Journal: eLife

Article Title: An arms race between 5’ppp-RNA virus and its alternative recognition receptor MDA5 in RIG-I-lost teleost fish

doi: 10.7554/elife.94898

Figure Lengend Snippet: Figure 1. A genetic compensation response model for RIG loss. (A) Loss of RIG-I in vertebrates. Each branch tip represents one species. Red lines show lineages where loss of RIG-I. The red circle indicates the occurrence of an independent loss event. Branch lengths scale in millions of years (MYA). (B) Comparative analysis of gene synteny of RIG-I in vertebrate genomes. (C and D) SCRV-RNA and VSV-RNA were extracted from SCRV and VSV virus and dephosphorylated them with Calf Intestinal Alkaline Phosphatase (CIAP). Then, MKC cells were transfected with SCRV-RNA and SCRV-RNA +CIAP, and DF-1 cells were transfected with VSV-RNA and VSV-RNA +CIAP. IRF3 dimerization was analyzed by native gel electrophoresis. (E) MKC cells were transfected with different concentrations of SCRV-RNA and SCRV-RNA +CIAP, then the expression of IFN-1 was detected by qRT-PCR. As a control, the expression level of IFN-1 in MKC cells transfected with the same volume of water was set to ‘1’. (F) Predicted protein structures of MDA5 and LGP2 of M. miiuy and RIG-I of H. sapiens. (G) The expression of MDA5 and LGP2 plasmids of M. miiuy were detected by western Blot. (H) MKC cells were co- transfected with MDA5 plasmids and LGP2 plasmids for 24 hr and then treated with SCRV (MOI = 5) for 24 hr. The expression of IFN-1 was determined by qPCR. (I) MKC cells were co-transfected with MDA5 plasmids and si-LGP2 for 24 hr and then treated with SCRV (MOI = 5) for 24 hr. The expression of LGP2 and IFN-1 was determined by qPCR. (J) A genetic compensation response model for RIG loss. All data presented as the means ± SE from three independent triplicated experiments. **, p<0.01, as determined by Student’s t test.

Article Snippet: The antibody against MDA5 was diluted at 1: 500 (Beyotime, Cat# AF7164); The antibody against IRF3 was diluted at 1: 500 (Boster, Cat# BA4351- 2); anti- Flag, anti- Myc, anti- HA and anti- Tubulin monoclonal antibody were diluted at 1: 1,000 (Beyotime); and HRP- conjugated anti- rabbit IgG or anti- mouse IgG (Abbkine) at 1: 5,000.

Techniques: Virus, Transfection, Nucleic Acid Electrophoresis, Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: eLife

Article Title: An arms race between 5’ppp-RNA virus and its alternative recognition receptor MDA5 in RIG-I-lost teleost fish

doi: 10.7554/elife.94898

Figure Lengend Snippet: Figure 2. MDA5 promotes host antiviral innate immunity. (A) Silence efficiency of si-MDA5 measured by qRT-PCR and Western blotting. Two siRNAs of MDA5 were transfected into MPC cells for 48 hr, respectively. (B) MKC cells were transfected with MDA5 plasmids for 48 hr, then the overexpression efficiency was detected through MDA5 endogenous antibodies. (C) Subcellular localization of MDA5 in MKCs by immunofluorescence. (D) MDA5 knockdown suppresses NF-κB, IRF3, IRF7, and IFN-1 signaling. MPC cells were transfected with pRL-TK Renilla luciferase plasmid, luciferase reporter genes, together with MDA5 expression plasmid. At 48 hr post-transaction, the luciferase activity was measured and normalized to renilla luciferase activity. (E) Knockdown of MDA5 attenuates the expression of antiviral genes. MPC cells were transfected with si-Ctrl or si-MDA5 for 24 hr and then treated with SCRV (MOI = 5) or poly(I:C)-HMW for 24 hr. The expression of IFN-1, Mx1, ISG15, and Viperin were determined by qPCR. (F and G) Overexpression of MDA5 promotes the expression of antiviral genes. MKC cells were transfected with vector or MDA5 expression plasmids for 24 hr and then treated with SCRV (MOI = 5) or poly (I:C)-HMW for 24 hr. The expression of MDA5 (F), and antiviral genes, including IFN-1, Mx1, ISG15, and Viperin (G) were determined by qPCR. (H and J) MPC cells transfected with si-Ctrl or si-MDA5 and MKC cells transfected with pcDNA3.1 vector or MDA5 plasmid 24 hr and then treated with SCRV at the dose indicated for 48 hr. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. (I and K) MDA5 inhibits SCRV replication. MPC cells were transfected with si-Ctrl or si-MDA5 and MKC cells were transfected with pcDNA3.1 vector or MDA5 expression plasmid for 24 hr, then infected with SCRV (MOI = 5) for 12, 24, or 48 hr. The qPCR analysis was conducted for SCRV-M and SCRV-G RNA levels. (L and M) MDA5 immunoprecipitates with MAVS (L) and STING (M). EPC cells (~1 × 107) were co- transfected with MAVS-Myc or STING-Myc and MDA5-Flag expression plasmids for 24 hr, followed by immunoprecipitation (IP) with anti-Myc. (N and O) MKC cells were transfected with MDA5 plasmids and STING plasmids (N) or si-STING (O) for 24 hr and then treated with SCRV (MOI = 5) for 24 hr. The expression of IFN-1 was determined by qPCR. (P and Q) MKC cells were transfected with si-MDA5 for 24 hr and then transfected with SCRV-RNA

