ViralORFeome: an integrated database to generate
a versatile collection of viral ORFs
J. Pellet1,2,3, L. Tafforeau1,2,3, M. Lucas-Hourani4, V. Navratil2,3,5, L. Meyniel1,2,3, G. Achaz6,
A. Guironnet-Paquet1,2,3,7, A. Aublin-Gex1,2,3, G. Caignard4, P. Cassonnet8,
A. Chaboud2,3,9, T. Chantier1,2,3, A. Deloire1,2,3, C. Demeret8, M. Le Breton1,2,3,
G. Neveu8, L. Jacotot6, P. Vaglio6, S. Delmotte2,3,7, C. Gautier2,3,7, C. Combet2,3,10,
G. Deleage2,3,10, M. Favre8, F. Tangy4, Y. Jacob8, P. Andre1,2,3,11, V. Lotteau1,2,3,11,
C. Rabourdin-Combe1,2,3,* and P. O. Vidalain4,*
1INSERM U851, Lyon,2IFR128-BioSciences, Universite ´ Lyon 1,3Universite ´ de Lyon, Lyon,4Laboratoire de
Ge ´nomique Virale et Vaccination, Institut Pasteur, CNRS URA3015, Paris,5INRA UMR754, Lyon,6Modul-Bio,
Marseille,7CNRS, UMR5558, Laboratoire de Biologie et de Biome ´trie E´volutive, Lyon,8Unite ´ de Ge ´ne ´tique,
Papillomavirus et Cancer Humain, Institut Pasteur, Paris,9RDP, UMR 5667 INRA/CNRS/ENSL/UCB-Lyon I,
10IBCP, CNRS UMR5086 and11Hospices Civils de Lyon, Ho ˆpital de la Croix-Rousse, Laboratoire de virologie,
Received August 5, 2009; Revised September 18, 2009; Accepted October 18, 2009
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 inte-
grated set of bioinformatic tools to clone viral
ORFs in the Gateway?
through virus genome sequences, to design ORF-
specific cloning primers, to validate the sequence
of generated constructs and to browse estab-
lished 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 con-
structs generated with ViralORFeome platform has
been tested with success for heterologous protein
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.
The number of viral genomic sequences available in public
databases has increased exponentially, opening new
perspectives to understand genetic basis and functional
mechanisms that underlie virus replication, pathogenesis
and evolution. In particular, this enabled to establish for
each virus a list of potential regulatory and expressed
sequences, a framework often referred as the ‘parts list’
of biological systems (1). Current investigations aim at
understanding how these viral components act upon
each other and interact with host macromolecules to
carry on viral replication and spreading. To reach such
a system view of virus cycles, more functional analyses
of viral components are necessary, especially in the field
of virus–host molecular interactions (2,3). To address this
question, a large collection of viral open reading frames
(ORFs) established in a recombination-based cloning
*To whom correspondence should be addressed. Tel: +33 4 37 28 23 42; Fax: +33 4 37 28 23 21; Email: email@example.com
Correspondence may also be addressed to P.O. Vidalain. Tel: +33 1 45 68 87 73; Fax: +33 1 40 61 31 67; Email: firstname.lastname@example.org
V. Navratil, Universite ´ de Lyon, CNRS/ENSL/UCB-Lyon I, Centre de RMN a ` Tre ` s Hauts Champs, Villeurbanne, France.
The authors wish it to be known that, in their opinion, the first three authors and the last two authors should be regarded as joint First Authors.
Published online 8 December 2009Nucleic Acids Research, 2010, Vol. 38, Database issue D371–D378
? The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
system allowing their mass transfer into various functional
assays would be extremely helpful. Such ORF collections,
often referred as ‘ORFeomes’, have been developed for
human and few other organisms and represent great
resources to explore protein functions in a large-scale
setting (4–9). Recombination-based cloning technologies
like the Gateway?system enable the mass cloning of
polymerase chain reaction (PCR)-amplified ORFs into a
‘donor’ vector to create ‘entry’ clones. Once entry clones
have been established, ORFs can be easily recombined
into different ‘destination’ vectors that allow protein
expression. So far, only few viral ORFeomes have been
built and they are dedicated to a single virus, arguing that
much more needs to be achieved (10–13).
