RESUMO
Bluetongue virus (BTV) is the etiologic agent of a non-contagious arthropod-borne disease transmitted to wild and domestic ruminants. BTV induces a large panel of clinical manifestations ranging from asymptomatic infection to lethal hemorrhagic fever. Despite the fact that BTV has been studied extensively, we still have little understanding of the molecular determinants of BTV virulence. In our report, we have performed a comparative yeast two-hybrid (Y2H) screening approach to search direct cellular targets of the NS4 virulence factor encoded by two different serotypes of BTV: BTV8 and BTV27. This led to identifying Wilms' tumor 1-associated protein (WTAP) as a new interactor of the BTV-NS4. In contrast to BTV8, 1, 4 and 25, NS4 proteins from BTV27 and BTV30 are unable to interact with WTAP. This interaction with WTAP is carried by a peptide of 34 amino acids (NS422-55) within its putative coil-coiled structure. Most importantly, we showed that binding to WTAP is restored with a chimeric protein where BTV27-NS4 is substituted by BTV8-NS4 in the region encompassing residue 22 to 55. We also demonstrated that WTAP silencing reduces viral titers and the expression of viral proteins, suggesting that BTV-NS4 targets a cellular function of WTAP to increase its viral replication.
Assuntos
Vírus Bluetongue/metabolismo , Bluetongue/metabolismo , Bluetongue/virologia , Doenças dos Bovinos/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Fatores de Virulência/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Bluetongue/genética , Vírus Bluetongue/química , Vírus Bluetongue/genética , Vírus Bluetongue/patogenicidade , Bovinos , Doenças dos Bovinos/genética , Doenças dos Bovinos/virologia , Interações Hospedeiro-Patógeno , Ligação Proteica , Fatores de Processamento de RNA/genética , Alinhamento de Sequência , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Fatores de Virulência/genética , Replicação ViralRESUMO
Bluetongue virus (BTV), an arbovirus transmitted by Culicoides biting midges, is a major concern of wild and domestic ruminants. While BTV induces type I interferon (alpha/beta interferon [IFN-α/ß]) production in infected cells, several reports have described evasion strategies elaborated by this virus to dampen this intrinsic, innate response. In the present study, we suggest that BTV VP3 is a new viral antagonist of the IFN-ß synthesis. Indeed, using split luciferase and coprecipitation assays, we report an interaction between VP3 and both the mitochondrial adapter protein MAVS and the IRF3-kinase IKKε. Overall, this study describes a putative role for the BTV structural protein VP3 in the control of the antiviral response.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Vírus Bluetongue/metabolismo , Bluetongue/metabolismo , Proteína DEAD-box 58/metabolismo , Receptores Imunológicos/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Bluetongue/genética , Bluetongue/virologia , Vírus Bluetongue/genética , Proteína DEAD-box 58/genética , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Ligação Proteica , Receptores Imunológicos/genética , Transdução de Sinais , Proteínas do Core Viral/genéticaRESUMO
BACKGROUND The bluetongue virus (BTV) is the prototype virus in the genus Orbivirus within the family Reoviridae. Recent studies indicate that BTVs are capable of infecting and selectively lysing human hepatic carcinoma cells (Hep-3B) and prostate carcinoma cells (pc-3). This study was designed to evaluate the oncolytic potential of BTV in experimental models of human renal cancer in vitro and in vivo. MATERIAL AND METHODS Five human renal cancer cell lines, ACHN, CAKI-1, OS-RC-2, 786-O, and A498, were used in this study to analyze BTV replication. These cells were lysed by oncolysis compared to normal control. Xenograft models were used to assess the efficacy and toxicity of BTVs in vivo. Data were analyzed by one-way ANOVA or two-sided unpaired t tests. RESULTS The results showed HPTEC cells to be relatively resistant to cytotoxic effects of BTVs and exhibited normal growth rate even at high dose of BTVs. Nonetheless, the renal cancer cells showed a remarkably higher sensitivity to BTVs. Moreover, the ultramicroscopic subcellular changes were also detected in the renal cells. The viral particles were observed in all the RCC cell lines, but not in HPTEC cells. Intratumoral injections of BTVs significantly decreased the tumor volume as compared to animals that received no virus treatment. Infection with BTVs significantly increased the percentage of apoptotic renal cancer cells but not the HPTEC cells. Moreover, BTV triggered apoptosis in renal cancer cells via a mitochondria-mediated pathway. CONCLUSIONS This study for the first time demonstrated the oncolytic potential of BTV in experimental models of human renal cancer. BTV exhibits the potential to inhibit human renal cancer cell growth in vitro and in vivo.
