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

Text S1 Text for supporting figures. Descriptive text for Figures S4 and S5 Validation of HSV-1-host Y2H interactors. A subset of protein interactions identified in the HSV-1-host Y2H screen were validated using the LUMIER pull-down assay in a mammalian cell system. Strength of interaction was determined by Z-score, where a score 1 to 2 represents a weak interaction and score .2 represents a strong interaction. (h) Distribution of direct HSV-1 targets in the RNAi screen. The proteins directly targeted by HSV-1 were taken from the Y2H data set and from the literature curation. An enrichment of literature-derived targets could be observed in the top 5% most inhibiting knockdowns (2.4-fold enrichment; p = 0.008), but the same could not be observed for the Y2H-detected direct targets. (TIF) Figure S2 Validation of HF identified by RNAi. HFs identified in the HSV-1 perturbation screen were validated with deconvoluted siRNAs and qPCR: (a) chromoboxes, (b) homeoboxes, (c) general transcription factors, (d) nuclear receptors, (e) proteasome family members, (f) topoisomerases, (g) mediator complex subunits, (h) proteins involved in vesicle transport, (i) integrins, (j) Y2H interactors, (k) ubiquitin E2 ligases, (l) vesicle transport -further candidates, (m) interferon-stimulated membrane proteins and (n) others. HSV replication is presented as normalized replication slope, and is the mean of six individual assay points. Error bars represent standard deviation of the six data points. Deconvoluted siRNAs which had a sequence different to that in the original screen are highlighted in red. Genes for which the primary screen phenotype was not confirmed in the deconvolution assay are shown in bold text. (TIF) Figure S3 HSV-1 HFs are involved in diverse cellular pathways and at multiple stages of the HSV life cycle. Enrichment of protein functions among the HSV-1 HFs. The enrichment for gene ontology (GO) terms and KEGG, BIO-CARTA or REACTOME pathway annotations among the HSV-1 HFs identified by (a) RNAi and (b) Y2H assay was performed using DAVID bioinformatics software. (c) Direct interactions between human and HSV-1 proteins. The protein-protein interactions (PPIs) depicted are from the high confidence Y2H data set and from literature curation. Circles and diamonds correspond to human and HSV-1 proteins, respectively. Human proteins detected in the Y2H screen are drawn with red borders. Human genes that showed the strongest effects in the RNAi screen are colored yellow (extreme 10%) and orange (extreme 5%). (d) Highly interconnected regions in the human interaction network composed of HFs. Highly interconnected regions in the human interaction network composed of HFs. We assembled a human interaction network using data from the major PPI databases. A subnetwork consisting of HFs was then defined by limiting the network to the HFs detected in the Y2H screen, the RNAi (extreme 5%) screen, and the literature curation. Highly interconnected regions in the subnetwork were sought out using the MCODE algorithm. The top six scoring regions are shown. Proteins displayed in red correspond to HFs that are known only from the literature, and those in green are those that were detected in either of the screens performed in this study. The three boolean values beside each gene symbol represent whether the HF is present in the Y2H screen, the RNAi screen (extreme 5%), or the literature curated set, in that order. Dominant functional categories could be observed i