RESUMO
Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that is classified as the type species of the genus Ephemerovirus. In addition to the five canonical rhabdovirus structural proteins (N, P, M, G, and L), the large and complex BEFV genome contains several open reading frames (ORFs) between the G and L genes (α1, α2/α3, ß, and γ) encoding proteins of unknown function. We show that the 10.5-kDa BEFV α1 protein is expressed in infected cells and, consistent with previous predictions based on its structure, has the properties of a viroporin. Expression of a BEFV α1-maltose binding protein (MBP) fusion protein in Escherichia coli was observed to inhibit cell growth and increase membrane permeability to hygromycin B. Increased membrane permeability was also observed in BEFV-infected mammalian cells (but not cells infected with an α1-deficient BEFV strain) and in cells expressing a BEFV α1-green fluorescent protein (GFP) fusion protein, which was shown by confocal microscopy to localize to the Golgi complex. Furthermore, the predicted C-terminal cytoplasmic domain of α1, which contains a strong nuclear localization signal (NLS), was translocated to the nucleus when expressed independently, and in an affinity chromatography assay employing a GFP trap, the full-length α1 was observed to interact specifically with importin ß1 and importin 7 but not with importin α3. These data suggest that, in addition to its function as a viroporin, BEFV α1 may modulate components of nuclear trafficking pathways, but the specific role thereof remains unclear. Although rhabdovirus accessory genes occur commonly among arthropod-borne rhabdoviruses, little is known of their functions. Here, we demonstrate that the BEFV α1 ORF encodes a protein which has the structural and functional characteristics of a viroporin. We show that α1 localizes in the Golgi complex and increases cellular permeability. We also show that BEFV α1 binds importin ß1 and importin 7, suggesting that it may have a yet unknown role in modulating nuclear trafficking. This is the first functional analysis of an ephemerovirus accessory protein and of a rhabdovirus viroporin.
Assuntos
Vírus da Febre Efêmera Bovina/metabolismo , Febre Efêmera/metabolismo , Carioferinas/metabolismo , Proteínas Virais/metabolismo , beta Carioferinas/metabolismo , Motivos de Aminoácidos , Animais , Bovinos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Febre Efêmera/genética , Febre Efêmera/virologia , Vírus da Febre Efêmera Bovina/química , Vírus da Febre Efêmera Bovina/genética , Carioferinas/genética , Sinais de Localização Nuclear , Ligação Proteica , Transporte Proteico , Proteínas Virais/química , Proteínas Virais/genética , beta Carioferinas/genéticaRESUMO
Recombinant baculoviruses expressing the BEFV envelope glycoprotein G and non-structural glycoprotein G(NS) were constructed. The G protein expressed in insect cells was located on the cell surface and induced spontaneous cell fusion at mildly acidic pH. The expressed G protein reacted with MAbs to continuous and conformational neutralization sites (G1, G2, G3b and G4), but not to conformational site G3a. The expressed G(NS) protein was also located on the cell surface but did not exhibit fusogenic activity. The G(NS) protein reacted with polyclonal antiserum produced from vaccinia-virus-expressed recombinant G(NS) but did not react with G protein antibodies. A His(6)-tagged, soluble form of the G protein was expressed and purified by Ni(2+)-NTA chromatography. The purified G protein reacted with BEFV-neutralizing MAbs to all continuous and conformational antigenic sites. The highly protective characteristics of the native BEFV G protein suggest that the secreted, baculovirus-expressed product may be a useful vaccine antigen.
Assuntos
Antígenos Virais/imunologia , Baculoviridae/metabolismo , Vírus da Febre Efêmera Bovina/imunologia , Febre Efêmera/virologia , Glicoproteínas/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas não Estruturais Virais/imunologia , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/isolamento & purificação , Baculoviridae/genética , Bovinos , Linhagem Celular , Febre Efêmera/imunologia , Vírus da Febre Efêmera Bovina/química , Vírus da Febre Efêmera Bovina/genética , Expressão Gênica , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/isolamento & purificação , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Spodoptera , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificação , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificaçãoRESUMO
Bovine ephemeral fever is a viral disease of cattle and buffaloes besides subclinical involvement of a variety of ruminant species. The subtropical and temperate regions of Africa, Asia and Australia have experienced the major epidemic of the bovine ephemeral fever but the occurrence in the tropics can not be overlooked. Although the substantial role played by the vectors viz., mosquitoes and culicoides in bovine ephemeral fever perpetuation and dissemination, other vector involvement if any should be extensively studied. The clinical severity of the disease is not apparent and the mortality is low. However, high morbidity, enormous economic losses in terms of significant reduction in production, disruption of national and international trade and finally a variety of complications resulting from the disease have drawn appreciable attention from the researchers around the world to resolve the unsolved questions in this area. In this review, detailed informations of all the aspects of the disease has been provided in a simple, lucid and easily understandable manner.
