Figure 2

The MC signalling pathway is anti-viral to HSV-1. (a) The MC signalling pathway. Unliganded MR forms a cytoplasmic complex with the chaperone protein HSP90 (H) and the immunophilin FKBP5 (F5). Binding of the steroid hormone aldosterone (A) induces a structural change replacing FKBP5 with FKBP4 (F4), enabling nuclear translocation via the dynein complex and activation of transcription from the hormone response element (HRE) to regulate sodium reabsorption. (b) Protein-protein interaction networks of the MR. Human interaction information was compiled from public protein-protein interactions databases and curated pathways (KEGG and REACTOME) and combined with RNAi screen data for the top 2.5% most enhancing and most inhibiting genes (ranked by distance from the median replication slope). White nodes, not present in siRNA library; grey nodes, weak effect; red nodes, 2.5% most inhibiting; green nodes, 2.5% most enhancing. (c) Components of the MC signalling pathway are anti-viral to HSV-1. Virus growth curves in HSV-1-infected HeLa cells (MOI 0.5) depleted for members of the MC signalling pathway were plotted and compared to negative (mock-transfected) and positive controls (ICP4, an essential HSV-1 gene). (d) HSV-1 is inhibited by overexpression of MC signalling pathway members. 293T cells transiently over-expressing members of the MC signalling pathway were infected with HSV-1-eGFP at MOI 0.5 and replication monitored by GFP fluorescence. Replication slopes over the linear phase were calculated and normalised to controls (pCR3-transfected cells). Error bars represent the standard error of the mean of at least three independent experiments, carried out in triplicates. (e) The MR signalling complex is translocated to the nucleus via the dynein intermediate chain DYNC1I1. HeLa cells were transfected with positive control siRNA (FKBP4, essential for MR nuclear translocation), negative control siRNA (RISC-free, RSCF) or siRNA targeting the intermediate dynein chains DYNC1I1 or DYNC1I2 before transiently overexpressing HA-tagged MR and stimulating with aldosterone (1 μM). MR was detected with an anti-HA antibody (red) and nuclei visualised using a mounting medium containing DAPI (blue) by confocal microscopy.