Because of their small-sized genomes, most viruses
exhibit a limited number of ORFs. Therefore, building a
viral ORFeome collection covering a significant number
of viral pathogens can appear like a manageable project.
However, this eventually becomes a daunting task when
considering virus strains and polymorphisms that affect
viral proteins (Table 1). Nonetheless, these variations
cannot be neglected since they often determine virulence
and/or host adaptation. For example, a unique amino
acid mutation in the polymerase of poliovirus can alter
its processivity, turning a highly pathogenic virus into an
attenuated strain that can eventually be used as a vaccine
(14). Similarly, a Semliki Forest virus strain encoding an
nsP2 protein, with a single amino acid mutation in its
nuclear localization signal, is impaired for the control of
type I interferon response and strongly attenuated in vivo
(15). Thus, specific care must be taken when cloning
a virus ORFeome, since an accurate identification of the
strain that is used as a template is absolutely critical,
both in terms of genotype and phenotype. For this
reason, wild-type virus strains corresponding to primary
isolates will be generally preferred to culture-adapted
laboratory strains that often exhibit an altered phenotype
in vivo. Such biological samples are often difficult to
obtain, since it requires access to patients, medical-care
facilities and laboratories of an appropriate biosafety
level. With such constraints to obtain suitable viral
RNA or DNA templates, a collaborative effort between
virology laboratories is needed to build a comprehensive
viral ORFeome resource. This motivated the development
of ViralORFeome 1.0, an open-access database and man-
agement system designed to assist academic laboratories
in the development of a viral ORFeome collection using
a recombination-based cloning technology.
DESIGN AND IMPLEMENTATION
ViralORFeome is a database for creating and managing
viral ORF clones. It includes a web interface to search
and display viral sequences and annotations downloaded
from GenBank. When users have identified the viral
ORF sequences that they want to clone, clicking on
target sequences automatically creates virtual clones in
ViralORFeome database together with suitable cloning
primers. These primers are designed for PCR amplifica-
tion and cloning of viral ORFs using the Gateway?
system. To determine if ‘entry’ clones produced at the
bench exhibit expected sequences, sequence traces can
be uploaded in ViralORFeome, automatically aligned,
and compared to corresponding virtual clones. Finally,
ViralORFeome database allows users to manage their
collection of viral ORF clones, keep track of expression
plasmids derived from ‘entry’ clones and share plasmids
with other laboratories. System architecture and data flow
are depicted in Figure 1.
The World Wide Web server for ViralORFeome is
Apache (http://www.apache.org/) with Hypertext Pre-
processor (PHP, http://www.php.net) and Asynchronous
database management system for ViralORFeome is
PostgreSQL (http://www.postgresql.org/). ViralORFeome
Entity-Relationship (ER) model was divided into five
‘ORFeome’, ‘Interactome’ and ‘Users’ schemes (Supple-
mentary Figure S1).
ViralORFeome data source
ViralORFeome 1.0 is based on conventional sequence and
virus databases. Virus and host organism classification
was retrieved from the taxonomy database at National
Center for Biotechnology Information [NCBI; ftp://ftp
.ncbi.nih.gov/pub/taxonomy/; (16)]. Complete viral CDS
and associated annotations were downloaded from the
CoreNucleotide division of GenBank [http://www.ncbi
Entrez Programming Utilities (eUtils) at NCBI (see
Supplementary Data). ICTV names were obtained from
themost recent publication
Committee on Taxonomy of Viruses using Perl scripts
(18). Finally, host gene and protein sequence annotations
were extracted from Ensembl database (19).
of the International
ViralORFeome database can be accessed at: http://www
.viralorfeome.com. When selecting ‘Virus’ option in
the menu bar, ViralORFeome web interface allows
users to navigate throughout viral sequences retrieved
from GenBank by entering criteria such as virus species,
taxon IDs oraccession
Figure S2). ViralORFeome can also execute a BLASTn
or BLASTp search among available viral sequences (20).
