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

h before 50 ml infection media was added and plates returned to the incubator prior to monitoring virus replication. 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. d) SFV replication assays. Selected siRNA SMARTpools were diluted to 0.5 mM in 16siRNA buffer and dispensed in 10 ml volumes in black 96-well plates (Corning). To this, 10 ml Dharmafect 1 diluted in HBSS to a final concentration of 0.15% was manually added. Following a 20 min incubation to enable complex formation, 4610 4 Hela cells in 80 ml transfection media (DMEM/5% FCS/L-glu) were seeded on to the complexes bringing the final volume of transfection to 100 ml in each well. Plates were incubated for 48 h at 37uC in a humidified incubator with 5% CO 2 before infection. To infect, media was removed from plates by inversion, and 25 ml media (as for transfection, but containing penicillin-streptomycin; Lonza) or virus (SFV4(3H)-Rluc [89] diluted in phosphate buffered saline (PBS)/0.75% bovine serum albumin to an MOI 0.01) was manually added. Plates were incubated at 37uC for 1 h before media (as for transfection media) was added manually to increase the volume to 100 ml per well. Plates were incubated at 37uC for 24 h before cells were lysed using a passive lysis buffer and Renilla luciferase levels measured with a microplate reader (Promega) using a dual luciferase reporter assay kit (Promega). Luciferase activity, which is representative of virus genome replication, was normalized to mock-transfected cells and mean luciferase activity from six replicates used for subsequent data analyses. e) Vaccinia virus replication assays. Hela cells were transfected as described in primary siRNA screen. Plates were incubated for 48 h at 37uC in a humidified incubator with 5% CO 2 before infection. To infect, media was removed from plates by inversion, and 15 ml media (as for transfection, but containing penicillin-streptomycin) or 15 ml media containing Vaccinia virus strain WR with eGFP-tagged A5 protein [90] , diluted to MOI 0.05, was added using the Multidrop 384. Plates were incubated at 37uC for 1 h before 50 ml of media was added to each well, the plates inverted to remove the media and virus, and a final volume of 50 ml of media added to the plates before they were returned to the incubator. Replication was calculated as a function of eGFP fluorescence at 48 h post-infection using the POLARstar OPTI-MA plate reader (BMG Labtech). Virus replication was normalized to mock transfected wells on individual assay plates, and the mean replication from eight replicates used for subsequent data analyses.