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

n each of the regions, including transcription (e.g., Mediator complex, RNA polymerase II and associated genes), translation initiation, splicing, and intracellular transport. (TIF) Figure S4 HFs involved in viral entry and capsid transport. (a) Diagrammatic summary of the role of dynein chains in HSV-1 infection. (b) Microtubule transport is required for HSV-1 infection. The role of dynein microtubule transport components in HSV-1 replication was analysed by comparing the replication slope of HSV-1-eGFP (C12)-infected cells depleted for a range of dynein chains from the primary siRNA perturbation screen. Error bars represent the mean of three independent experiments done in duplicate. (c) Depletion of dyneins inhibits virus particle release. The effect of dynein chain depletion on HSV-1 particle release was determined by quantifying virus titer in supernatants of Hela cells depleted of DYNC1H1 (heavy chain), DYNC1I2 (intermediate chain) and DYNC2LI1 (light intermediate chains) in a high multiplicity (MOI 5; HSV-1 KOS) growth assay. Titers were compared to control transfected cells (NT, nontargeting siRNA). (d) Depletion of dynein chains prevents immediate-early gene expression. Immediate-early (ICP0) and late (VP16) viral protein expression in cells depleted of DYNC1H1, DYNC1I2 or DYNC2LI1 was analysed and quantified by Western blot. Levels were compared to control transfected cells (NT, non-targeting siRNA). (e) Quantification of ICP0 and VP16 protein expression. Protein levels of ICP0 and VP16 in cells depleted of DYNC1H1, DYNC1I2 or DYNC2LI1 were quantified with an Odyssey Imager and normalized to protein levels in control transfected cells (Non-targeting siRNA). (TIF) Figure S5 E2 ubiquitin conjugating enzymes in HSV-1 immune evasion. (a) Depletion of E2 ubiquitin ligases inhibits PML degradation following HSV-1 infection. Hela cells mounted on coverslips were depleted for a range of E2 ubiquitin ligases for 24 h before infecting with HSV-1 17+. Cells were fixed and stained for ICP0 (green) and PML (red), analysed by confocal microscopy and PML-positive cells were counted (5 fields of view per coverslip) and expressed as a mean percentage of PMLpositive cells remaining (3 independent experiments). Error bars represent the standard deviation over 3 independent experiments. (b) Inhibition of PML degradation by E2s is ICP0-dependent. Hela cells were seeded on coverslips, transfected as above and infected with an ICP0 RING-finger deletion mutant (FXE). Remaining PML-positive cells were quantified as above. (c) Immunofluorescence staining for PML bodies in cells transfected with control siRNA not incorporated into the RISC complex (RSCF) or cells depleted of the E2 ubiquitin conjugating enzymes E2D1 or E2L3. The arrows highlight cells that have wt PML levels remaining in them whilst containing wt ICP0. (TIF) Figure S6 Med23 is an anti-viral component of the largely pro-viral multi-protein Mediator complex. (a) Diagrammatic summary of the role of Mediator complex subunits in virus replication. Subunits are coloured according to whether HSV-1 replication was unchanged (grey), inhibited (top 5%, red; top 10%, orange) or enhanced upon gene knockdown (top 5%, light green; top 10%, dark green). Subunits not included are white; herpesvirus proteins reported to target Mediator subunits are violet; Mediator subunits detected in other viral RNAi screens are highlighted by coloured diamonds. (b) Mediator complex subunits influence HSV-1 replicatio