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

For inter-viral comparison, siRNAs were considered inhibitory or enhancing if normalized replication was 626STDEV of the controls a) HSV-1 replication assays. Selected siRNA SMARTpools were diluted to 0.3 mM in 16siRNA buffer and dispensed in black 96-well plates (Corning). To this 10 ml Dharmafect 1, diluted in HBSS to a final concentration of 0.15%, was added using the Multidrop 384. Following a 20 min incubation to enable complex formation, 1610 4 Hela cells in 80 ml transfection media 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 penicillinstreptomycin; Lonza) or virus (strain C12, diluted to MOI 0.5 in infection media) was added using the Multidrop 384. Plates were incubated at 37uC for 1 h before virus was removed by plate inversion and 100 ml infection media was added. Plates were returned to the incubator before replication was monitored as a function of eGFP fluorescence from 24 h to 80 h post-infection. 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. b) VZV replication assays. Selected siRNA SMARTpools were diluted to 0.3 mM in 16siRNA buffer and dispensed in black 384-well plates (Corning). To this, 10 ml Dharmafect 1 diluted in HBSS to a final concentration of 0.07% was added using the Multidrop 384. Following a 20 min incubation to enable complex formation, 2.5610 3 MeWo cells (ATCC, HTB-65 TM ) in 40 ml media (EMEM/10% FCS/1% non-essential amino acids) were seeded on to the complexes bringing the final volume of transfection to 60 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 colony forming units of VZV-eGFP-infected MeWo cells (vaccine strain Oka) [87] diluted in MeWo growth media were seeded on to the complexes using the Multidrop 384. Virus growth was measured in 3 h intervals as a function of eGFP fluorescence from 44 to 72 h post-infection. 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. c) hCMV replication assays. Selected siRNA SMARTpools were diluted to 0.5 mM in 16siRNA buffer and dispensed in 10 ml volumes in black 384-well plates (Corning). To this, 10 ml Dharmafect, 1 diluted in HBSS to a final concentration of 0.2%, was added using the Multidrop 384. Following a 20 min incubation to enable complex formation, 3610 3 MRC-5 (ATCC, CCL-171 TM ) in 40 ml growth medium (phenol red-free DMEM/ 10%FBS/L-glutamine/1% non-essential amino acids were seeded on to the complexes bringing the final volume of transfection to 60 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 10 ml media (as for transfection, but containing penicillin-streptomycin) or virus hCMV-GFP (strain AD169) [88] , diluted to MOI 0.5 in infection media, was added using the Multidrop 384. Plates were incubated at 37uC for 1