Assuntos
Vírus Bluetongue/metabolismo , Neoplasias Renais/terapia , Neoplasias Renais/virologia , Animais , Apoptose/fisiologia , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/terapia , Carcinoma de Células Renais/virologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Humanos , Neoplasias Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Vírus Oncolíticos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Bluetongue virus (BTV) is an Orbivirus that has a profound economic impact due to direct loss of livestock as well as movement bans in an attempt to prevent the spread of the disease to susceptible areas. BTV VP7, along with VP3, forms the inner capsid core of the virus where it acts as the barrier between the outer layer and the inner core housing the genetic material. Purification of BTV VP7 has proven to be problematic and expensive mainly due to its insolubility is several expression systems. To overcome this, in this paper we present a protocol for the solubilisation of BTV VP7 from inclusion bodies expressed in E.coli, and subsequent purification using nickel affinity chromatography. The purified protein was then characterised using native PAGE, far ultraviolet circular dichroism (far-UV CD) and intrinsic fluorescence and found to have both secondary and tertiary structure even in the presence of 5â¯M urea. Both tertiary and secondary structure was further shown to be to be maintained at least to 42⯰C in 5â¯M urea.
Assuntos
Vírus Bluetongue/metabolismo , Corpos de Inclusão Viral/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas do Core Viral/metabolismo , Vírus Bluetongue/genética , Dicroísmo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Desdobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Solubilidade , Espectrometria de Fluorescência , Temperatura , Proteínas do Core Viral/química , Proteínas do Core Viral/genéticaRESUMO
VP7 is the major core protein of orbiviruses and is essential for virion assembly. African horse sickness virus (AHSV) VP7 self-assembles into highly insoluble crystalline particles - an attribute that may be related to the role of AHSV VP7 in virus assembly but also prevents crystallization. Given that this inherent insolubility is unique to AHSV VP7, we use amino acid sequence conservation analysis between AHSV VP7 and other orbiviruses to identify putative key residues that drive AHSV VP7 self-assembly. A homology model of the AHSV VP7 trimer was generated to analyze surface properties of the trimer and to identify surface residues as candidates for the AHSV VP7 trimer-trimer interactions that drive AHSV VP7 self-assembly. Nine regions were identified as candidate residues for future site-directed mutagenesis experiments that will likely result in a soluble AHSV VP7 protein. Additionally, we identified putative residues that function in the intermolecular interactions within the AHSV VP7 trimer as well as several epitopes. Given the many previous efforts of solubilizing AHSV VP7, we propose a useful strategy that will yield a soluble AHSV VP7 that can be used to study AHSV assembly and increase yield of recombinant vaccine preparations.
Assuntos
Vírus da Doença Equina Africana/química , Anticorpos Monoclonais/química , Antígenos Virais/química , Vírus Bluetongue/química , Proteínas do Core Viral/química , Vírus da Doença Equina Africana/imunologia , Vírus da Doença Equina Africana/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Sítios de Ligação , Vírus Bluetongue/imunologia , Vírus Bluetongue/metabolismo , Mapeamento de Epitopos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Alinhamento de Sequência , Homologia Estrutural de Proteína , Proteínas do Core Viral/imunologia , Proteínas do Core Viral/metabolismo , Montagem de VírusRESUMO
Bluetongue is an economically important viral disease of small ruminants. The present/current diagnostic kits and methods to diagnose BTV are laborious, time consuming and expensive. In the present study, we have attempted to develop a novel approach to detect BTV antibodies in sera that in future can be harnessed for developing a pen side diagnostic test. Briefly, we identified the immunodominant regions of the VP7 protein of BTV and synthesized them in the multiple antigenic peptide (MAP) format with cysteine at C-terminal of the lysine mosaic, which elicited highly ordered conformation as well as ELISA reactivity. Finally, we coated the MAP peptides on the gold nanoparticles that can be used to detect BTV specific antibodies in the sera using a spot test.
