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

Combined bioinformatic analyses of protein interaction and siRNA depletion screens found a significant functional enrichment for proteins involved in transcription, and identified multi-protein complexes enriched for pro-viral HFs which strongly inhibited HSV-1 upon depletion, including the RNA-polymerase II, eIF3 and Mediator complexes ( Figure 3a) . The Mediator complex links the cellular transcription machinery (RNA polymerase II) to specific transcription factors, and the identification of many Mediator subunits as HFs in other viral siRNA depletion screens highlights its significant role in viral genome transcription [7, 9, 11, 40] (Table S2 in Text S2) . Further, several Mediator subunits (Med25, 29, 17 and 8) are known to interact with the HSV-1 transactivator VP16 (UL48) and other herpesviral proteins [41] ( Figure S6a) . Consistently, the Mediator complex was found to be strongly required for HSV-1 replication, with depletion of the majority of subunits (Med 4, 6, 7, 8, 14, 16, 17, 21, 25, 26, 27 and 28) leading to a severe reduction in virus replication in the primary screen ( Figure S6b ) or in confirmatory deconvolution assays ( Figure 3b) . However, depletion of the Med23 subunit was striking in that it led to a significant enhancement of virus growth ( Figure 3b ). Flow cytometry quantification found that removal of Med23 not only increased the total number of infected cells (combination of GFP lo and GFP hi cells; 75.5% in comparison to 49.6% in mock-transfected cells) but also the copy number of virus genomes (GFP hi cells; 44.8% in comparison to 24.4% in mocktransfected cells) (Figure 3c Med23 could exert anti-viral effects either by having an inhibitory effect on viral transactivators or by interacting with and having a positive effect on an existing anti-viral factor. We first tested whether Med23 directly affects viral gene expression using luciferase reporters with HSV-1 promoters, however observed no inhibitory effect (data not shown). Since the Mediator complex and Med23 in particular is known to be involved in Jak/Statmediated interferon signaling [42] , we used the lung epithelial cell line A549 and its Stat-1-deficient derivative A549-V [43] to determine if Med23 influences HSV-1 replication by modulating innate immunity. In the parental A549 cells the phenotype of HSV-1 replication was the same as that observed in Hela cells, where depletion of Med23 enhanced replication and overexpression inhibited virus growth. However, in the Stat1-deficient A549-V cells HSV-1 replication was unaffected by both depletion and over-expression of Med23 (Fig. 4a) , indicating that Med23 requires an intact Jak/Stat signalling pathway to exert its anti-viral effects.