Article Snippet: The antibody against MDA5 was diluted at 1: 500 (Beyotime, Cat# AF7164); The antibody against IRF3 was diluted at 1: 500 (Boster, Cat# BA4351- 2); anti- Flag, anti- Myc, anti- HA and anti- Tubulin monoclonal antibody were diluted at 1: 1,000 (Beyotime); and HRP- conjugated anti- rabbit IgG or anti- mouse IgG (Abbkine) at 1: 5,000.

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Over Expression, Immunofluorescence, Knockdown, Luciferase, Plasmid Preparation, Expressing, Activity Assay, Staining, Infection, Immunoprecipitation

Journal: eLife

Article Title: An arms race between 5’ppp-RNA virus and its alternative recognition receptor MDA5 in RIG-I-lost teleost fish

doi: 10.7554/elife.94898

Figure Lengend Snippet: Figure 3. RD domain is required for MDA5 to recognize SCRV RNA. (A) The association of MDA5 proteins with SCRV. (B) Schematics and expression of MDA5 and truncated MDA5 (MDA5-△RD). (C) The association of MDA5 and MDA5-△RD proteins with SCRV. (D) MKC cells transfected with pcDNA3.1 vector, MDA5, or MDA5-△RD plasmids for 24 hr and then treated with SCRV at the dose indicated for 48 hr. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. (E) MKC cells were transfected with pcDNA3.1 vector, MDA5 or MDA5-△RD expression plasmids for 24 hr, then infected with SCRV for 24 hr, then the qPCR analysis was conducted for SCRV-M and SCRV-G RNA levels. (F–I) Expression of IFN-1 (F), Mx1 (G), ISG15 (H), and Viperin (I) in Mock-infected, SCRV-infected, and poly(I:C)-HMW-stimulated MKC cells transfected with pcDNA3.1 vector, MDA5, or MDA5-△RD expression plasmids. All data presented as the means ± SE from three independent triplicated experiments. **, p<0.01, as determined by Student’s t test.

Article Snippet: The antibody against MDA5 was diluted at 1: 500 (Beyotime, Cat# AF7164); The antibody against IRF3 was diluted at 1: 500 (Boster, Cat# BA4351- 2); anti- Flag, anti- Myc, anti- HA and anti- Tubulin monoclonal antibody were diluted at 1: 1,000 (Beyotime); and HRP- conjugated anti- rabbit IgG or anti- mouse IgG (Abbkine) at 1: 5,000.

Techniques: Expressing, Transfection, Plasmid Preparation, Staining, Infection

Journal: eLife

Article Title: An arms race between 5’ppp-RNA virus and its alternative recognition receptor MDA5 in RIG-I-lost teleost fish