Assuntos
Antígenos Virais/metabolismo , Vírus Bluetongue/metabolismo , Bluetongue/diagnóstico , Ouro/química , Compostos de Sulfidrila/química , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/química , Bluetongue/virologia , Epitopos , Regulação Viral da Expressão Gênica , Nanopartículas Metálicas , Ruminantes , Proteínas ViraisRESUMO
Bluetongue virus (BTV), an economically important orbivirus of the Reoviridae family, is a non-enveloped, dsRNA virus that causes a haemorrhagic disease mainly in sheep, but little is known of the cellular immunity elicited against BTV. We observed that vaccination of interferon type I-deficient mice (IFNAR((-/-))), or inoculation of the wild type C57BL/6 strain with BTV-8, induced a strong T cell response. Therefore, we proceeded to identify some of the T cell epitopes targeted by the immune system. We selected, using H-2(b)-binding predictive algorithms, 3 major histocompatibility complex (MHC)-class II-binding peptides and 7 MHC-class I binding peptides from the BTV-8 core protein VP7, as potential T cell epitopes. Peptide binding assays confirmed that all 7 MHC-class I predicted peptides bound MHC-class I molecules. Three MHC-class I and 2 MHC-class II binding peptide consistently elicited peptide-specific IFN-γ production (as measured by ELISPOT assays) in splenocytes from C57BL/6 BTV-8-inoculated mice and IFNAR((-/-))-vaccinated mice. The functionality of these T cells was confirmed by proliferation and cytotoxicity assays. Flow cytometry analysis demonstrated that CD8(+) T cells responded to MHC-class I binding peptides and CD4(+) T cells to MHC-class II binding peptides. Importantly, these 5 epitopes were also able to induced IFN-γ production in sheep inoculated with BTV-8. Taken together, these data demonstrate the activation of BTV-specific T cells during infection and vaccination. The characterisation of these novel T cell epitopes may also provide an opportunity to develop DIVA-compliant vaccination approach to BTV encompassing a broad-spectrum of serotypes.
Assuntos
Vírus Bluetongue/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Proteínas do Core Viral/imunologia , Algoritmos , Animais , Vírus Bluetongue/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Epitopos de Linfócito T/metabolismo , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Imunidade Celular/imunologia , Interferon gama/biossíntese , Interferon gama/imunologia , Interferon gama/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica/imunologia , Ovinos/imunologia , Ovinos/metabolismo , Proteínas do Core Viral/metabolismoRESUMO
In the present study, group-specific antigen VP7 of bluetongue virus (BTV) serotype 21 isolated from cattle in Tochigi prefecture in Japan in 1994 was characterized by sequencing and expression. Gene was amplified from cDNA synthesized on viral dsRNA using reverse-transcriptase-PCR. Nucleotide sequence of this isolate showed high similarity with other published BTV VP7 sequences. Full-length and C-terminal truncated forms of VP7 were expressed in insect cells by a baculovirus gene expression system under control of the viral polyhedrin promoter. Expression of full-length recombinant VP7 was confirmed by immunoprecipitation with VP7 specific monoclonal antibody (8A3B.6, ATCC). Recombinant proteins expressed with or without 6x His-tag showed good expression levels in TN5 cells and reacted well with the monoclonal antibody in the indirect ELISA. However C-terminal truncated VP7 with His-tag failed to react with this monoclonal antibody, while poor antigenicity was evident when it was reacted with infected bovine serum. Reduced antigenicity of the latter suggested that C-terminal truncation affects 8A3B.6 epitope construction probably via inhibition of VP7 trimer structure formation.
Assuntos
Vírus Bluetongue/metabolismo , Bluetongue/virologia , Doenças dos Bovinos/virologia , Regulação Viral da Expressão Gênica/fisiologia , Proteínas do Core Viral/metabolismo , Animais , Antígenos Virais/genética , Antígenos Virais/metabolismo , Bluetongue/epidemiologia , Vírus Bluetongue/classificação , Vírus Bluetongue/genética , Vírus Bluetongue/imunologia , Bovinos , Doenças dos Bovinos/epidemiologia , Linhagem Celular , Cricetinae , Japão/epidemiologia , Sorotipagem , Proteínas do Core Viral/genéticaRESUMO
Bluetongue virus (BTV) is transmitted by blood-feeding insects (Culicoides sp.) and causes hemorrhagic diseases in livestock. BTV is a nonenveloped, double-stranded RNA (dsRNA) virus with two capsids: a well-studied, stable core enclosing the dsRNA genome and a highly unstable, poorly studied coat responsible for host cell attachment and entry. Here, based on cryo-electron microscopy (cryoEM), we report a 7-A resolution structure of the infectious BTV virion, including the coat proteins. We show that unlike other dsRNA viruses, the VP2 attachment trimer has a triskelion shape composed of three tip domains branching from a central hub domain. We identify three putative sialic acid-binding pockets in the hub and present supporting biochemical data indicating sugar moiety binding is important for BTV infection. Despite being a nonenveloped virus, the putative VP5 membrane penetration trimer, located slightly inward of the VP2 attachment trimer, has a central coiled-coil alpha-helical bundle, similar to the fusion proteins of many enveloped viruses (e.g., HIV, herpesviruses, vesicular stomatitis virus, and influenza virus). Moreover, mapping of the amino acid sequence of VP5 to the secondary structural elements identified by cryoEM locates 15 amphipathic alpha-helical regions on the external surface of each VP5 trimer. The cryoEM density map also reveals few, weak interactions between the VP5 trimer and both the outer-coat VP2 trimer and the underlying core VP7 trimer, suggesting that the surface of VP5 could unfurl like an umbrella during penetration and shedding of the coat to release the transcriptionally active core particle.
