Journal: The Journal of Biological Chemistry

Article Title: Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity

doi: 10.1074/jbc.M117.812784

Figure Lengend Snippet: Characterization of MxA G domain variants. A, antiviral activity of the G domain variants in a FLUAV minireplicon system. 293T cells were co-transfected with expression plasmids for the MxA variants (300 ng) and the minireplicon system of VN/04, including a reporter construct encoding firefly luciferase under the control of the viral promoter. After 24 h, firefly luciferase activity was determined and normalized to the activity of constitutively co-expressed Renilla luciferase. Results are presented relative to the activity in the absence of MxA, the vector control (see “Experimental procedures” for calculation), and as arithmetic means ± S.D. of three independent experiments. Protein expression of FLAG-tagged MxA, viral NP, and actin was determined by Western blot analysis. B, restriction of FLUAV replication by G domain variants in tissue culture. A549 cells were transfected with MxA expression plasmids (500 ng) and 24 h later infected with SC35MNS1_2A_GFP-NEP (H7N7) at an MOI of 0.5. After fixation of the cells at 10 h post-infection, MxA was stained, and cells were analyzed by FACS. MxA-positive cells were selected, and the percentage of infected (GFP-positive) cells was determined. The percentage of GFP-positive cells expressing the inactive mutant T103A was set to 100%. Arithmetic means ± S.D. (error bars) of four independent experiments are shown. C, G domain dimer of GMPPCP-bound (gray) stalkless MxA (4P4S, G domain A in yellow (residues 70–340), G domain B in blue (residues 68–340)) (8). Positions of MxA G domain variations are highlighted in red. Amino acid residues of WT MxA are shown in stick representations. D, analytical gel-filtration analysis of the indicated mutants in the presence and absence (apo) of GDP-AlFx. E, nucleotide binding analysis of monomeric MxA G domain variants by ITC at 8 °C. GTPγS was titrated stepwise into the protein solution. The resulting heat changes were integrated, and the obtained values were fitted to a quadratic binding equation (one-site binding model). The following KD values were derived from the fittings: M527D (black), KD = 13 ± 5 μm, n = 0.83 ± 0.08; M527D/N220D (red), KD = 13 ± 2 μm, n = 0.50 ± 0.04; M527D/G255E (blue), KD = 5 ± 2 μm, n = 0.48 ± 0.18; M527D/V268M (green), KD = 17 ± 3 μm, n = 0.54 ± 0.04. The MxA constructs showed a varying degree of precipitation in these assays, which may explain the reduced binding numbers. F, protein concentration-dependent GTPase activities of monomeric M527D (□) and M527D/N220D (○) were determined at 37 °C by an HPLC-based assay. The mean kobs was calculated from two independent experiments for each concentration. The error bars show the range of the two data points. mAU, milliabsorbance units.

Article Snippet: The following primary antibodies were used: anti-FLAG (clone M2, mouse monoclonal, F3165, Sigma-Aldrich), anti-HA (rabbit polyclonal, H6908, Sigma-Aldrich), anti-FLUAV NP (clone AA5H, mouse monoclonal, MCA400, Bio-Rad), anti-THOV NP (rabbit polyclonal ( 5 )), anti-β-actin (rabbit polyclonal, ab8227, Abcam), and anti-MxA (mouse monoclonal, M143 ( 44 )).

Techniques: Activity Assay, Transfection, Expressing, Construct, Luciferase, Plasmid Preparation, Western Blot, Infection, Staining, Mutagenesis, Filtration, Binding Assay, Derivative Assay, Protein Concentration, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity

doi: 10.1074/jbc.M117.812784

Figure Lengend Snippet: MxA G-interface variants have no dominant-negative effect on WT MxA. A, co-immunoprecipitation of WT MxA with G domain variants. 293T cells were co-transfected with HA-tagged WT MxA and FLAG-tagged MxA mutants. At 24 h post-transfection, cell lysates were subjected to FLAG-specific immunoprecipitations (IP). Precipitates and whole-cell lysates (WCL) were analyzed by Western blotting. B, effect of MxA G domain variants on the antiviral activity of WT MxA in the FLUAV minireplicon system of VN/04, as described in the legend to Fig. 2A. HA-tagged WT MxA (300 ng) was co-transfected with the components of the minireplicon and increasing amounts (50, 100, and 200 ng) of the indicated FLAG-tagged MxA variants. Protein expression was monitored by Western blot analysis. Data are presented as described in the legend to Fig. 2A. C, GTPase activity of M527D can be stimulated by the monomeric G domain mutant N220D. M527D (2.5 μm) was incubated with increasing concentrations of M527D/N220D, and GTPase activity was measured as described in the legend to Fig. 2F. Vertical lines in the Western blots indicate cuts combining two blots of one experiment run in parallel. Error bars, S.D.

