RESUMO
The bulk of the major core protein VP7 in African horse sickness virus (AHSV) self-assembles into flat, hexagonal crystalline particles in a process appearing unrelated to viral replication. Why this unique characteristic of AHSV VP7 is genetically conserved, and whether VP7 aggregation and particle formation have an effect on cellular biology or the viral life cycle, is unknown. Here we investigated how different small peptide and enhanced green fluorescent protein (eGFP) insertions into the VP7 top domain affected VP7 localization, aggregation, and particle formation. This was done using a dual laser scanning confocal and transmission electron microscopy approach in conjunction with analyses of the solubility, aggregation, and fluorescence profiles of the proteins. VP7 top domain modifications did not prevent trimerization, or intracellular trafficking, to one or two discrete sites in the cell. However, modifications that resulted in a misfolded and insoluble VP7-eGFP component blocked trafficking, and precluded protein accumulation at a single cellular site, perhaps by interfering with normal trimer-trimer interactions. Furthermore, the modifications disrupted the stable layering of the trimers into characteristic AHSV VP7 crystalline particles. It was concluded that VP7 trafficking is driven by a balance between VP7 solubility, trimer forming ability, and trimer-trimer interactions.
Assuntos
Vírus da Doença Equina Africana/metabolismo , Microscopia Confocal/métodos , Microscopia Eletrônica de Transmissão/métodos , Proteínas do Core Viral/fisiologia , Proteínas do Core Viral/ultraestrutura , Vírus da Doença Equina Africana/genética , Animais , Baculoviridae/genética , Regulação Viral da Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde , Estágios do Ciclo de Vida , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/ultraestrutura , Células Sf9 , Proteínas do Core Viral/química , Proteínas do Core Viral/genética , Proteínas Virais de Fusão/fisiologia , Proteínas Virais de Fusão/ultraestrutura , Replicação ViralRESUMO
A unique characteristic of the African horse sickness virus (AHSV) major core protein VP7 is that it is highly insoluble, and spontaneously forms crystalline particles in AHSV-infected cells and when expressed in vitro. The aggregation of AHSV VP7 into these crystals presents many problems in AHSV vaccine development, and it is unclear whether VP7 aggregation affects AHSV assembly or contributes to AHSV pathogenesis. Here, we set out to abolish VP7 self-assembly by targeting candidate amino acid regions on the surface of the VP7 trimer via site-directed mutagenesis. It was found that the substitution of seven amino acids resulted in the complete disruption of AHSV VP7 self-assembly, which abolished the formation of VP7 crystalline particles and converted VP7 to a fully soluble protein still capable of interacting with VP3 to form core-like particles. This work provides further insight into the formation of AHSV VP7 crystalline particles and the successful development of AHSV vaccines. It also paves the way for future research by drawing comparisons with similar viral phenomena observed in human virology.
Assuntos
Vírus da Doença Equina Africana , Proteínas do Core Viral , Vírus da Doença Equina Africana/genética , Animais , Antígenos Virais , Proteínas do Core Viral/metabolismoRESUMO
The VP5 outer capsid protein of African horse sickness virus (AHSV) is cytotoxic when expressed in Spodoptera frugiperda (Sf-9) cells. Secondary structure analysis of the VP5 amino acid sequence of AHSV-9 identified two N-terminal amphipathic α-helices within the first 43 amino acids. Baculovirus expression of N- and C-terminal truncated VP5 proteins in Sf-9 cells indicated that the N-terminal 43 amino acids correlated with low levels of protein expression and with increased membrane permeabilization and cytotoxicity. Exogenous addition of chemically synthesized VP5 peptides indicated that both N-terminal amphipathic α-helices are required for membrane permeabilization of Sf-9 cells. These findings suggest that AHSV VP5 is a membrane-destabilizing protein.
Assuntos
Vírus da Doença Equina Africana/patogenicidade , Proteínas do Capsídeo/metabolismo , Permeabilidade da Membrana Celular , Fatores de Virulência/metabolismo , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Linhagem Celular , Estrutura Secundária de Proteína , Spodoptera/virologia , Fatores de Virulência/química , Fatores de Virulência/genéticaRESUMO
The role of African horsesickness virus (AHSV) nonstructural membrane protein NS3 in determining the effects of AHSV infection on Vero cells was examined. NS3 protein sequences are highly variable and cluster into three phylogenetic groups, alpha, beta, and gamma. Three AHSV strains, with NS3 from alpha, beta, or gamma, were shown to have quantitatively different phenotypes in Vero cells. Reassortants between these strains, in which the S10 genome segment encoding NS3 was exchanged alone or with other segments, were generated and compared to parental strains. Exchange of the NS3 gene resulted in changes in virus release, membrane permeability and total virus yield, indicating an important role for NS3 in these viral properties. Differences in the cytopathicity and the effect on cell viability between the parental strains could not be associated with NS3 alone, and it is likely that a number of viral and host factors play a role.
