Journal: Journal of Virology

Article Title: Mumps Virus SH Protein Inhibits NF-κB Activation by Interacting with Tumor Necrosis Factor Receptor 1, Interleukin-1 Receptor 1, and Toll-Like Receptor 3 Complexes

doi: 10.1128/JVI.01037-17

Figure Lengend Snippet: Generation of rMuV with mutated SH genes. (A) Scheme of the MuV genome and the modified SH fragments of the generated rMuVs. For the SH-deficient viruses, the positions of introduced stop codons starting at the ATG of SH (rMuV-SHstop-C) and the FLAG epitope sequence (rMuV-SHstop) are stated. (B) BSR-T7 cells were transfected with pG-MuV-FL_SH-N-flag, pG-MuV-FL_SH-3stop-N-flag, pG-MuV-FL_SH-C-flag, or pG-MuV-FL_SH-3stop-C-flag plus pMuV-L, pMuV-N, and pMuV-P. As a negative control (mock), cells were transfected with pMuV-EGFP. Pictures of syncytia were taken at 7 days posttransfection. (C) Vero76 cells were infected with rMuV-SH, rMuV-SHstop, rMuV-SH-C, or rMuV-SHstop-C at an MOI of 0.01 or left untreated (mock). At 2 days p.i., cells were fixed and stained using an MuV F-specific Cy3-coupled antibody and DAPI. Images were generated using a Zeiss cLSM780 confocal laser scanning microscope. (D) At 3 days p.i., rMuV-infected Vero76 cells (MOI, 0.01) were lysed and subjected to Western blot analysis. MuV N and GAPDH were detected by specific monoclonal antibodies, and MuV SH was detected by an anti-FLAG antibody.

Article Snippet: 293G (kindly provided by C. Uhlenhaut, Berlin, Germany), Vero76, and A549 cells (both from ATCC) were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 5% fetal bovine serum (FBS; PAN Laboratories GmbH), 2 mM l -glutamine (Lonza), 100 U/ml penicillin (Gibco), and 100 μg/ml streptomycin (Gibco).

Techniques: Modification, Generated, FLAG-tag, Sequencing, Transfection, Negative Control, Infection, Staining, Laser-Scanning Microscopy, Western Blot, Bioprocessing

Journal: Journal of Virology

Article Title: Mumps Virus SH Protein Inhibits NF-κB Activation by Interacting with Tumor Necrosis Factor Receptor 1, Interleukin-1 Receptor 1, and Toll-Like Receptor 3 Complexes

doi: 10.1128/JVI.01037-17

Figure Lengend Snippet: rMuV replication and SH protein expression. (A) Vero76 cells were infected with rMuV-SH, rMuV-SHstop, rMuV-SH-C, or rMuV-SHstop-C at an MOI of 0.01. Supernatants were collected at the indicated time points and titrated in triplicate on Vero76 cells. The detection limit due to assay procedure is 102 (dashed line). The graph depicts means ± standard deviations (SD) from one representative experiment for which three independent infections were performed at the same time. A549 (B and D) and Vero76 (C and E) cells were infected with rMuVs at an MOI of 5. Cells were harvested at the indicated time points and analyzed by flow cytometry. Total SH protein (B and C) was detected using an APC-conjugated FLAG-specific antibody. The values of rMuV-SHstop or rMuV-SHstop-C were subtracted from the values of rMuV-SH or rMuV-SH-C. Total N protein (D and E) was detected using an N-specific primary antibody and an Alexa Fluor 488-conjugated secondary antibody. The values of mock-infected cells were subtracted from the values of rMuV-infected cells. Graphs depict means ± SD from three individual experiments. *, P < 0.05; results were calculated by unpaired t tests with a two-tailed P value. (F) A549 cells were infected with rMuVs at an MOI of 5 and harvested at 20 h postinfection. Cells were treated with 0.5% saponin (permeabilized) for intra- and extracellular SH detection or not treated (nonpermeabilized) for extracellular SH detection by flow cytometry. Staining and calculation for total SH protein were carried out as described for panels B and C. The graph depicts means ± SD from three individual experiments. ***, P < 0.001; results were calculated by unpaired t tests with a two-tailed P value.

Article Snippet: 293G (kindly provided by C. Uhlenhaut, Berlin, Germany), Vero76, and A549 cells (both from ATCC) were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 5% fetal bovine serum (FBS; PAN Laboratories GmbH), 2 mM l -glutamine (Lonza), 100 U/ml penicillin (Gibco), and 100 μg/ml streptomycin (Gibco).

Techniques: Expressing, Infection, Flow Cytometry, Two Tailed Test, Staining

Journal: bioRxiv

Article Title: The Sequence Basis for Selectivity of Ephrin-B2 Ligand for Eph Receptors and Pathogenic Henipavirus G Glycoproteins

doi: 10.1101/2023.04.26.538420

Figure Lengend Snippet: (A) Magnified views of the G-H binding loop bound to Eph receptor (magenta) and NiV-G (green) are shown. The difference of the conservation scores for the NiV-G-High sorts from the two EFNB2 deep mutational scans are mapped onto the RBD of EFNB2 and colored as in . (B) Expi293F cells expressing full-length NiV-G fused at the C-terminus to sfGFP were incubated with increasing concentrations of sEFNB2-Fc protein. Binding of sEFNB2-Fc to membrane-displayed NiV-G-sfGFP was measured by flow cytometry. Data are mean ± SEM, N = 3 biological replicates. (C) Mixtures of rCedV-GFP chimeric viruses pre-incubated with increasing concentrations of sEFNB2-Fc protein were added to pre-seeded Vero 76 cells. Neutralizing activity of sEFNB2-Fc was measured by counting the reduction in GFP fluorescent foci. Data are mean ± SD, N = 2 independent experiments, each performed in technical triplicate. (D) Proposed mechanism by which the Q130L and V167L mutations affect the conformational selectivity of the G-H binding loop. In wild-type EFNB2, the G-H binding loop is dynamic and in equilibrium between conformations for binding both Eph receptors (magenta) and NiV-G (green). The side chain of EFNB2-Q130 is fully exposed to solvent in the ‘open’ loop structure bound to Eph receptors but is packed against neighboring side chains in the ‘closed’ loop structure bound by NiV-G. In EFNB2-D62Q-Q130L-V167L, the conformational equilibrium shifts to favor the ‘closed’ state, which minimizes interactions between solvent and the hydrophobic Q130L substitution. Additionally, a leucine substitution at EFNB2-V167 acts as a cavity-filling mutation and increases the hydrophobic packing around EFNB2-F129 in the ‘closed’ state.

Article Snippet: Vero 76 cells were seeded at a density of 2 × 10 4 cells/well in black walled clear bottom 96-well plates (Corning Life Sciences). sEFNB2-Fc proteins were serially diluted 3-fold for 7-point dose response curves.

Techniques: Binding Assay, Expressing, Incubation, Protein Binding, Membrane, Flow Cytometry, Activity Assay, Solvent, Mutagenesis