Assuntos
Vírus Bluetongue/química , Proteínas do Capsídeo/química , Ácido N-Acetilneuramínico/química , Multimerização Proteica , Proteínas Virais de Fusão/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Vírus Bluetongue/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/ultraestrutura , Linhagem Celular , Cricetinae , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ácido N-Acetilneuramínico/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Virais de Fusão/metabolismo , Proteínas Virais de Fusão/ultraestrutura , Replicação ViralRESUMO
Bluetongue virus (BTV) non-structural protein 2 (NS2) belongs to a class of highly conserved proteins found in members of the orbivirus genus of the reoviridae. NS2 forms large multimeric complexes, localizes to cytoplasmic inclusion bodies in the infected cells and binds non-sequence specifically single-stranded RNA (ssRNA). Due to its ability to bind ssRNA, it has been suggested that the protein is involved in the selection and condensation of the BTV ssRNA segments prior to genome encapsidation. We have previously determined the crystal structure of the 177 amino acid N-terminal domain, sufficient for ssRNA binding ability of NS2, to 2.4A resolution. The C-terminal domain, as determined at low resolution using small-angle X-ray scattering, is an elongated dimer. This domain expressed in insect cells is phosphorylated at S249 and S259. Electron microscopy of the full-length protein shows a variety of species with the largest having a ring-like appearance. Based on the electron micrographs, the crystal structure of the N-terminal domain and the structure of the C-terminal domain reported here, we propose a model for a decamer of the full-length protein. This decamer changes conformation upon binding of a non-hydrolysable ATP analogue.
Assuntos
Vírus Bluetongue/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Trifosfato de Adenosina/química , Animais , Clonagem Molecular , Reagentes de Ligações Cruzadas/farmacologia , Dimerização , Insetos , Microscopia Eletrônica , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , RNA/química , Espalhamento de Radiação , Raios XRESUMO
The development of recombinant capripoxviruses for protective immunization of ruminants against bluetongue virus (BTV) infection is described. Sheep (n=11) and goats (n=4) were immunized with BTV recombinant capripoxviruses (BTV-Cpox) individually expressing four different genes encoding two capsid proteins (VP2 and VP7) and two non-structural proteins (NS1, NS3) of BTV serotype 2 (BTV-2). Seroconversion was observed against NS3, VP7 and VP2 in both species and a lymphoproliferation specific to BTV antigens was also demonstrated in goats. Finally, partial protection of sheep challenged 3 weeks after BTV-Cpox administration with a virulent strain of BTV-2, was observed.
Assuntos
Vírus Bluetongue/imunologia , Vírus Bluetongue/metabolismo , Capripoxvirus/imunologia , Capripoxvirus/metabolismo , Expressão Gênica , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Animais , Vírus Bluetongue/genética , Vírus Bluetongue/patogenicidade , Capripoxvirus/genética , Células Cultivadas , Chlorocebus aethiops , Vetores Genéticos/genética , Cabras , Ovinos , Taxa de Sobrevida , Proteínas Virais/genética , Vacinas Virais/efeitos adversos , Vacinas Virais/imunologiaRESUMO
Minor structural protein VP6 is the putative helicase of African horse sickness virus (AHSV), of the genus Orbivirus in the Reoviridae family. We investigated how the protein interacts with double-stranded (ds) RNA and other nucleic acids. Binding was assayed using an electrophoretic migration retardation assay and a nucleic acid overlay protein blot assay. VP6 bound double and single stranded RNA and DNA in a NaCl concentration sensitive reaction. Of six truncated VP6 peptides investigated, two partially overlapping peptides were found to bind dsRNA at pH 7.0, while other peptides with the same overlap did not. The distinction between the peptides appeared to be the pI which ranged from more than 8.0 to just above 6.0. Changing the pH of the binding buffer modified the binding activity. Regardless of assay conditions, only peptides with a specific region of amino acids in common, showed evidence of binding activity. No sequence homology was identified with other binding domains, however, the presence of charged amino acids are assumed to be important for binding activity. The results suggested dsRNA binding in the blot assay was strongly affected by the net charge on the peptide.