Virus genomes and coding sequences of interest are
visualized with an integrated genome browser adapted
from GBrowse that provides a synthetic view of genomic
sequence features (21). When users have identified a viral
ORF sequence that they want to clone, clicking on this
sequence automatically generates cloning primers and
Table 1. Viral sequences available in ViralORFeome 1.0 (GenBank
Viral speciesVirus strains Complete CDS Annotated proteins
D372 Nucleic AcidsResearch, 2010, Vol.38,Database issue
virtual clones in ViralORFeome database (Figure 2).
ORF-specific Gateway?primers are designed using the
adapted for recombinational cloning (22). By default,
primers are designed to clone full-length ORFs from
ATG to STOP codon according to GenBank annotations.
However, users can specify 50- and 30-coordinates to clone
ORF fragments corresponding to specific domains or even
upload manually designed primers. Once primers have
been validated, virtual clones are generated and used
as entry points in ViralORFeome to store and access
all information relative to the viral ORF constructs
(e.g. primers, sequencing traces, potential mutations, and
Importantly, when users want to clone some variant of
an ORF that is not defined in GenBank, and therefore is
not available in ViralORFeome, an interface allows for
the creation of a ‘variant’ clone that is anchored to
the most similar annotated sequence found by BLAST
in the database (Supplementary Figure S3). This ensures
a maximum of flexibility when viral sequences are known
and characterized but not readily available in GenBank,
a frequent situation when working with primary isolates.
This also facilitates the management of mutants generated
on purpose to study the role of specific residues in a
In this recombination-based cloning pipeline, viral
ORFs amplified by PCR or RT-PCR are cloned by
in vitro recombination into donor vectors such as
pDONR207 or pDONR223 (see Supplementary Data
for detailed protocols). After transformation, a construct
can be purified either from a mini-pool of bacteria
colonies to keep the sequence diversity of the original
template (e.g. when working with viral quasi-species)
or from a single isolated colony. These two cloning
strategies, referred as 1.0 and 2.0, respectively (5), are
manageable using ViralORFeome interface (Figure 2).
ViralORFeome automatically aligns uploaded sequence
traces onto virtual clones using either an extended
Libalign Perl tool or Phred and T-Coffee programs that
build first a contig of sequence traces before the alignment
is performed (23,24). Validated ‘entry’ constructs can be
subsequently used to recombine viral ORFs into various
Gateway?-compatible destination vectors to achieve
protein expression. ViralORFeome allows users to keep
track of the different expression constructs that have
been established for each viral ORF.
Users can browse the set of viral ORF clones already
established in ViralORFeome by selecting the ‘clones’
option in the menu bar. Queries can be performed by
entering criteria such as virus species, GenBank protein
accession numbers or ViralORFeome clone IDs. Viral
ORF clones can also be searched by nucleotide or protein
BLAST. Until now, three laboratories involved in the
ViralORFeome interface and generated a collection of
528 viral ORFs cloned into pDONR vectors. Among
them, 145 have already been stored in a dedicated reposi-
tory for viral ORFeome resource (ORFeotheque; Hospices
Civils de Lyon), and 134 are available upon request under
material transfer agreement via ViralORFeome. This set of
‘ORFeotheque’ option in the menu bar.
validate ‘entry’ constructsby sequencing,
To validate the viral ORF clone collection built using
ViralORFeome pipeline, we tested a subset of 66 ORFs
(Figure 3a) from three Alphaviruses (Chikungunya,
Sindbis and Semliki Forest viruses), one Flavivirus
(yellow fever virus), one Hepadnavirus (hepatitis B virus)
and four Paramyxoviruses (measles virus wild-type and
vaccine strains, mumps, Tioman and Nipah viruses).
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.