doi: 10.7554/elife.94898

Figure Lengend Snippet: Figure 4. MDA5 recognizes 5’ppp-RNA in M. miiuy and G. gallus. (A) Schematic representation of 5’ppp-SCRV and dsRNA. The product of in vitro transcription runs as a single product degraded by RNase I. (B) Pull-down of biotinylated or non-biotinylated dsRNA and 5’ppp-SCRV by mmiMDA5. MKC cells were transfected with mmiMDA5-Flag plasmid, and the input and immunoprecipitated MDA5 proteins were analyzed by Western blot. (C) MKC cells were transfected with 5’ppp-SCRV, 5’OH-SCRV (5’ppp-SCRV dephosphorylated by CIAP), or 5’pppGG-SCRV (5’ppp-SCRV capped by m7G cap analog), then IRF3 dimerization was analyzed by native gel electrophoresis. (D) WT and si-MDA5 MKC cells were transfected with 5’ppp- SCRV and poly(I:C)-HMW, then IRF3 dimerization was analyzed by native gel electrophoresis. (E) The cytoplasmic fraction of MKC cells transfected with Flag-tagged mmiMDA5 was incubated with biotinylated 5’ppp-SCRV, 5’OH-SCRV, or 5’pppGG-SCRV. RNA-protein complexes were pulled down using streptavidin affinity beads. Input and pull-down samples were analyzed by SDS-PAGE and immunoblotting using anti-Flag antibody. (F) Purity of recombinant mmiMDA5 and mmiMDA5-△RD were determined by SDS-PAGE and immunoblotting using anti-His antibody. (G) EMSA of 5’ppp-SCRV with mmiMDA5. For binding competition, indicated non-biotinylated RNAs (50-fold molar excess over the probe) were included. The asterisk marks the binding bands between mmiMDA5 protein and different RNA probes. (H) EMSA of 5’ppp-SCRV with mmiMDA5 or mmiMDA5-△RD. The asterisk marks the binding bands between mmiMDA5 or mmiMDA5-△RD proteins and 5’ppp-SCRV RNA probes. (I) Schematic representation of 5’ppp-VSV. The product of in vitro transcription runs as a single product degraded by RNase I. (J) Pull-down of dsRNA and 5’ppp-VSV by ggaMDA5. DF-1 cells were transfected with ggaMDA5-Flag plasmids, and the input and immunoprecipitated MDA5 proteins were analyzed by Western blot. (K) DF-1 cells were transfected with 5’ppp-VSV, 5’OH-VSV (5’ppp-VSV dephosphorylated by CIAP), or 5’pppGG-VSV (5’ppp-VSV capped by m7G cap analog), then IRF3 dimerization was analyzed by native gel electrophoresis. (L) The cytoplasmic fraction of DF-1 cells transfected with Flag-tagged ggaMDA5 was incubated with biotinylated 5’ppp-VSA, 5’OH-VSV, or 5’pppGG-RNA. RNA-protein complexes were pulled down using streptavidin affinity beads. Input and pull-down samples were analyzed by SDS-PAGE and immunoblotting using anti-Flag antibody. (M) EMSA of 5’ppp-RNA with ggaMDA5. For binding competition, indicated non-biotinylated RNAs (50-fold molar excess over the probe) were included. The asterisk marks the binding bands between ggaMDA5 protein and different RNA probes.

Article Snippet: The antibody against MDA5 was diluted at 1: 500 (Beyotime, Cat# AF7164); The antibody against IRF3 was diluted at 1: 500 (Boster, Cat# BA4351- 2); anti- Flag, anti- Myc, anti- HA and anti- Tubulin monoclonal antibody were diluted at 1: 1,000 (Beyotime); and HRP- conjugated anti- rabbit IgG or anti- mouse IgG (Abbkine) at 1: 5,000.

Techniques: In Vitro, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Nucleic Acid Electrophoresis, Incubation, SDS Page, Recombinant, Binding Assay

Journal: eLife

Article Title: An arms race between 5’ppp-RNA virus and its alternative recognition receptor MDA5 in RIG-I-lost teleost fish

doi: 10.7554/elife.94898

Figure Lengend Snippet: Figure 5. Increased m6A modification and expression of MDA5 upon SCRV infection. (A) Schematic of the MeRIP-seq protocol used to identify differential m6A methylation following infection of M. miiuy spleen tissues with SCRV. (B) Circos plots showing differentially m6A-methylated peaks identified from normal and SCRV-infected spleen tissues of M. miiuy. The green and red lines on the innermost ring represented the m6A peaks identified from normal and SCRV-infected spleen tissues of M. miiuy, respectively. The orange lines represent the log2 fold change values for each differentially m6A peak. The blue spots represent the fold change of gene expression. Chromosomes were shown on the outermost ring, the ruler on which represented the physical distance is in millions of bases (Mb). (C) The m6A abundance in MDA5 mRNA transcripts detected by MeRIP-seq, the m6A peak of MDA5 is circled in the green box. (D) The exon1 sequence of MDA5 was submitted to the SRAMP website, and then the predicted m6A site was displayed. Four predicted methylation sites located on the exon1 of MDA5 were marked by red box, then we designed two m6A specific primers (MDA5-m6A-1 and MDA5-m6A-2). (E) m6A abundance on MDA5 exon1 detected by MeRIP-qPCR and emiquantitative PCR in MKC cells. (F) MeRIP-qPCR analysis of relative m6A level of MDA5 in Mock, SCRV-infected, and poly(I:C)-HMW-stimulated MKC cells. (G) Relative luciferase activity of pmirGLO- MDA5-exon1 firefly luciferase reporters in Mock and SCRV-infected (MOI = 5) MKC cells that treated with DMSO or Cycloleucine (CL, 20 mM). (H) mRNA levels of MDA5 in spleen and kidney tissues measured by qRT-PCR at indicated time after SCRV infection (MOI = 5). (I and J) mRNA and protein levels of MDA5 in MKC cells measured by qRT-PCR and Western blotting at indicated time after SCRV infection (MOI = 5) (I) and poly(I:C)-HMW stimulation (J). All data presented as the means ± SE from three independent triplicated experiments. **, p<0.01, as determined by Student’s t test (E-G) and one- way ANOVA with Dunnett’s multiple comparisons test (H-J).

