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

infection. To gain further mechanistic insight into host factors involved in HSV-1 infection, in parallel to the siRNA depletion screen we carried out a yeast two-hybrid protein interaction screen to identify cellular interaction partners of viral proteins. We generated a collection of 107 partial and full-length HSV-1 cDNA constructs and tested them for interactions with proteins encoded by a library of 12,381 human cDNA clones [35] . 231 HSV-1human protein interactions were detected once (low confidence), and 63 more than once (high-confidence)( Table S3 in Text S2) . Using these high-confidence interactions, the previously reported HSV-1 interactome [36] was connected into a human interactome (62,310 published protein interactions) to generate a combined pathogen-host interactome ( Figure S1a ). Both degree centrality (which indicates the number of interactions a protein has, where high values represent highly interactive 'hubs') and betweenness centrality (which indicates the number of shortest paths between (3 replicates) or the capacity to influence replication of the HSV-1 GFP reporter virus C12 (6 replicates) from 24 to 80 h post-infection. Virus replication slopes during the linear phase were calculated and normalized to mock-transfected cells. Replication slopes were then compared to replication upon knockdown of essential (ICP4, VP16) or non-essential (VP11/12) viral genes, a cellular receptor for HSV-1 (HVEM) or control RISC-free siRNA (RSCF). (c) Overlap between the HSV-1 HFs identified in this study with those published in HIV-1 [7, 8, 9] , Hepatitis C Virus (HCV) [13] and Influenza A virus [10, 11, 12] . doi:10.1371/journal.ppat.1003514.g001 any pair of proteins passing through the protein considered) were significantly increased for HSV-1 interactors, particularly in the high-confidence network (Figure S1b-e). These data suggest HSV-1 proteins preferentially target highly connected central human proteins in the cellular interaction network, similar to other viruses [23] .