Nucleic Acids Research, 2010,Vol.38, Database issueD373
ORFs were expressed in fusion downstream of the red
fluorescent protein Cherry and constructs transfected
inhuman cellsto achieve
(Figure 3b). We observed a specific localization pattern
distinct from Cherry alone for most constructs, demon-
strating that viral ORFs are properly expressed in this
system. In addition, and even if the N-terminal tag was
expected to alter the localization of proteins like viral
envelopes, observed localization patterns were generally
consistent with literature. For example, expression of
nsP1 not only from Semliki Forest virus and Sindbis
virus (25) but also Chikungunya virus induced character-
istic filopodia-like extensions in transfected cells. The
capsid of yellow fever virus (C) was found in the nucleus
where it accumulated in a dot-like pattern, reminiscent of
nucleoli, as reported for C of dengue and Japanese
encephalitis viruses (26,27).
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 pathwayswere inhibited
Chikungunya and Semliki forest viruses. This is consistent
with previous reports that used mutant viruses or
replicons to demonstrate that nsP2 from Old World
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 50- and 30-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).
D374 Nucleic AcidsResearch, 2010, Vol.38,Database issue
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 24h 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-a/b (pISRE-Luc) or NF-kB (pNF-kB-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 24h 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.
Nucleic Acids Research, 2010,Vol.38, Database issueD375
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-scale functional assays.
SUMMARY AND FUTURE DEVELOPMENTS
In conclusion, ViralORFeome is the first open-access
database that provides an integrated set of bioinformatic
Figure 3. Continued.
D376 Nucleic AcidsResearch, 2010, Vol.38,Database issue
tools to build a collection of viral ORFs clones in a versa-
tile system suitable for reverse proteomic experiments. In
this perspective, ViralORFeome was especially designed to
handle the diversity of virus strains, variants and species.
As shown here, our cloning pipeline has been validated
using functional assays and a collection of 528 viral
ORFs has been generated in the Gateway?system. As
collaborative efforts between virology laboratories are
required to establish an ORFeome collection covering
most viral genera and species, we believe ViralORFeome
will provide the community with a framework to sustain
this global effort.
In the near future, our objective will be to motivate
more laboratories to join this program. In addition,
we would like to implement new modules allowing users
to store and visualize all kind of functional data generated
with viral ORFs from the collection. This virus clone
collection was primarily developed to map virus–host
protein–protein interactions using the yeast two-hybrid
(Y2H) system as a collaborative effort between co-authors
of this manuscript. Current storage and display of virus–
host interaction data obtained by Y2H screening of
HCV proteins constitute a first attempt to reach this
goal. The ‘Interaction’ option of the menu bar allows
users to select HCV ORFs and access all interacting
cellular cDNA clones identified by Y2H. Whereas
filtered data sets have already been published (12),
ViralORFeome interface allows users to access Y2H raw
data to filter these results according to their own quality
criteria. Other functional data such as subcellular localiza-
tion or interference with immune response pathways
should be implemented.
ACCESS TO THE DATABASE
Public access to the ViralORFeome is available at:
http://www.viralorfeome.com. Registration is not neces-
sary, and use of the database is free. Users who want to
generate viral ORF clones that are tagged with their
institutional name can create an account to register.
Supplementary Data are available at NAR Online.
sequencing core facility, in particular C. Bouchier and
C. Gouyette. We thank Drs Marie-Louise Michel,
Charles M. Rice, Kaoru Takeuchi, T. Fabian Wild and
Ali Amara for providing RNA or DNA templates used to
build viral ORF clones. We also thank Eric Coissac for
providing SQL codes.
thank allmembersof PF1-PasteurGenopole
The Institut National de la Sante ´ et de la Recherche
Me ´ dicale;the InstitutNational
Agronomique; the Association de la Recherche contre le
Cancer (3731XA0531F and 4867); the Ligue Nationale
Contre le Cancer (RS07/75-75); the Agence Nationale de
la Recherche (EPI-HPV-3D); the French Ministry of
Industry; the Institut Pasteur; the Centre National de la
Recherche Scientifique (Maladies Infectieuses Emergentes
to P.O.V., G.C. and F.T.). Funding for open access
charge: Institut Pasteur.
Conflict of interest statement. None declared.
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