Article Snippet: The antibody against MDA5 was diluted at 1: 500 (Beyotime, Cat# AF7164); The antibody against IRF3 was diluted at 1: 500 (Boster, Cat# BA4351- 2); anti- Flag, anti- Myc, anti- HA and anti- Tubulin monoclonal antibody were diluted at 1: 1,000 (Beyotime); and HRP- conjugated anti- rabbit IgG or anti- mouse IgG (Abbkine) at 1: 5,000.

Techniques: Modification, Expressing, Infection, Methylation, Gene Expression, Sequencing, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot

Journal: eLife

Article Title: An arms race between 5’ppp-RNA virus and its alternative recognition receptor MDA5 in RIG-I-lost teleost fish

doi: 10.7554/elife.94898

Figure Lengend Snippet: Figure 6. m6A-modification weakens MDA5 mRNA stability and antiviral ability. (A) Silence efficiency of si-METTL3 and si-METTL14 measured. Three siRNAs of METTL3 and METTL14 were transfected into MPC cells for 48 hr, respectively. (B) METTL3 immunoprecipitates with METTL14. EPC cells (1×107) were co-transfected with METTL3-Myc and METTL14-Flag expression plasmids for 24 hr, followed by immunoprecipitation (IP) with anti- Myc. (C) METTL3 and METTL14 significantly increased the m6A content. MPCs were transfected with si-Ctrl or si-METTL3 or si-METTL14 and MKCs were transfected with vector or METTL3 or METTL14 plasmids for 48 hr, then the m6A level was measured by colorimetry. (D) METTL3 and METTL14 overexpressed MKC cells seeded in 48-well plates overnight were treated with SCRV at the dose indicated for 48 hr. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. (E) MKC cells were transfected with pcDNA3.1 vector and METTL3 or METTL14 expression plasmid for 24 hr, then infected with SCRV (MOI = 5) for 24 hr. The qPCR analysis was conducted for SCRV-M and SCRV-G RNA levels. (F) The m6A level alteration of MDA5 upon METTL3 or METTL14 knockdown or overexpression was examined by MeRIP-qPCR. MPC cells were transfected with si-Ctrl or si-METT3 and MKC cells were transfected with vector or METTL3 or METTL14 plasmids for 48 hr. (G and H) MPC cells were transfected with si-Ctrl, si-METTL3 or METTL14 and MKC cells were transfected with vector, METTL3, or METTL14 plasmids for 48 hr, the expression of MDA5 was detected by qRT-PCR and western blotting. (I) MPC cells were transfected with si-Ctrl, si-METTL3, or si-METTL14, and MKC cells were transfected with vector, METTL3, or METTL14 plasmids, then 5 µg/ml actinomycin D was added to the cells for 0 hr, 2 hr, and 4 hr. The half-life of MDA5 was analyzed by qRT-PCR. (J) MKC cells were transfected with vector or METTL3&14 plasmids for 24 hr and then treated with Cycloleucine (CL) for 24 hr in a final concentration of 20 mM. The expression of MDA5 was detected by qRT-PCR and western blotting. (K) MKC cells were transfected with vector or METTL3&14 plasmids for 24 hr and then treated with CL for 24 hr at 20 mM, then 5 µg/ml actinomycin D was added to the cells for 0 hr, 2 hr, and 4 hr. The half-life of MDA5 was analyzed by qRT-PCR. (L) MKC cells were transfected with vector or METTL3&14 plasmids for 24 hr and stimulated with SCRV (MOI = 5) for 24 hr, then the expression of MDA5 was detected by qRT-PCR and western blotting. (M) MKC cells seeded in 48-well plates overnight were transfected with MDA5 or MDA5 + METTL3&14 plasmids for 48 hr, then the expression of MDA5 was detected by qRT-PCR. (N) MKC cells seeded in 48-well plates overnight were transfected with MDA5 or MDA5 + METTL3&14 plasmids were treated with SCRV at the dose indicated for 48 hr. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. (O) MKC cells were transfected with MDA5 or MDA5 +