Article Snippet: The following primary antibodies were used: anti-FLAG (clone M2, mouse monoclonal, F3165, Sigma-Aldrich), anti-HA (rabbit polyclonal, H6908, Sigma-Aldrich), anti-FLUAV NP (clone AA5H, mouse monoclonal, MCA400, Bio-Rad), anti-THOV NP (rabbit polyclonal ( 5 )), anti-β-actin (rabbit polyclonal, ab8227, Abcam), and anti-MxA (mouse monoclonal, M143 ( 44 )).

Techniques: Dominant Negative Mutation, Immunoprecipitation, Transfection, Western Blot, Activity Assay, Expressing, Mutagenesis, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity

doi: 10.1074/jbc.M117.812784

Figure Lengend Snippet: Antiviral activity of MxA stalk variants. A, antiviral activity against FLUAV in the VN/04 minireplicon system, as described in the legend to Fig. 2A. The vector control and the antiviral activity of WT MxA and T103A have already been shown in Fig. 2A. B, antiviral activity against THOV in the SiAr126 minireplicon system. MxA variants (100 ng of expression plasmids) and the components of the minireplicon system, including a reporter construct encoding firefly luciferase under the control of the THOV promoter and Renilla luciferase to monitor transfection efficiency, were co-expressed in 293T cells. Firefly luciferase activity was measured in the cell lysates at 24 h post-transfection and normalized to the activity of the Renilla luciferase. Data are presented as described in the legend to Fig. 2A. C, restriction of FLUAV replication by stalk variants in A549 cells analyzed by FACS, as described in the legend to Fig. 2B. The antiviral activities of WT MxA and T103A have already been shown in Fig. 2B. D and E, antiviral activity against VSV. D, 293T cells were co-transfected with FLAG-tagged MxA variants (300 ng) and VSV-G (300 ng). At 24 h post-transfection, cells were infected with VSV*ΔG(Luc) at an MOI of 1. Another 24 h later, supernatants were collected, the cells were harvested, and firefly luciferase activity was measured (VLP infection). E, the supernatants containing newly produced VLPs were used to infect naive 293T cells, which were lysed 24 h later to determine firefly luciferase activity (VLP titration). The values are presented relative to the activity in the absence of MxA, the vector control (see “Experimental procedures” for calculation). Arithmetic means ± S.D. (error bars) of three biological replicates are shown. F, restriction of VSV replication by stalk variants in tissue culture. 24 h after transfection with MxA expression plasmids (500 ng), A549 cells were infected with VSV-GFP at an MOI of 0.5 for 6.5 h. Fixed cells stained with an MxA-specific antibody were analyzed by FACS. MxA-positive cells were selected, and the percentage of infected (GFP-positive) cells was determined. The percentage of GFP-positive cells expressing the inactive mutant T103A was set to 100%. Results are displayed as arithmetic means ± S.D. of three independent experiments. Protein expression of FLAG-tagged MxA, actin, FLUAV, and THOV NP was verified by Western blot analyses. Vertical lines in the Western blots indicate cuts combining two blots of one experiment run in parallel. The color code of the bars is explained under “Results.”

Article Snippet: The following primary antibodies were used: anti-FLAG (clone M2, mouse monoclonal, F3165, Sigma-Aldrich), anti-HA (rabbit polyclonal, H6908, Sigma-Aldrich), anti-FLUAV NP (clone AA5H, mouse monoclonal, MCA400, Bio-Rad), anti-THOV NP (rabbit polyclonal ( 5 )), anti-β-actin (rabbit polyclonal, ab8227, Abcam), and anti-MxA (mouse monoclonal, M143 ( 44 )).

Techniques: Activity Assay, Plasmid Preparation, Expressing, Construct, Luciferase, Transfection, Infection, Produced, Titration, Staining, Mutagenesis, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity

doi: 10.1074/jbc.M117.812784

Figure Lengend Snippet: Characterization of the MxA stalk variants V470G and E516del. A, antiviral activity against RVFV. MxA (100 ng) and RVFV-N and -L (50 ng each) were co-expressed in 293T cells. 24 h post-transfection, cells were infected with Rift Valley fever VLPs encoding firefly luciferase as a reporter. Luciferase activities were measured 24 h later and are presented relative to the activity in the absence of MxA, the vector control (see “Experimental procedures” for calculation). Arithmetic means ± S.D. (error bars) of four biological replicates are shown. Western blot analysis was performed to control protein expression of FLAG-tagged MxA and actin. Significance was calculated using one-way analysis of variance with Dunnett's post hoc test. ns, not significant; **, p ≤ 0.01; ****, p ≤ 0.0001. B, protein concentration–dependent GTPase activities of WT MxA (gray; same control as in Fig. 5A) and V470G (green), as in Fig. 5A. C, analytical gel filtration of WT MxA, M527D (gray and black; same controls as in Fig. 5B), and V470G (green) in the absence of nucleotide, as described in the legend to Fig. 2D. D, co-immunoprecipitation of HA-tagged WT MxA (500 ng) with the FLAG-tagged variants (IP) V470G and E516del (500 ng) as described in the legend to Fig. 5C. Precipitates and whole-cell lysates (WCL) were analyzed by Western blotting. E, nuclear co-translocation of the MxA stalk variants V470G and E516del with WT MxA. Artificial nuclear forms of WT and MxA variants carrying an HA tag and the NLS of the SV40 large T antigen (HA-NLS-MxA) were co-expressed with FLAG-tagged WT MxA or MxA variants in HeLa cells. At 24 h post-transfection, cells were fixed and stained with antibodies directed against the HA tag (red) and the FLAG tag (green). The right column displays the overlay of the two signals. F, effect of different amino acid substitutions at position 470 on the antiviral activity of MxA. The antiviral activity of the mutants against FLUAV was determined in the VN/04 minireplicon system, as described in the legend to Fig. 2A.

Article Snippet: The following primary antibodies were used: anti-FLAG (clone M2, mouse monoclonal, F3165, Sigma-Aldrich), anti-HA (rabbit polyclonal, H6908, Sigma-Aldrich), anti-FLUAV NP (clone AA5H, mouse monoclonal, MCA400, Bio-Rad), anti-THOV NP (rabbit polyclonal ( 5 )), anti-β-actin (rabbit polyclonal, ab8227, Abcam), and anti-MxA (mouse monoclonal, M143 ( 44 )).

Techniques: Activity Assay, Transfection, Infection, Luciferase, Plasmid Preparation, Western Blot, Expressing, Protein Concentration, Filtration, Immunoprecipitation, Translocation Assay, Staining, FLAG-tag

Journal: Journal of Virology

Article Title: Evolution and Antiviral Specificities of Interferon-Induced Mx Proteins of Bats against Ebola, Influenza, and Other RNA Viruses

doi: 10.1128/JVI.00361-17

Figure Lengend Snippet: Antiviral activity of bat Mx1 against orthomyxoviruses. 293T cells were cotransfected with plasmids encoding the components of different orthomyxoviral polymerase reconstitution systems, including the polymerase subunits PB1, PB2, and PA, the NP, and a viral minigenome coding for firefly luciferase reporter under the control of a viral promoter (pPol-I FF-Luc). Expression plasmids for the indicated Mx1 constructs and for Renilla luciferase under the control of a constitutive promoter (RLuc) were cotransfected. At 24 h posttransfection, cells were lysed and firefly and Renilla luciferase activities were determined. (A and B) FLUAV H5N1 (A/Vietnam/1203/04) minireplicon system using 10 ng of PB2, 10 ng of PB1, 10 ng of PA, 100 ng of NP, 50 ng of Pol-I FF-Luc, 10 ng of RLuc, and 300 ng of Mx1 expression plasmids or the corresponding inactive mutants with a K-to-A (A) or T-to-A (B) change. Mx1 protein and NP expression was monitored by Western blotting using Mx-specific M143 (A) or FLAG-specific (B) antibody. (C) To measure the bat Mx1 effect on viral growth, 293T cells (6-well format) were transfected with 800 ng of Mx1 expression plasmids 16 h prior to infection with SC35M (H7N7, Kan1 NP) with an MOI of 0.001. At 48 h after infection, the supernatants were harvested and titrated. Viral titer of the empty vector control was set to 100% (corresponding to 105 to 106 PFU/ml in four independent experiments). (D) THOV minireplicon system using 10 ng of PB2, 10 ng of PB1, 10 ng of PA, 25 ng of NP, 50 ng of Pol-I FF-Luc, 10 ng of RLuc, and 300 ng of Mx1 expression plasmids. At 24 h posttransfection, the cell lysates were analyzed for luciferase activity and for Mx1 protein and NP expression by Western blotting. Firefly luciferase activity was normalized to Renilla luciferase activity (FF/RL). The empty vector control was set to 100%. Significance was calculated with a one-sided Student t test (n = 3). ns, nonsignificant. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). The numbers above the columns indicate fold changes in viral polymerase activity (A, B, and D) or viral titer (C) in the presence of wild-type Mx1 compared to the inactive control.

Article Snippet: Western blot analyses were performed with specific antibodies directed against FLAG (Sigma-Aldrich) or Mx-specific M143 ( 35 ), FLUAV-NP (Serotec), THOV-NP ( 44 ), and β-actin (Sigma-Aldrich).