Assuntos
Vírus da Doença Equina Africana/fisiologia , Doença Equina Africana/virologia , Permeabilidade da Membrana Celular , Proteínas não Estruturais Virais/fisiologia , Doença Equina Africana/metabolismo , Vírus da Doença Equina Africana/genética , Animais , Chlorocebus aethiops , Genoma Viral/genética , Vírus Reordenados , Células Vero , Proteínas não Estruturais Virais/genética , Replicação ViralRESUMO
In an effort to simplify and expand the utility of African horse sickness virus (AHSV) reverse genetics, different plasmid-based reverse genetics systems were developed. Plasmids containing cDNAs corresponding to each of the full-length double-stranded RNA genome segments of AHSV-4 under control of a T7 RNA polymerase promoter were co-transfected in cells expressing T7 RNA polymerase, and infectious AHSV-4 was recovered. This reverse genetics system was improved by reducing the required plasmids from 10 to five and resulted in enhanced virus recovery. Subsequently, a T7 RNA polymerase expression cassette was incorporated into one of the AHSV-4 rescue plasmids. This modified 5-plasmid set enabled virus recovery in BSR or L929 cells, thus offering the possibility to generate AHSV-4 in any cell line. Moreover, mutant and cross-serotype reassortant viruses were recovered. These plasmid DNA-based reverse genetics systems thus offer new possibilities for investigating AHSV biology and development of designer AHSV vaccine strains.
Assuntos
Vírus da Doença Equina Africana/genética , Genoma Viral , Plasmídeos/genética , Genética Reversa , Animais , Linhagem Celular , Cricetinae , DNA Complementar , Expressão Gênica , Ordem dos Genes , RNA Viral , TransfecçãoRESUMO
As a means to develop African horse sickness (AHS) vaccines that are safe and DIVA compliant, we investigated the synthesis of empty African horse sickness virus (AHSV) particles. The emphasis of this study was on the assembly of the major viral core (VP3 and VP7) and outer capsid proteins (VP2 and VP5) into architecturally complex, heteromultimeric nanosized particles. The production of fully assembled core-like particles (CLPs) was accomplished in vivo by baculovirus-mediated co-synthesis of VP3 and VP7. The two different outer capsid proteins were capable of associating independently of each other with preformed cores to yield partial virus-like particles (VLPs). Complete VLPs were synthesized, albeit with a low yield. Crystalline formation of AHSV VP7 trimers is thought to impede high-level CLP production. Consequently, we engineered and co-synthesized VP3 with a more hydrophilic mutant VP7, resulting in an increase in the turnover of CLPs.
Assuntos
Vírus da Doença Equina Africana/genética , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Vacinas de Partículas Semelhantes a Vírus/metabolismo , Virossomos/isolamento & purificação , Virossomos/metabolismo , Baculoviridae , Vetores Genéticos , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vacinas de Partículas Semelhantes a Vírus/genética , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Virossomos/genéticaRESUMO
Bluetongue virus (BTV), the type species of the genus Orbivirus within the family Reoviridae, has a genome consisting of 10 linear double-stranded RNA genome segments. Current reverse genetics approaches for engineering the BTV genome rely upon in vitro synthesis of capped RNA transcripts from cloned cDNA corresponding to viral genome segments. In an effort to expand the utility of BTV reverse genetics, we constructed a reverse genetics vector containing a T7 RNA polymerase promoter, hepatitis delta ribozyme sequence and T7 RNA polymerase terminator sequence. Viable virus was recovered following transfection of mammalian cells, expressing T7 RNA polymerase, with 10 plasmid constructs representing the cloned BTV-1 genome. Furthermore, the plasmid-based reverse genetics system was used successfully to isolate viable cross-serotype reassortant viruses and a mutant virus containing a defined mutation in the replicating viral genome. The new reverse genetics platform established here for BTV is likely applicable to other orbiviruses.