Article Snippet: The antibody against MDA5 was diluted at 1: 500 (Beyotime, Cat# AF7164); The antibody against IRF3 was diluted at 1: 500 (Boster, Cat# BA4351- 2); anti- Flag, anti- Myc, anti- HA and anti- Tubulin monoclonal antibody were diluted at 1: 1,000 (Beyotime); and HRP- conjugated anti- rabbit IgG or anti- mouse IgG (Abbkine) at 1: 5,000.

Techniques: Modification, Transfection, Expressing, Immunoprecipitation, Plasmid Preparation, Colorimetric Assay, Staining, Infection, Knockdown, Over Expression, Quantitative RT-PCR, Western Blot, Concentration Assay

Journal: eLife

Article Title: An arms race between 5’ppp-RNA virus and its alternative recognition receptor MDA5 in RIG-I-lost teleost fish

doi: 10.7554/elife.94898

Figure Lengend Snippet: Figure 7. Detailed m6A regulatory mechanism of MDA5. (A) The binding relationship between YTHDF1, YTHDF2, or YTHDF3 and MDA5 mRNA was validated using a RIP assay. MKC cells were transfected with YTHDF1-Flag, YTHDF2-Flag, YTHDF3-Flag, or pcDNA3.1-Flag for 48 hr. (B) SiRNA silencing effect test of YTHDF1, YTHDF2, and YTHDF3. MPC cells were transfected with si-YTHDF1, si-YTHDF2, or si-YTHDF3 for 48 h. (C and D) MPC cells were transfected with si-YTHDF1, si-YTHDF2, si-YTHDF3 or si-Ctrl (C), and MKC cells were transfected with YTHDF1, YTHDF2, YTHDF3 or vector for 48 hr

Article Snippet: The antibody against MDA5 was diluted at 1: 500 (Beyotime, Cat# AF7164); The antibody against IRF3 was diluted at 1: 500 (Boster, Cat# BA4351- 2); anti- Flag, anti- Myc, anti- HA and anti- Tubulin monoclonal antibody were diluted at 1: 1,000 (Beyotime); and HRP- conjugated anti- rabbit IgG or anti- mouse IgG (Abbkine) at 1: 5,000.

Techniques: Binding Assay, Transfection, Plasmid Preparation

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

Article Title: Loss of RIG-I leads to a functional replacement with MDA5 in the Chinese tree shrew

doi: 10.1073/pnas.1604939113

Figure Lengend Snippet: The function of tMDA5 and tLGP2 in response to RNA virus infections in TSPRCs. (A) Up-regulation of the tMDA5 and tLGP2 mRNA expression levels after challenging with different viruses. TSPRCs (1 × 105/well) were grown in a 12-well plate overnight and were harvested after infection with different viruses at the indicated times for total RNA isolation. The tMDA5 and tLGP2 mRNA expression levels (relative to β-actin) were quantified using qRT-PCR. Statistical difference was performed between the uninfected (0 h) and infected cells at the indicated time points. (B) Overexpression and knockdown of tMDA5 and tLGP2 in TSPRCs. The TSPRCs (1 × 105/well) were grown in a 12-well plate overnight and were transfected with Flag-tagged tMDA5 expression vector, Flag-tagged tLGP2 expression vector or siRNA (sitMDA5, sitLGP2, and sitMDA5/sitLGP2; 50 nM each), and were harvested at 36 h after transfection. Cell lysates were analyzed by immunoblot using the anti-Flag antibody. The endogenous tMDA5 and tLGP2 mRNA and protein expression levels were analyzed by using qRT-PCR and immunoblot (using anti-MDA5 antibody and anti-LGP2 antibody), respectively. (C) Overexpression of tMDA5 or tLGP2 inhibited VSV-GFP replication. The TSPRCs (1 × 105/well) were grown in a 12-well plate overnight and were transfected with the empty vector (pCMV-3Tag-8 vector/Vector; 1 μg) or indicated expression vector (hRIG-I, tMDA5, tLGP2; each 1 μg) for 12 h, followed by infection with VSV-GFP (MOI = 0.01) for 12 h. Cells were photographed under an Olympus microscopy (original magnification, ×10). (D) Up-regulation of the tIFNB1 mRNA expression in response to EMCV (MOI = 1) infection at the indicated times. (E) Effects of overexpression of tMDA5, tMDA5 K188Q, and tMDA5 A402K on the IFN-β-Luc, NF-κB-Luc, and ISRE-Luc reporters upon EMCV infection. The TSPRCs (1 × 104/well) were grown in a 24-well plate overnight and were transfected with the indicated expression vector or empty vector (400 ng), together with the respective luciferase vector (100 ng), TK (10 ng, as an inner control) for 36 h, followed by infection with EMCV (MOI = 1) for 12 h before the harvest for luciferase assay. Statistical difference was performed between cells transfected with vector and tMDA5 or between cells transfected with vector and tMDA5 mutant, unless otherwise marked. (F) Knockdown of tLGP2 (sitLGP2), tMDA5 (sitMDA5), and tLGP2/tMDA5 (sitMDA5/sitLGP2) caused an inhibition of EMCV-induced up-regulation of the tIFNB1 mRNA expression. Statistical difference was performed between the scramble cells and cells transfected with siRNA. Bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student t test. All experiments were repeated for three times, with similar results.