Assuntos
Vírus da Doença Equina Africana/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Proteínas Estruturais Virais/metabolismo , Vírus da Doença Equina Africana/genética , Vírus da Doença Equina Africana/patogenicidade , Animais , Sítios de Ligação/genética , Vírus Bluetongue/genética , Vírus Bluetongue/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Genes Virais , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , RNA de Cadeia Dupla/genética , RNA Viral/genética , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genéticaRESUMO
The VP6 protein of bluetongue virus possesses a number of activities, including nucleoside triphosphatase, RNA binding, and helicase activity (N. Stauber, J. Martinez-Costas, G. Sutton, K. Monastyrskaya, and P. Roy, J. Virol. 71:7220-7226, 1997). Although the enzymatic functions of the protein have been documented, a detailed structure and function study has not been completed and the oligomeric form of the protein in solution has not been described. In this study, we have characterized VP6 activity by creating site-directed mutations in the putative functional helicase domains. Mutant proteins were expressed at high levels in an insect cell by using recombinant baculoviruses purified and analyzed for ATP binding, ATP hydrolysis, and RNA unwinding activities. UV cross-linking experiments indicated that the lysine residue in the conserved motif AXXGXGK(110)V is directly involved in ATP binding, whereas mutant R(205)Q in the arginine-rich motif ER(205)XGRXXR bound ATP at a level comparable to that of the wild-type protein. The RNA binding activity was drastically altered in the R(205)Q mutant and was also affected in the K(110)N mutant. Helicase activity was altered in both mutants. The mutation E(157)N in the DEXX sequence, presumed to act as a Walker B motif, showed an intermediate activity, implying that this motif does not play a crucial role in VP6 function. Purified protein demonstrated stable oligomers with a ring-like morphology in the presence of nucleic acids similar to those shown by other helicases. Gel filtration chromatography, native gel electrophoresis, and glycerol gradient analysis clearly indicated multiple oligomeric forms of VP6.
Assuntos
Vírus Bluetongue/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , RNA Helicases/química , RNA Helicases/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas do Capsídeo/genética , Células Cultivadas , Dados de Sequência Molecular , Mutação , Nucleopoliedrovírus/genética , RNA Helicases/genética , RNA Viral/metabolismo , Proteínas Recombinantes/genética , Spodoptera , Relação Estrutura-AtividadeRESUMO
Bluetongue virus, an arbovirus of the Orbivirus genus, infects and replicates in both insect and mammalian cells. However, the cytopathic effect (cpe) on each host is very different. Mammalian cells show substantial cpe, most likely a result of the mechanism of virus release, whereas insect cells show little cpe and appear to release virus without cell lysis. Expression analysis of each infected cell type shows one protein, the nonstructural (NS) protein NS3, to be differentially expressed in the different cell types, suggesting it may act in the virus egress pathway. The molecular basis of such an interaction, however, has never been clear. Here, by using yeast two-hybrid analysis, we show that NS3 interacts with a cellular protein p11 (calpactin light chain), part of the annexin II complex that is involved in exocytosis. We map the NS3 region of interaction with p11 to a 13-residue peptide found at the N terminus of the protein and show it effectively competes with p36 (annexin II heavy chain) for p11 ligand binding. Further, we show that the C-terminal domain of NS3 interacts with VP2, the outermost protein of the fully assembled virus particle, suggesting that NS3 forms a bridging molecule that draws assembled virus into contact with the cellular export machinery. Our data describe the first host protein involvement in orbivirus egress and provide new insights into understanding arbovirus interactions with their hosts.
