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

b) Yeast two-hybrid assays. Haploid yeast strains AH109 and Y187 were transformed with 1 mg prey (pGADT7 or pGADT7-IRF1, -IRF2, -IRF3, -IRF4, -IRF5, -IRF7 or -IRF9) or bait (pGBKT7 or pGBKT7-Med23) plasmid DNA, respectively, and grown overnight in synthetic defined (SD) medium lacking either leucine (-L; Prey) or tryptophan (-W; Bait) before prey-and bait-expressing haploid yeast cells were mated overnight in SD-LW/5% YPDA medium. Haploids were selected in SD-LW for 48 h before transferring to triple-knockout, histidine-deficient SD-LWH liquid medium containing 3-aminotriazol (3-AT) and 4methylumbelliferyl-b-D-galactopyranoside (4-MuX) for 5-7 days. Interactions were tested in quadruplicates, detected by growth of colonies on SD-LWH agar with 3-AT, and quantified by measurement of fluorescence released from a-galactosidase cleavage of 4-MuX upon protein interaction. Relative fluorescence (RFU) was normalized to negative control interaction (empty pGADT7 mated with empty pGBKT7).