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-Flag (Abmart; M20008), mouse anti-c-Myc (9E11) (Life Technologies; MA1-16637), mouse monoclonal anti-GFP (EnoGene; E12-152), rabbit monoclonal anti-IRF3 (Cell Signal Technology; 4302), rabbit monoclonal anti-phospho-IRF3 (Ser396) (Cell Signal Technology; 4947), rabbit polyclonal anti-MDA5 (Merck Millipore; ABF210), rabbit polyclonal anti-LGP2 (IBL; 29030), rabbit polyclonal anti-MITA (LifeSpan BioSciences; LS-B7237), and mouse monoclonal anti β-actin (EnoGene; E1C602-2).

Techniques: Expressing, Infection, Isolation, Quantitative RT-PCR, Over Expression, Transfection, Plasmid Preparation, Western Blot, Microscopy, Luciferase, Mutagenesis, Inhibition

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

Article Title: Loss of RIG-I leads to a functional replacement with MDA5 in the Chinese tree shrew

doi: 10.1073/pnas.1604939113

Figure Lengend Snippet: RNA immunoprecipitation assay showed that the tMDA5- or tMDA5/tLGP2-associated RNAs are SeV RNAs. (A) Sequencing electrophoregrams showing the MDA5- or tMDA5/tLGP2-associated RNA as the SeV RNA. The tMDA5 or tMDA5/tLGP2 immunoprecipitates were extracted for RNA and were reversely transcribed to cDNA, followed by PCR amplification and sequencing. The sequencing results matched to the SeV genome fragment (GenBank accession no. M30202.1). Input Sev RNA: viral RNA isolated from whole cell lysate infected with SeV. IP SeV RNA: viral RNA isolated from the immunoprecipitated protein-RNA complexes. (B) Quantification of the amount of EMCV RNA in hRIG-I, tMDA5, and IgG immunoprecipitates by using the strand-specific RT-PCR. (Top) Immunoblot showing Flag-tagged hRIG-I and tMDA5 in IP. (Bottom) The EMCV RNA levels were calculated by comparison with a dilution curve of cDNA prepared from the EMCV-infected cells. (C) Up-regulation of the tIFNB1 mRNA expression by the tMDA5-associated EMCV RNAs was dependent on tMDA5. TSPRCs (1 × 105) were first transfected with sitMDA5 (50 nM) and siRNA control (Scramble, 50 nM) for 24 h and then transfected with the indicated amount EMCV RNAs for 9 h before the harvest. The relative tIFNB1 mRNA levels were measured by using qRT-PCR. (D) Effects of the overexpression of the tLGP2 and tMDA5 on the induction of the IFN-β-Luc reporter in TSPRCs. Cells (1 × 104 cells per well) were grown in 24-well plate overnight and were transfected with the IFN-β-Luc reporter vector (100 ng) and TK (10 ng, as an inner control). The tMDA5 expression vector was transfected at a constant quantity of 50 ng plasmid per well, whereas the amount of tLGP2 was titrated. After 24 h, cells were transfected with low-molecular-weight poly I:C (1 μg/mL) or without low-molecular-weight poly I:C (Control) for 12 h before the harvest for luciferase assay. (E) Both high-molecular-weight poly I:C (poly I:C H) and low molecular weight poly I:C (poly I:C L) up-regulated the tIFNB1 mRNA levels in TSPRCs. The TSPRCs (1 × 105) were transfected with poly I:C H (100 ng/mL) or poly I:C L (100 ng/mL) or without transfection (NC) and were harvested at the indicated time points after transfection. (F) Knockdown of tLGP2 (sitLGP2) or tMDA5 (sitMDA5) caused an inhibition of poly I:C-induced tIFNB1 mRNA expression. The TSPRCs (1 × 105/well) were grown in 12-well plate overnight and were transfected with the siRNA negative control (Scramble, 50 nM) or indicated siRNA (50 nM) for 36 h, followed by transfection with poly I:C H or poly I:C L (100 ng/mL) for 12 h, respectively, before the harvest. The relative tIFNB1 mRNA levels were measured using qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001, relative to the NC or Scramble, Student t test. Bars represent mean ± SEM. All experiments were repeated for three times with similar results.

