ViralORFeome: an integrated database to generate a versatile collection of viral ORFs

Pellet, J.; Tafforeau, L.; Lucas-Hourani, M.; Navratil, V.; Meyniel, L.; Achaz, G.; Guironnet-Paquet, A.; Aublin-Gex, A.; Caignard, G.; Cassonnet, P.; Chaboud, A.; Chantier, T.; Deloire, A.; Demeret, C.; Le Breton, M.; Neveu, G.; Jacotot, L.; Vaglio, P.; Delmotte, S.; Gautier, C.; Combet, C.; Deleage, G.; Favre, M.; Tangy, F.; Jacob, Y.; Andre, P.; Lotteau, V.; Rabourdin-Combe, C.; Vidalain, P. O.

As numerous viral proteins are known to inhibit the host immune response, the 66 ORFs were also tested for their ability to block signaling downstream of two key antiviral cytokines: Interferon-b (IFN-b) and tumor necrosis factor-a (TNF-a). Each ORF was expressed in fusion downstream of the 3xFLAG tag and, using luciferase reporter constructs, tested for its ability to inhibit IFN-b or TNF-a signaling (Figure 3c and d) . Both pathways were inhibited by nsP2 from Chikungunya and Semliki forest viruses. This is consistent with previous reports that used mutant viruses or replicons to demonstrate that nsP2 from Old World Alphaviruses induces a transcriptional shutoff and Figure 2 . Building a viral ORF collection using ViralORFeome interface. Viral sequences and annotations from GenBank are visualized with a genome browser that provides a synthetic view of sequence features (1) . CDS are shown in blue and proteins in green. Users can design a new clone by clicking on a viral protein of interest (2) . By default, ViralORFeome will anchor cloning primers at the extremities of selected ORFs (Method 1), but user can specify 5 0 -and 3 0 -coordinates and clone ORF fragments corresponding to specific domains. Users can also upload manually designed primers (Method 2). ViralORFeome will automatically design Gateway Õ cloning primers (3) and after validation (4), a virtual clone is created in the database (5) . Users need to select between two cloning strategies, 1.0 ('in pool') or 2.0 ('individual clone'), before they can access a webpage where all information relative to the construct are stored (6) . This includes clone coordinates, primers, sequence and comments (upper panel), sequencing traces and alignments (middle panel), and available entry and destination vectors to achieve viral ORF expression (lower panel). When back to the genome browser (1), viral ORF clones are displayed in red (1.0 constructs) or purple (2.0 constructs). A CMV-Renilla plasmid was also co-transfected and used as an internal control for transfection efficiency and cell viability. After transfection, cells were incubated for 24 h in the presence of IFN-b (c) or TNF-a (d) to activate ISRE or NF-kB response elements, respectively. Cells in position A1 and B1 correspond to negative and positive controls that were respectively left untreated or stimulated with IFN-b or TNF-a. Relative luciferase activity was determined using a chemiluminescent substrate, and results expressed in relative percentage to positive control. Data show one representative experiment out of two. controls the host antiviral response (15, 28) . In contrast, nsP2 derived from a Sindbis virus infectious cDNA clone (29) did not localize in the nucleus and failed to block signaling. This supports previous reports showing that nsP2 from Alphaviruses must be nuclear to control the antiviral response (15) . We also confirmed that V proteins of measles and mumps viruses and V and W proteins of Nipah virus block IFN-b signaling (30) . Interestingly, the V protein of Tioman virus was unable to do so, suggesting that this virus infecting flying foxes (Pteropus genus) is not adapted to human cells. Altogether, observed localization patterns and functional assays validate the clone collection that was generated with ViralORFeome platform. Furthermore, these results illustrate how large collections of viral ORF clones established in a versatile cloning system provide access to reverse proteomic platforms and large