Assuntos
Anexinas/química , Vírus Bluetongue/metabolismo , Proteínas não Estruturais Virais/química , Motivos de Aminoácidos , Animais , Anexina A2/química , Anexinas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , DNA Complementar/metabolismo , Exocitose , Biblioteca Gênica , Células HeLa , Humanos , Insetos , Mutagênese Sítio-Dirigida , Peptídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais/metabolismoRESUMO
Double-stranded RNA (dsRNA) viruses conceal their genome from the host to avoid triggering unfavorable cellular responses. The crystal structure of the core of one such virus, bluetongue virus, reveals an outer surface festooned with dsRNA. This may represent a deliberate strategy to sequester dsRNA released from damaged particles to prevent host cell shutoff.
Assuntos
Vírus Bluetongue/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Proteínas do Core Viral/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , RNA de Cadeia Dupla/química , RNA Viral/química , Proteínas do Core Viral/química , Difração de Raios XRESUMO
Bluetongue virus (BTV) is an arthropod-borne virus transmitted by Culicoides species to vertebrate hosts. The double-capsid virion is infectious for Culicoides vector and mammalian cells, while the inner core is infectious for only Culicoides-derived cells. The recently determined crystal structure of the BTV core has revealed an accessible RGD motif between amino acids 168 to 170 of the outer core protein VP7, whose structure and position would be consistent with a role in cell entry. To delineate the biological role of the RGD sequence within VP7, we have introduced point mutations in the RGD tripeptide and generated three recombinant baculoviruses, each expressing a mutant derivative of VP7 (VP7-AGD, VP7-ADL, and VP7-AGQ). Each expressed mutant protein was purified, and the oligomeric nature and secondary structure of each was compared with those of the wild-type (wt) VP7 molecule. Each mutant VP7 protein was used to generate empty core-like particles (CLPs) and were shown to be biochemically and morphologically identical to those of wt CLPs. However, when mutant CLPs were used in an in vitro cell binding assay, each showed reduced binding to Culicoides cells compared to wt CLPs. Twelve monoclonal antibodies (MAbs) was generated using purified VP7 or CLPs as a source of antigen and were utilized for epitope mapping with available chimeric VP7 molecules and the RGD mutants. Several MAbs bound to the RGD motif on the core, as shown by immunogold labeling and cryoelectron microscopy. RGD-specific MAb H1.5, but not those directed to other regions of the core, inhibited the binding activity of CLPs to the Culicoides cell surface. Together, these data indicate that the RGD motif present on BTV VP7 is responsible for Culicoides cell binding activity.
Assuntos
Vírus Bluetongue/metabolismo , Ceratopogonidae/metabolismo , Ceratopogonidae/virologia , Oligopeptídeos/metabolismo , Proteínas do Core Viral/química , Proteínas do Core Viral/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , Vírus Bluetongue/genética , Vírus Bluetongue/imunologia , Vírus Bluetongue/ultraestrutura , Linhagem Celular , Ceratopogonidae/citologia , Microscopia Crioeletrônica , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mutação , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologia , Montagem de VírusRESUMO
Bluetongue virus (BTV) infection of ruminants is endemic throughout tropical and subtropical regions of the world. The S7 gene segments of prototype Chinese strains of BTV serotypes 1, 2, 3, 4, 12, 15, and 16 were sequenced and compared to the same genes of prototype strains of BTV from the US, Australia, and South Africa. The S7 genes and predicted VP7 proteins of the Chinese viruses were relatively conserved, with the notable exception of serotype 15. Furthermore, phylogenetic analysis of the S7 genes did not predict geographic origin of the various strains of BTV.
Assuntos
Vírus Bluetongue/classificação , Bluetongue/virologia , Filogenia , Proteínas do Core Viral/genética , Animais , Austrália , Vírus Bluetongue/genética , Vírus Bluetongue/metabolismo , Bovinos , China , Dados de Sequência Molecular , Ovinos , África do Sul , Estados UnidosRESUMO
The core of bluetongue virus (BTV) is a multienzyme complex composed of two major proteins (VP7 and VP3) and three minor proteins (VP1, VP4, and VP6) in addition to the viral genome. The core is transcriptionally active and produces capped mRNA from which all BTV proteins are translated, but the relative role of each core component in the overall reaction process remains unclear. Previously we showed that the 76-kDa VP4 protein possesses guanylyltransferase activity, a necessary part of the RNA capping reaction. Here, through the use of highly purified (>95%) VP4 and synthetic core-like particles containing VP4, we have investigated the extent to which this protein is also responsible for other activities associated with cap formation. We show that VP4 catalyzes the conversion of unmethylated GpppG or in vitro-produced uncapped BTV RNA transcripts to m7GpppGm in the presence of S-adenosyl-L-methionine. Analysis of the methylated products of the reaction by HPLC identified both methyltransferase type 1 and type 2 activities associated with VP4, demonstrating that the complete BTV capping reaction is associated with this one protein.
