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 transient over-expression, 1.5610 4 HEK293 cells were seeded in black 96-well plates. The following day, cells were transfected with 100 ng pCR3-Med23 using Lipofectamine TM LTX (Invitrogen) and incubated for 48 h before infection with the recombinant HSV-1 reporter viruses C12 and VP26-YFP at MOI 0.5. Replication growth curves were monitored, and endpoint replication (as determined by fluorescence) was normalized to untransfected cells. For stable expression, Hela cells were transduced with pLenti-Med23, generated using the ViraPower TM Lentiviral Expression System (Invitrogen), as per manufacturers' instructions. Stable cells were infected, and replication monitored, as above. For confirmation of overexpression, RNA was extracted (QIAGEN RNA-easy kit) and expression quantified by one-step RT-qPCR (Thermofisher), with gene-specific primers (Table S9 in Text S2), and probes from the Universal Probe Library (Roche). Expression levels were normalized to the housekeeping cellular gene hypoxanthine phosphoribosyltransferase 1 (HPRT) and calibrated to mock-transfected cells. qPCR was carried out in duplicate for each sample, and normalized expression levels averaged. The VP26-YFP reporter virus was generated from a KOS BAC kindly provided by David Leib using the Red-mediated recombination system, where the first 4 amino acids of VP26 were replaced with YFP [91, 92, 93] .