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

Select siRNAs SMARTpools were diluted to 500 nM in HBSS and 100 ml was incubated with 100 ml Dharmafect 1 diluted in HBSS to a final concentration of 0.15% in individual wells of a 12well plate. After 20 min incubation, 2610 5 Hela cells in 800 ml transfection medium were added. Plates were incubated for 48 h at 37uC in a humidified incubator with 5% CO 2 before infection by removing medium and adding 500 ml HSV-1-eGFP at a MOI of 1. After incubation for 1 h at 37uC, virus was removed and replaced with 2 ml growth medium. After 48 h, medium was removed, cells rinsed in PBS and dislodged by trypsinisation. Cells were washed in PBS and pelleted by centrifugation for 10 min at 199 g. Supernatant was removed and cells fixed in 4% paraformaldehyde before analysing for eGFP expression by flow cytometry (FACS DiVa, BD Biosciences) using the CellQuest software package.