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

INSERM U851, Lyon, IFR128-BioSciences, Université Lyon 1, Université de Lyon, Lyon, France.
Nucleic Acids Research (Impact Factor: 9.11). 12/2009; 38(Database issue):D371-8. DOI: 10.1093/nar/gkp1000
Source: PubMed


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 (, 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.

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    • "In addition, this study demonstrated the phenotypic expression of 67 viral genes transfected in HEK-293T cells 15. Unfortunately, the overall progress of viral cloning has not progressed much beyond this due to the high cost of HT cloning and the difficulty in managing a large repository in a general lab 2, 15, 16. "
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    ABSTRACT: Throughout the long history of virus-host co-evolution, viruses have developed delicate strategies to facilitate their invasion and replication of their genome, while silencing the host immune responses through various mechanisms. The systematic characterization of viral protein-host interactions would yield invaluable information in the understanding of viral invasion/evasion, diagnosis and therapeutic treatment of a viral infection, and mechanisms of host biology. With more than 2,000 viral genomes sequenced, only a small percent of them are well investigated. The access of these viral open reading frames (ORFs) in a flexible cloning format would greatly facilitate both in vitro and in vivo virus-host interaction studies. However, the overall progress of viral ORF cloning has been slow. To facilitate viral studies, we are releasing the initiation of our panviral proteome collection of 2,035 ORF clones from 830 viral genes in the Gateway® recombinational cloning system. Here, we demonstrate several uses of our viral collection including highly efficient production of viral proteins using human cell-free expression system in vitro, global identification of host targets for rubella virus using Nucleic Acid Programmable Protein Arrays (NAPPA) containing 10,000 unique human proteins, and detection of host serological responses using micro-fluidic multiplexed immunoassays. The studies presented here begin to elucidate host-viral protein interactions with our systemic utilization of viral ORFs, high-throughput cloning, and proteomic technologies. These valuable plasmid resources will be available to the research community to enable continued viral functional studies.
    Full-text · Article · Jun 2014 · Theranostics
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    • "ession vector encoding CHIKV structural proteins ( CHIKVsp ) was previously described ( Akahata et al . , 2010 ) and kindly provided by Dr . Gary Nabel of National Institute of Allergy Infectious Diseases . mCherry tagged plasmids encoding various CHIKV proteins ( nsP1 , nsP2 , nsP3 , nsP4 , E1 , E2 , E3 , Capsid , K6 ) were previously described ( Pellet et al . , 2010 ) and generously provided by Dr . Pierre - Olivier Vidalain of Unité de Gé nomique Virale et Vaccination , Institut Pasteur , France through Dr . Deborah Lenschow of Washington University School of Medicine , St . Louis , MO ."
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    ABSTRACT: Chikungunya virus (CHIKV) is a re-emerging alphavirus transmitted by Aedes mosquitoes. Infection with CHIKV elicits a type I interferon response that facilities virus clearance, probably through the action of down-stream effectors such as antiviral IFN-stimulated genes (ISGs). Bone marrow stromal antigen 2 (BST-2) is an ISG shown to restrict HIV-1 replication by preventing the infection of bystander cells by tethering progeny virions on the surface of infected cells. Here we show that enrichment of cell surface BST-2 results in retention of CHIKV virus like particles (VLPs) on the cell membrane. BST-2 was found to co-localize with CHIKV structural protein E1 in the context of VLPs without any noticeable effect on BST-2 level. However, CHIKV nonstructural protein 1 (nsP1) overcomes BST-2-mediated VLPs tethering by down-regulating BST-2 expression. We conclude that BST-2 tethers CHIKV VLPs on the host cell plasma membrane and identify CHIKV nsP1 as a novel BST-2 antagonist.
    Full-text · Article · Mar 2013 · Virology
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    • "TioV-V (NP_665866), TioV-P (NP_665865), TioV-W (NP_665867), TioV-VCT (AA 150-228 of TioV-V), MuV-V (ABG48763, Mumps virus isolate “Sophie”, GenBank accession number: DQ660370) and NiV-V (NP_112023.1) coding sequences were amplified by RT-PCR (Titan One tube; Roche) from RNA samples kindly provided by Dr. TF Wild, and cloned into pDONR207 (Invitrogen) using an in vitro recombination-based cloning system (Gateway system; Invitrogen) as previously described [30]. Chikungunya virus (CHIKV) nsP4 construct was previously described [30]. "
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    ABSTRACT: The capacity of a virus to cross species barriers is determined by the development of bona fide interactions with cellular components of new hosts, and in particular its ability to block IFN-α/β antiviral signaling. Tioman virus (TioV), a close relative of mumps virus (MuV), has been isolated in giant fruit bats in Southeast Asia. Nipah and Hendra viruses, which are present in the same bat colonies, are highly pathogenic in human. Despite serological evidences of close contacts between TioV and human populations, whether TioV is associated to some human pathology remains undetermined. Here we show that in contrast to the V protein of MuV, the V protein of TioV (TioV-V) hardly interacts with human STAT2, does not degrade STAT1, and cannot block IFN-α/β signaling in human cells. In contrast, TioV-V properly binds to human STAT3 and MDA5, and thus interferes with IL-6 signaling and IFN-β promoter induction in human cells. Because STAT2 binding was previously identified as a host restriction factor for some Paramyxoviridae, we established STAT2 sequence from giant fruit bats, and binding to TioV-V was tested. Surprisingly, TioV-V interaction with STAT2 from giant fruit bats is also extremely weak and barely detectable. Altogether, our observations question the capacity of TioV to appropriately control IFN-α/β signaling in both human and giant fruit bats that are considered as its natural host.
    Full-text · Article · Jan 2013 · PLoS ONE
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