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

An extended literature and database search identified 599 cellular proteins that interact with or are involved in infection with human herpesviruses. The overlap between the high-confidence Y2H cellular interactors (63) and HFs (358) with this set was statistically significant (p = 0.008) (Figure 2a; Figure S1h ; Table S5 in Text S2). From this combined analysis, a subset of HFs was chosen for further validation. Protein interactions were tested in a mammalian cell system by LUMIER pull-down assay [37] . Of the 45 interactions tested, 26 (57.8%) were confirmed, with 15 strongly positive (z-score .2) and 11 weakly positive (zscore 1-2) ( Figure S1g ). siRNA deconvolution (4 siRNAs per gene tested individually) was used to further validate 72 HFs (Figure 2b ; Figure S2 ). The replication phenotype could be confirmed ($2 or more siRNAs gave the same or better replication slope than observed in the primary screen) in a high proportion (83.3%) of candidates, highlighting the reliability of the primary screen dataset. Quantitative RT-PCR analysis of mRNA expression levels found a minimum depletion of 60% (mean 88%) in a subset of 52 genes (data not shown; Table S6 in Text S2) confirming the observed effects on HSV-1 replication are genuine and not due to 'off-target' effects or insufficient gene knockdown. To further investigate the virus-specificity of our identified HFs, we tested this subset for their effect on the replication of an additional a-herpesvirus (Varicella-Zoster virus, VZV), the bherpesvirus Cytomegalovirus (CMV), and a completely unrelated RNA virus, Semliki Forest Virus (SFV). None of the three proteins which enhanced HSV-1 replication upon knockdown had an effect on either VZV or CMV, and one (NR3C2) was even inhibitory for SFV ( Figure 2c ). Of the 64 siRNAs which inhibited HSV-1, 27 (42.2%) were also inhibitory for VZV, 60 for CMV (93.8%) and 23 (35.9%) for SFV replication ( Table S7 in Text S2) . Some functional groups (transcriptional regulators) were required by most viruses, but there were notable differences between other proteins. For example IFITM-1, previously identified as an inhibitor of Influenza A, Dengue virus and WNV [10] , inhibited VZV yet had a positive effect on HSV-1 replication. These data suggest that whilst there are some HFs which are broad in their effects on virus replication, a large proportion are species-specific. Table S8 in Text S2). Pathways included those involved in gene expression, transcription, splicing and translational regulation (RNAi screen), and protein transport, cell cycle, and transcriptional repressor activity (Y2H screen). A combined analysis of HFs from both screens found dominant functional categories centred on the regulation of transcription (RNA polymerase II-associated genes, splicing factors, transcription activation and the Mediator complex) ( Figure S3c, d) . The physiological relevance of some HFs and pathways was confirmed by further biological validation. Protein transport pathways (in the form of dynein microtubule networks) are exploited by HSV-1 early after infection to shuttle viral capsids to the nucleus. These screens confirmed known interactions between dynein subunits and viral proteins, and identified additional previously unknown interactions (Text S1 and Figure S4a ). Several dynein chain subunits were found to be essential for virus replication, whilst the moderate effect of depletion of other subunits demonstrated a level of functional redundancy in HS