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-Flag (Abmart; M20008), mouse anti-c-Myc (9E11) (Life Technologies; MA1-16637), mouse monoclonal anti-GFP (EnoGene; E12-152), rabbit monoclonal anti-IRF3 (Cell Signal Technology; 4302), rabbit monoclonal anti-phospho-IRF3 (Ser396) (Cell Signal Technology; 4947), rabbit polyclonal anti-MDA5 (Merck Millipore; ABF210), rabbit polyclonal anti-LGP2 (IBL; 29030), rabbit polyclonal anti-MITA (LifeSpan BioSciences; LS-B7237), and mouse monoclonal anti β-actin (EnoGene; E1C602-2).

Techniques: Immunoprecipitation, Sequencing, Amplification, Isolation, Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Quantitative RT-PCR, Over Expression, Plasmid Preparation, Molecular Weight, Luciferase, Inhibition, Negative Control

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

Article Title: Loss of RIG-I leads to a functional replacement with MDA5 in the Chinese tree shrew

doi: 10.1073/pnas.1604939113

Figure Lengend Snippet: Analysis of positive selection for the Chinese tree shrew MDA5 and LGP2 genes

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-Flag (Abmart; M20008), mouse anti-c-Myc (9E11) (Life Technologies; MA1-16637), mouse monoclonal anti-GFP (EnoGene; E12-152), rabbit monoclonal anti-IRF3 (Cell Signal Technology; 4302), rabbit monoclonal anti-phospho-IRF3 (Ser396) (Cell Signal Technology; 4947), rabbit polyclonal anti-MDA5 (Merck Millipore; ABF210), rabbit polyclonal anti-LGP2 (IBL; 29030), rabbit polyclonal anti-MITA (LifeSpan BioSciences; LS-B7237), and mouse monoclonal anti β-actin (EnoGene; E1C602-2).

Techniques: Selection

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

Article Title: Loss of RIG-I leads to a functional replacement with MDA5 in the Chinese tree shrew

doi: 10.1073/pnas.1604939113

Figure Lengend Snippet: Evolutionary conservation of the positively selected sites in the tree shrew MDA5 and its impact on the predicted protein structure. (A) Sequence alignment of the second CARD domain of the MDA5 and DECH Box Helicase domain in six mammalian species [human, rat, mouse, dog, Chinese tree shrew, macaque; sequence data were retrieved from Ensemble (asia.ensembl.org/index.html)]. PSSs are marked in red. (B) Sequence alignment of the second CARD domain of the MDA5 and the DECH Box Helicase domain in eight primates (white-tufted-ear marmoset, Sumatran orangutan, gorilla, human, chimpanzee, northern white-cheeked gibbon, macaque, olive baboon) and the Chinese tree shrew. (C) Diagram illustrating domain structure of the tree shrew MDA5. The tMDA5 protein is composed of three critical domains: (1) tandem CARDs at its N-terminal, which are essential for interacting with MAVS to mediate the downstream signaling; (2) a central DECH-box helicase domain that encompasses conserved helicase subdomains, Hel1 (surrounding helicase motifs Q, I, II, and III), Hel2 (surrounding helicase motifs IV, V, and VI), and the helicase insert domain Hel2i; and (3) a C-terminal domain (CTD) for auto-regulation and RNA terminus recognition. (D) Structure of human MDA5 binding to dsRNA. The positively selected site (Ala-402; shown in red) in tree shrew was equivalent to Lys-405 in hMDA5.

