection, Expressing, Infection, Immunocytochemistry, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response

doi: 10.1073/pnas.1621508114

Figure Lengend Snippet: MCL1 limits cytoplasmic accumulation of TIPARP. (A) Wild-type MEFs stably expressing TIPARP-FLAG with BCL2, BCLW, BCLXL, or MCL1 protein were infected with SINV (MOI = 5) for 12 h. Fixed samples were subjected to immunocytochemistry analysis of FLAG-tagged protein and Hoechst 33342 staining of genomic DNA. Frequencies of MEFs with cytoplasmic accumulation of TIPARP were determined. (B) Wild-type MEFs were infected with SINV (MOI = 0.5) for 12 h. Levels of viral protein, MCL1 protein, and β-actin in cell lysates were measured by immunoblotting analysis. (Scale bar, 20 μm.) Experiments were performed three times, and representative data are shown (means ± SD of three independent samples). *P < 0.05.

Article Snippet: Anti-SINV E2 antibody was originally prepared by D. E. Griffin (Johns Hopkins University School of Medicine) and was obtained from Y. Yoshinaka (Tokyo Medical and Dental University).

Techniques: Stable Transfection, Expressing, Infection, Immunocytochemistry, Staining, Western Blot