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

As IFN-l expression is induced following activation of pathogen recognition receptors (PRRs) by virus infection [45, 46, 47, 48] , we tested whether Med23 induced IFN-l by directly interacting with an interferon-responsive transcription factor (IRF). Y2H and confirmatory co-immunoprecipitation experiments in mammalian cells with a panel of IRFs found that Med23 interacted with IRF4 and IRF7 (Figure 5a ; Figure S7b ). We also observed a weak interaction with IRF9, which may explain the previously observed effect of Med23 on Jak/Stat signalling [42] . To determine if this interaction had a functional effect, we looked at whether Med23 influenced IRF-mediated induction of IFN-l. In a luciferase reporter assay, neither IRF4 nor IRF9 led to a significant induction of the IFN-l1 promoter, either alone or in conjunction with Med23 (data not shown). IRF7 induced expression from the IFN-b and IFN-l1 promoters to similar levels (,7-fold and 9-fold higher than background, respectively), whilst the ISRE, induced by IRF7 and also present in the IRF7 promoter, was induced ,15-fold ( Figure 5b ). Whilst co-expression of Med23 with IRF7 had no further effect on IFN-b expression, a synergistic induction of the IFN-l1 promoter and, to a lesser extent, the ISRE, was observed (IFN-l1 doubled to ,18-fold, p = 0.02) ( Figure 5b ). Interestingly, a Med23 mutant unable to induce immediate early gene expression via jun/fos (R617Q, or R611Q in Med23 transcript variant 1 used here) synergistically induced ISRE expression with IRF7, yet was unable to further enhance IRF7mediated induction of IFN-l1 (data not shown). A similar synergistic effect of Med23 and IRF7 was seen at the protein level, where co-expression increased supernatant levels of IFN-l3 more than 2-fold those seen with Med23 or IRF7 alone ( Figure S7c , d).