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

siRNA SMARTpools (4 siRNAs per gene) at 0.3 mM were dispensed in 10 ml volumes using a Rapidplate384 liquid handler (Qiagen) into triplicate black 384-well plates (Corning), sealed with adhesive seals (ThermoFisher) and plastic lids. Plates were stored at 280uC until needed (minimum 24 h, maximum 48 h). On the day of transfection, assay plates were thawed at room temperature and 10 ml transfection reagent (Dharmafect 1, Dharmacon), diluted in Hank's buffered saline solution (HBSS, ThermoFisher) to give a final concentration of 0.1%, was added using a Multidrop 384 (ThermoFisher). Plates were incubated for 20 min at room temperature to allow formation of transfection complexes. During complex formation, low-passage (p20-22) Hela cells (ECACC) from ,50% confluent flasks were washed in PBS and trypsinised in Trypsin-EDTA (Lonza) before diluting in phenol red-free, antibiotic-free transfection medium (DMEM/F-12 1:1/5% FCS with 15 mM Hepes and L-glu; Gibco). Cells were counted and 3610 3 cells in 40 ml were added to each well using the Multidrop 384. Plates were incubated for 48 h at 37uC in a humidified incubator with 5% CO 2 . To infect, media was removed from plates by inversion, and 10 ml media (as for transfection, but containing penicillin-streptomycin; Lonza) or virus (HSV-1-eGFP strain C12, diluted to MOI 0.5 in infection media) [34] was added using the Multidrop 384. Plates were incubated at 37uC for 1 h before 50 ml infection media was added and plates returned to the incubator. Replication was monitored as a function of eGFP fluorescence from 24 h to 80 h post-infection using the POLARstar OPTIMA plate reader (BMG Labtech). Virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses.