A Systematic Analysis of Host Factors Reveals a Med23-Interferon-? Regulatory Axis against Herpes Simplex Virus Type 1 Replication

Griffiths, Samantha J.; Koegl, Manfred; Boutell, Chris; Zenner, Helen L.; Crump, Colin M.; Pica, Francesca; Gonzalez, Orland; Friedel, Caroline C.; Barry, Gerald; Martin, Kim; Craigon, Marie H.; Chen, Rui; Kaza, Lakshmi N.; Fossum, Even; Fazakerley, John K.; Efstathiou, Stacey; Volpi, Antonio; Zimmer, Ralf; Ghazal, Peter; Haas, Jürgen

Proteins were transiently expressed in HEK293 cells as hybrid proteins with the Staphylococcus aureus protein A tag or Renilla reniformis luciferase fused to their amino termini. 20 ng of each expression construct were transfected into 1610 4 HEK293 cells using 0.05 ml of lipofectamine 2000 (Invitrogen) in 96-well plates. After 40 h, medium was removed and cells were lysed on ice in 10 ml of ice-cold lysis buffer (20 mM Tris pH 7.5, 250 mM NaCl, 1% TritonX-100, 10 mM EDTA, 10 mM DTT, Protease Inhibitor Cocktail (Roche), Phosphatase Inhibitor Cocktail (Roche), Benzonase (Novagen) 25 units per ml final concentration) containing sheep-anti-rabbit IgGcoated magnetic beads (Invitrogen, Dynabeads M280, 2 mg/ml final concentration). Lysates were incubated on ice for 15 minutes. 100 ml of wash buffer (PBS, 1 mM DTT) were added per well, and 10% of the diluted lysate was removed to determine the luciferase activity present in each sample before washing. The remaining sample was washed 6 times in wash buffer in a Tecan Hydroflex plate washer. Luciferase activity was measured in the lysate as well as in washed beads. Negative controls were wells transfected with the plasmid expressing the luciferase fusion protein and a vector expressing two copies of protein A. For each sample, four values were measured: the luciferase present in 10% of the sample before washing (''input''), the luciferase activity present on the beads after washing (''bound''), and the same values for the negative controls (''input nc'', and ''bound nc''). Normalized interaction signals were calculated as follows: Log(bound)/log(input) -log(bound nc)/ log(input nc). Normalized interaction signals were z-transformed by subtracting the mean and dividing by the standard deviation. The mean and standard deviation were calculated from large datasets of protein pairs which were not expected to interact, i.e. from negative reference sets.