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

Abstract

Large collections of protein-encoding open reading frames (ORFs) established in a versatile recombination-based cloning system have been instrumental to study protein functions in high-throughput assays. Such 'ORFeome' resources have been developed for several organisms but in virology, plasmid collections covering a significant fraction of the virosphere are still needed. In this perspective, we present ViralORFeome 1.0 (http://www.viralorfeome.com), an open-access database and management system that provides an integrated set of bioinformatic tools to clone viral ORFs in the Gateway(R) system. ViralORFeome provides a convenient interface to navigate through virus genome sequences, to design ORF-specific cloning primers, to validate the sequence of generated constructs and to browse established collections of virus ORFs. Most importantly, ViralORFeome has been designed to manage all possible variants or mutants of a given ORF so that the cloning procedure can be applied to any emerging virus strain. A subset of plasmid constructs generated with ViralORFeome platform has been tested with success for heterologous protein expression in different expression systems at proteome scale. ViralORFeome should provide our community with a framework to establish a large collection of virus ORF clones, an instrumental resource to determine functions, activities and binding partners of viral proteins.

Figure 1.

Overview of ViralORFeome database architecture. Data from external public databases (left panel) have been integrated in ViralORFeome database (middle panel): genomic and taxonomic data have been extracted from NCBI and ICTV using Perl scripts. Host protein sequences and annotations have been obtained from Ensembl. ViralORFeome web interface (right panel) enables the design of ORF clones using viral CDS (Coding DNA Sequences) available in GenBank as templates. Corresponding plasmids are stored in ORFeotheque (i.e. viral ORFs physical library), and can be requested using the same interface. Raw data relative to Y2H experiments performed with viral ORF clones have been analyzed with an IST pipeline [pISTil, (31)] and are stored in ViralORFeome.

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′- and 3′-coordinates and clone ORF fragments corresponding to specific domains. Users can also upload manually designed primers (Method 2). ViralORFeome will automatically design Gateway® 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).

Figure 3.

Expression and functional validation of plasmid constructs generated with ViralORFeome database. (a) Matrix map of virus ORF constructs that have been tested in expression and functional assays. Clone IDs in ViralORFeome database are indicated between parentheses. CHIKV, Chikungunya virus; SIN, Sindbis virus; SFV, Semliki Forest virus; YF, Yellow Fever virus; MV Sch, Schwarz vaccine strain of measles virus; MV Ich, Ichinose wild-type strain of measles virus; TIV, Tioman virus; HBV, hepatitis B virus. (b) Virus ORFs were recombined from entry vectors pDONR207 or pDONR223 in a Gateway®-compatible expression vector to be expressed in fusion with the red-fluorescent protein Cherry. Plasmids were transfected in HEK-293T cells and subcellular localization determined 24 h later. (c and d) The same virus ORFs were recombined in an expression vector to be expressed in fusion with the 3×FLAG tag. Constructs were co-transfected in HEK-293T cells together with a reporter plasmid encoding luciferase downstream of a promoter containing either IFN-α/β (pISRE-Luc) or NF-κB (pNF-κB-Luc) response elements. 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-β (c) or TNF-α (d) to activate ISRE or NF-κB response elements, respectively. Cells in position A1 and B1 correspond to negative and positive controls that were respectively left untreated or stimulated with IFN-β or TNF-α. 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.

Figure 3.

Expression and functional validation of plasmid constructs generated with ViralORFeome database. (a) Matrix map of virus ORF constructs that have been tested in expression and functional assays. Clone IDs in ViralORFeome database are indicated between parentheses. CHIKV, Chikungunya virus; SIN, Sindbis virus; SFV, Semliki Forest virus; YF, Yellow Fever virus; MV Sch, Schwarz vaccine strain of measles virus; MV Ich, Ichinose wild-type strain of measles virus; TIV, Tioman virus; HBV, hepatitis B virus. (b) Virus ORFs were recombined from entry vectors pDONR207 or pDONR223 in a Gateway®-compatible expression vector to be expressed in fusion with the red-fluorescent protein Cherry. Plasmids were transfected in HEK-293T cells and subcellular localization determined 24 h later. (c and d) The same virus ORFs were recombined in an expression vector to be expressed in fusion with the 3×FLAG tag. Constructs were co-transfected in HEK-293T cells together with a reporter plasmid encoding luciferase downstream of a promoter containing either IFN-α/β (pISRE-Luc) or NF-κB (pNF-κB-Luc) response elements. 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-β (c) or TNF-α (d) to activate ISRE or NF-κB response elements, respectively. Cells in position A1 and B1 correspond to negative and positive controls that were respectively left untreated or stimulated with IFN-β or TNF-α. 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.