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-Flag (Abmart; M20008), mouse anti-c-Myc (9E11) (Life Technologies; MA1-16637), mouse monoclonal anti-GFP (EnoGene; E12-152), rabbit monoclonal anti-IRF3 (Cell Signal Technology; 4302), rabbit monoclonal anti-phospho-IRF3 (Ser396) (Cell Signal Technology; 4947), rabbit polyclonal anti-MDA5 (Merck Millipore; ABF210), rabbit polyclonal anti-LGP2 (IBL; 29030), rabbit polyclonal anti-MITA (LifeSpan BioSciences; LS-B7237), and mouse monoclonal anti β-actin (EnoGene; E1C602-2).

Techniques: Sequencing, Northern Blot, Binding Assay

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

Article Title: Loss of RIG-I leads to a functional replacement with MDA5 in the Chinese tree shrew

doi: 10.1073/pnas.1604939113

Figure Lengend Snippet: Antiviral activities of MDA5 and its mutants in TSPRCs and human cells. (A and B) Overexpression of tMDA5 and its mutants, tMDA5 K188Q and tMDA5 A402K. TSPRCs (1 × 104 cells per well) were grown in a 24-well plate overnight and were transfected with different amounts of expression vector (section A: 3.2, 16, 80, and 400 ng/well; section B: 400 ng/well) or empty vector (400 ng/well) for 36 h. Cell lysates were analyzed by immunoblot using the anti-Flag antibody. Sections A and B refer to the conditions described in Fig. 5 A and B, respectively. (C) Overexpression of tMDA5 and its mutants had different abilities to inhibit vesicular stomatitis virus (VSV) replication. TSPRCs (1 × 105 cells per well) were grown in a 12-well plate overnight and were transfected with the empty vector (pCMV-3Tag-8 vector, 1 μg) or indicated expression vector (1 μg) for 12 h, followed by infection with VSV-GFP (MOI = 0.01) for 12 h. Cells were photographed under an Olympus microscopy (original magnification, ×10). Data are representative of three independent experiments. (D) Overexpression of Flag-tagged hMDA5, hMDA5 Q187K, and hMDA5 K405A in the HEK293 cells. Cells (1 × 105 cells per well) were grown in 12-well plate overnight and were transfected for 48 h before the harvest. Cell lysates were analyzed by immunoblot using the anti-FLAG antibody. (E and F) Overexpression of hMDA5, hMDA5 Q187K, and hMDA5 K405A on the IFN-β-Luc, NF-κB-Luc, and ISRE-Luc reporter activities upon infection with different RNA viruses in HEK293 cells (E) and Hep G2 cells (F). Cells (1 × 104 cells per well) were grown in a 24-well plate overnight and were transfected with indicated expression vector and luciferase reporter for 24 h, followed by infection with SeV (20 HAU/mL), NDV (MOI = 10) or EMCV (MOI = 1) for 12 h before the harvest for luciferase assay. (G–I) tMDA5 K188Q RNA-IP assay. TSPRCs (1 × 108 cells) were grown in a 150-mm culture dish overnight and were transfected with Flag-tagged mutant tMDA5 K188Q overexpression vector (30 μg) for 24 h, and then cells were infected with SeV (20 HAU/mL) for 16 h. The subsequent procedure was similar to that of Fig. 3 D–F. Precipitation was verified by immunoblot with the anti-Flag antibody (G). RNAs from the SeV-infected TSPRCs overexpressing the Flag-tagged tMDA5 K188Q (Input), RNAs associated with tMDA5 K188Q or IgG (control) immunoprecipitates (IP), or RNAs remaining after tMDA5 K188Q or IgG immunoprecipitations (unbound) were tested for the abilities to stimulate the IFN-β-Luc in HEK293 cells (H) or to induce tIFNB1 mRNA (normalized to the β-actin) in TSPRCs (I). Same assays were performed for mutant tMDA5 K402A (J–L). *P < 0.05, **P < 0.01, ***P < 0.001, Student t test. Bars represent mean ± SEM. All experiments were repeated for three times, with similar results.

Article Snippet: The following antibodies were used in this study: mouse monoclonal anti-Flag (Abmart; M20008), mouse anti-c-Myc (9E11) (Life Technologies; MA1-16637), mouse monoclonal anti-GFP (EnoGene; E12-152), rabbit monoclonal anti-IRF3 (Cell Signal Technology; 4302), rabbit monoclonal anti-phospho-IRF3 (Ser396) (Cell Signal Technology; 4947), rabbit polyclonal anti-MDA5 (Merck Millipore; ABF210), rabbit polyclonal anti-LGP2 (IBL; 29030), rabbit polyclonal anti-MITA (LifeSpan BioSciences; LS-B7237), and mouse monoclonal anti β-actin (EnoGene; E1C602-2).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Western Blot, Infection, Microscopy, Luciferase, Mutagenesis