Assuntos
Vírus Bluetongue/metabolismo , Proteínas do Capsídeo , Capsídeo/genética , Capsídeo/metabolismo , RNA Mensageiro/metabolismo , Metilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The bluetongue virus (BTV) minor protein VP4, with molecular mass of 76 kDa, is one of the seven structural proteins and is located within the inner capsid of the virion. The protein has a putative leucine zipper near the carboxy terminus of the protein. In this study, we have investigated the functional activity of this putative leucine zipper by a number of approaches. The putative leucine zipper region (amino acids [aa] 523 to 551) was expressed initially as a fusion protein by using the pMAL vector of Escherichia coli, which expresses a maltose binding monomeric protein. The expressed fusion protein was purified by affinity chromatography, and its size was determined by gel filtration chromatography. Proteins of two sizes, 51 and 110 kDa, were recovered, one equivalent to the monomeric form and the other equivalent to the dimeric form of the fusion protein. To prove that the VP4-derived sequence was responsible for dimerization of this protein, a mutated fusion protein was created in which a VP4 leucine residue (at aa 537) within the zipper was replaced by a proline residue. Analyses of the mutated protein demonstrated that the single mutation indeed prevented dimerisation of the protein. The dimeric nature of VP4 was further confirmed by using purified full-length BTV-10 VP4 recovered from recombinant baculovirus-expressing BTV-10 VP4-infected insect cells. Using chemical cross-linking and gel filtration chromatography, we documented that the native VP4 indeed exists as a dimer in solution. Subsequently, Leu537 was replaced by either a proline or an alanine residue and the full-length mutated VP4 was expressed in the baculovirus system. By sucrose density gradient centrifugation and gel filtration chromatography, these mutant forms of VP4 were shown to lack the ability to form dimers. The biological significance of the dimeric forms of VP4 was examined by using a functional assay system, in which the encapsidation activity of VP4 into core-like particles (CLPs) was studied (H. LeBlois, T. French, P. P. C. Mertens, J. N. Burroughs, and P. Roy, Virology 189:757-761, 1992). We demonstrated conclusively that dimerization of VP4 was essential for encapsidation by CLPs.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Vírus Bluetongue/fisiologia , Proteínas do Capsídeo , Capsídeo/metabolismo , Proteínas de Escherichia coli , Zíper de Leucina/fisiologia , Leucina/metabolismo , Proteínas de Transporte de Monossacarídeos , Montagem de Vírus , Sequência de Aminoácidos , Animais , Sítios de Ligação , Vírus Bluetongue/genética , Vírus Bluetongue/metabolismo , Capsídeo/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Dimerização , Leucina/genética , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Prolina/genética , Prolina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Spodoptera , Proteínas do Core Viral/metabolismo , Vírion/metabolismoRESUMO
The importance of VP7 of bluetongue virus (BTV) in the binding of BTV to membrane proteins of the BTV vector Culicoides variipennis was investigated. Core BTV particles, prepared from whole viruses, lacked outer proteins VP2 and VP5 and had VP7 exposed. More core particles and whole viruses bound to membrane preparations of adults of C. variipennis and KC cells, which were cultured from this vector insect, than to membrane preparations of Manduca sexta larvae. More core particles than whole viruses bound to membrane preparations of adults of C. variipennis and KC cells. Polyclonal anti-idiotypic antibodies (anti-Id), which were made against an antigen-combining region of an anti-BTV-10 VP7 antibody and functionally mimicked VP7, bound more to the membrane preparations of adults of C. variipennis and KC cells, and less to cytosol preparations. In Western overalay analysis, the Culicoides plasma membrane preparation reduced binding of an anti-VP7 monoclonal antibody to VP7. Whole and core BTV particles and the anti-Id bound to a membrane protein with a molecular mass of 23 kDa that was present predominantly in membrane preparations of adults of C. variipennis and KC cells. This protein was present in much lower concentrations in membrane preparations of C6/36 and DM-2 insect cells.