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Identification and characterization of a novel non-structural protein of bluetongue virus.

ABSTRACT: Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77-79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell.

SUBMITTER: Ratinier M

PROVIDER: S-EPMC3248566 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Identification and characterization of a novel non-structural protein of bluetongue virus.

Ratinier Maxime M Caporale Marco M Golder Matthew M Franzoni Giulia G Allan Kathryn K Nunes Sandro Filipe SF Armezzani Alessia A Bayoumy Amr A Rixon Frazer F Shaw Andrew A Palmarini Massimo M

PLoS pathogens 20111229 12

Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 o ...[more]

PMID: 22241985

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Project description:Many viruses use specific viral proteins to bind calcium ions (Ca2+) for stability or to modify host cell pathways, however, to date no Ca2+ binding protein has been reported in Bluetongue virus (BTV), the causative agent of Bluetongue disease in livestock. Here, using a comprehensive bioinformatics screening, we identified a putative EF-hand-like Ca2+ binding motif in the carboxyl terminal region of BTV non-structural phosphoprotein 2 (NS2). Subsequently, using a recombinant NS2, we demonstrated that NS2 binds Ca2+ efficiently and that Ca2+ binding was perturbed when the Asp and Glu residues in the motif were substituted by Alanine. Using Circular dichroism analysis, we found that Ca2+ binding by NS2 triggered a helix-to-coil secondary structure transition. Further, cryo-electron microscopy in presence of Ca2+, revealed that NS2 forms helical oligomers which, when aligned with the N-terminal domain crystal structure, suggest an N-terminal domain which wraps around the C-terminal domain in the oligomer. Further, an in vitro kinase assay demonstrated that Ca2+ enhanced the phosphorylation of NS2 significantly. Importantly, mutations introduced at the Ca2+ binding site in the viral genome by reverse genetics failed to allow recovery of viable virus and that the NS2 phosphorylation level and assembly of VIBs were reduced. Together, our data suggest that NS2 is a dedicated Ca2+ binding protein and that calcium sensing acts as a trigger for VIB assembly, which in turn facilitates virus replication and assembly.IMPORTANCE After entering the host cells viruses use cellular host factors to ensure a successful virus replication process. For replication in infected cells members of Reoviridae family form an inclusion body like structure known as viral inclusion bodies (VIB) or viral factories. Bluetongue virus (BTV) forms VIBs in infected cells through non-structural protein 2 (NS2), a phosphoprotein. An important regulatory factor critical for VIB formation is phosphorylation of NS2. In our study, we discovered a characteristic calcium binding EF hand like motif in NS2 and found that the calcium binding preferentially affects phosphorylation level of the NS2 and has a role in regulating VIB assembly.

Project description:Bluetongue virus (BTV) causes non-contagious haemorrhagic disease in ruminants and is transmitted by Culicoides spp. biting midges. BTV encodes four non-structural proteins of which NS3/NS3a is functional in virus release. NS3/NS3a is not essential for in vitro virus replication. However, deletion of NS3/NS3a leads to delayed virus release from mammalian cells and largely reduces virus release from insect cells. NS3/NS3a knockout BTV in sheep causes no viremia, but induces sterile immunity and is therefore proposed to be a Disabled Infectious Single Animal (DISA) vaccine candidate. In the absence of viremia, uptake of this vaccine strain by blood-feeding midges would be highly unlikely. Nevertheless, unintended replication of vaccine strains within vectors, and subsequent recombination or re-assortment resulting in virulent phenotypes and transmission is a safety concern of modified-live vaccines.The role of NS3/NS3a in replication and dissemination of BTV1, expressing VP2 of serotype 2 within colonized Culicoides sonorensis midges was investigated. Virus strains were generated using reverse genetics and their growth was examined in vitro. A laboratory colony of C. sonorensis, a known competent BTV vector, was fed or injected with BTV with or without expressing NS3/NS3a and replication in the midge was examined using RT PCR. Crossing of the midgut infection barrier was examined by separate testing of midge heads and bodies.Although the parental NS3/NS3a expressing strain was not able to replicate and disseminate within C. sonorensis after oral feeding, this virus was able to replicate efficiently when the midgut infection barrier was bypassed by intrathoracic injection, whereas the NS3/NS3a knockout mutant was unable to replicate. This demonstrates that NS3/NS3a is required for viral replication within Culicoides.The lack of viremia and the inability to replicate within the vector, clearly demonstrate the inability of NS3/NS3a knockout DISA vaccine strains to be transmitted by midges.

Dataset Information

Identification and characterization of a novel non-structural protein of bluetongue virus.

Publications

Identification and characterization of a novel non-structural protein of bluetongue virus.

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