Fig 1. Strategy for creation of Semliki Forest Virus (SFV)–host protein-protein interaction map.

a, Schematic illustration of the genomic organisation of SFV. The first ORF encoding the non-structural proteins (nsp1, nsp2, nsp3, nsp4) is highlighted in green. The ZSG tag inserted within the nsp3 protein is also depicted. The second ORF encoding the structural proteins (capsid, E3,E2,6K,E1) is highlighted in orange. Other viral features depicted include the 5´ cap, poly(A) tail, as well as the position of the 26S subgenomic viral promoter. b, Flowchart outlining the experimental approach to transiently express N-terminally 3xFLAG-tagged (yellow rectangle) SFV proteins in mammalian cells in order to construct a SFV-host protein-protein interactome. Nsp3-Z refers to the nsp3 protein with the ZSG tag, C refers to the capsid, and E3 E2 6K E1 refer to the envelope proteins (Env) that were expressed as one polyprotein. c, Anti-FLAG western blot of SFV proteins after transient transfection and affinity purification from HeLa cells (without RNase A treatment). Red asterisks indicate 3xFLAG tagged SFV proteins at their expected sizes. Untransfected cells (Untr) and cells transfected with a plasmid encoding only the 3xFLAG tag with no additional coding region (empty) were included as controls. The expected sizes of the 3xFLAG-tagged proteins were: empty ~8kDa; nsp1 ~63kDa; nsp2 ~92kDa; nsp3-Z ~82kDa; nsp4 ~72kDa; capsid ~33kDa and Env (polyprotein ~111kDa, cleavage intermediates ~57kDa/ ~64kDa). The affinity purifications were conducted in triplicate (± RNase A treatment), and eluates analysed by mass spectrometry.