Techniques: Activity Assay, Luciferase, Expressing, Construct, Western Blot, Transfection, Infection, Plasmid Preparation

Journal: Journal of Virology

Article Title: Evolution and Antiviral Specificities of Interferon-Induced Mx Proteins of Bats against Ebola, Influenza, and Other RNA Viruses

doi: 10.1128/JVI.00361-17

Figure Lengend Snippet: Antiviral activity of bat Mx1 against orthomyxoviruses. 293T cells were cotransfected with plasmids encoding the components of different orthomyxoviral polymerase reconstitution systems, including the polymerase subunits PB1, PB2, and PA, the NP, and a viral minigenome coding for firefly luciferase reporter under the control of a viral promoter (pPol-I FF-Luc). Expression plasmids for the indicated Mx1 constructs and for Renilla luciferase under the control of a constitutive promoter (RLuc) were cotransfected. At 24 h posttransfection, cells were lysed and firefly and Renilla luciferase activities were determined. (A and B) FLUAV H5N1 (A/Vietnam/1203/04) minireplicon system using 10 ng of PB2, 10 ng of PB1, 10 ng of PA, 100 ng of NP, 50 ng of Pol-I FF-Luc, 10 ng of RLuc, and 300 ng of Mx1 expression plasmids or the corresponding inactive mutants with a K-to-A (A) or T-to-A (B) change. Mx1 protein and NP expression was monitored by Western blotting using Mx-specific M143 (A) or FLAG-specific (B) antibody. (C) To measure the bat Mx1 effect on viral growth, 293T cells (6-well format) were transfected with 800 ng of Mx1 expression plasmids 16 h prior to infection with SC35M (H7N7, Kan1 NP) with an MOI of 0.001. At 48 h after infection, the supernatants were harvested and titrated. Viral titer of the empty vector control was set to 100% (corresponding to 105 to 106 PFU/ml in four independent experiments). (D) THOV minireplicon system using 10 ng of PB2, 10 ng of PB1, 10 ng of PA, 25 ng of NP, 50 ng of Pol-I FF-Luc, 10 ng of RLuc, and 300 ng of Mx1 expression plasmids. At 24 h posttransfection, the cell lysates were analyzed for luciferase activity and for Mx1 protein and NP expression by Western blotting. Firefly luciferase activity was normalized to Renilla luciferase activity (FF/RL). The empty vector control was set to 100%. Significance was calculated with a one-sided Student t test (n = 3). ns, nonsignificant. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). The numbers above the columns indicate fold changes in viral polymerase activity (A, B, and D) or viral titer (C) in the presence of wild-type Mx1 compared to the inactive control.

Article Snippet: Western blot analyses of the VLP producer and indicator cell lysates were performed with antibodies directed against FLAG (Sigma-Aldrich), FLUAV NP (Thermo Fisher Scientific), and β-actin (Sigma-Aldrich).

Techniques: Activity Assay, Luciferase, Expressing, Construct, Western Blot, Transfection, Infection, Plasmid Preparation

Journal: Journal of Virology

Article Title: Evolution and Antiviral Specificities of Interferon-Induced Mx Proteins of Bats against Ebola, Influenza, and Other RNA Viruses

doi: 10.1128/JVI.00361-17

Figure Lengend Snippet: Influence of bat Mx1 on the polymerase activity of bat influenza (H17N10). (A) A FLUAV (H17N10) minireplicon system using 10 ng of PB2, 10 ng of PB1, 10 ng of PA, 10 ng of NP, and 50 ng of Pol-I FF-Luc was cotransfected with the helper plasmids encoding the additional structural proteins from H7N7 virus as well as 300 ng of Mx1 expression plasmids (producing cells). At 48 h posttransfection, supernatants were collected and the cell lysates were analyzed for firefly luciferase activity and for Mx1 protein and NP expression by Western blotting. (B) FLUAV (H17N10) VLP titration. Indicator cells (MDCK II) were coinfected with 50 μl of the VLP-producing cell supernatants and with the SC35M helper virus for 24 h. As a control, indicator cells were treated with a comparable amount of donor cell supernatant produced in the absence of the viral hemagglutinin (−HA). Firefly luciferase activity was measured in cell lysates, and infection of indicator cells with SC35M helper virus was monitored by Western blotting detecting NP. The empty vector control was set to 100%. Significance was calculated with a one-sided Student t test (n = 3). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). The numbers above the columns indicate fold changes in viral polymerase activity in the presence of wild-type Mx1 compared to the inactive control.

Article Snippet: Western blot analyses of the VLP producer and indicator cell lysates were performed with antibodies directed against FLAG (Sigma-Aldrich), FLUAV NP (Thermo Fisher Scientific), and β-actin (Sigma-Aldrich).

Techniques: Activity Assay, Expressing, Luciferase, Western Blot, Titration, Produced, Infection, Plasmid Preparation