Assuntos
Vírus Bluetongue/genética , Plasmídeos/genética , Genética Reversa/métodos , Animais , Linhagem Celular , Cricetinae , Genoma Viral , Replicação ViralRESUMO
African horse sickness virus (AHSV) VP7 is the major core protein of the virion. Apart from its role in virus assembly, VP7 forms crystalline-like particles during infection and when expressed in insect cells. The aim of this study was to investigate the process of VP7 crystalline-like particle formation. The intracellular distribution of VP7 was characterized in different systems and the association of VP7 with virus factories during AHSV infection was investigated. It was shown that the majority of VP7 is sequestered into these particles, and is therefore not available for new virion assembly. This is likely to have a negative impact on virus assembly and yield. By using specific markers and inhibitors of host trafficking pathways, VP7 localization was shown to be independent of host trafficking mechanisms and evaded host defenses against aggregation. Studying the process of VP7 crystalline-like particle formation will help us further understand AHSV replication and assembly.
Assuntos
Vírus da Doença Equina Africana/fisiologia , Antígenos Virais/metabolismo , Proteínas do Core Viral/metabolismo , Montagem de Vírus , Replicação Viral , Animais , Transporte Proteico , Células Sf9 , SpodopteraRESUMO
Infection of mammalian cell cultures with African horse sickness virus (AHSV) is known to result in dramatic cytopathic effects (CPE), but no CPE is observed in infected insect cell cultures despite productive virus replication. The basis for this phenomenon has not yet been investigated, but is suggestive of apoptosis being induced following virus infection of the mammalian cells. To investigate whether AHSV can induce apoptosis in infected mammalian cells, Culicoides variipennis (KC) insect cells and BHK-21 mammalian cells were infected with AHSV-9 and analyzed for morphological and biochemical hallmarks of apoptosis. In contrast to KC cells, infection of BHK-21 cells with AHSV-9 resulted in ultrastructural changes and nuclear DNA fragmentation, both of which are associated with the induction of apoptosis. Results also indicated that AHSV-9 infection of BHK-21 cells resulted in activation of caspase-3, a key agent in apoptosis, and in mitochondrial membrane depolarization. Cumulatively, the data indicate that the intrinsic pathway is activated in AHSV-induced apoptosis.
Assuntos
Vírus da Doença Equina Africana/patogenicidade , Apoptose , Vírus da Doença Equina Africana/crescimento & desenvolvimento , Animais , Caspase 3/metabolismo , Linhagem Celular , Ceratopogonidae , Cricetinae , Citoplasma/ultraestrutura , Fragmentação do DNA , Potencial da Membrana Mitocondrial , Microscopia Eletrônica de TransmissãoRESUMO
We have investigated the use of soluble chimeric trimers of the major capsid protein VP7 of African horse sickness virus (AHSV) as a vaccine delivery system by targeting some of the natural hydrophilic loops on the VP7 top domain for the insertion of foreign peptides. Key to this trimer display strategy is the solubility of AHSV VP7 and how the solubility of this hydrophobic protein can be manipulated by inserting peptides into the top domain. To investigate, we generated different cloning vectors by inserting multiple cloning sites at three different positions in the VP7 gene. These modifications inserted six amino acids at the cloning sites and in some cases this converted VP7 to a largely soluble protein without affecting the ability of the modified proteins to form trimers. The vectors were used to generate a number of soluble VP7 fusion proteins including a fusion with a 36 amino acid insert that overlaps important immunological domains on protein VP1 of foot and mouth disease virus (FMDV) as well as a 110 amino acid peptide derived from AHSV VP2. Soluble trimers of these fusion proteins were able to elicit a good insert-specific immune response in guinea pigs. l-Arginine was found to reverse protein aggregation and was employed as an effective strategy to isolate relatively pure soluble chimeric VP7 trimers. Another factor that increased VP7 solubility in both wild-type VP7 and one of the VP7 vector proteins was the substitution of the leucine residue in position 345 of the VP7 C-terminus with a hydrophilic arginine residue.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos Virais/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas do Core Viral/imunologia , Substituição de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Antígenos Virais/química , Antígenos Virais/genética , Arginina/metabolismo , Baculoviridae/genética , Feminino , Vetores Genéticos/genética , Cobaias , Imunidade Humoral/imunologia , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Solubilidade , Proteínas do Core Viral/química , Proteínas do Core Viral/genéticaRESUMO
RNA interference (RNAi) is the process by which double-stranded RNA directs sequence-specific degradation of homologous mRNA. Short interfering RNAs (siRNAs) are the mediators of RNAi and represent powerful tools to silence gene expression in mammalian cells including genes of viral origin. In this study, we applied siRNAs targeting the VP7 gene of African horse sickness virus (AHSV) that encodes a structural protein required for stable capsid assembly. Using a VP7 expression reporter plasmid and an in vitro model of infection, we show that synthetic siRNA molecules corresponding to the AHSV VP7 gene silenced effectively VP7 protein and mRNA expression, and decreased production of infectious virus particles as evidenced by a reduction in the progeny virion titres when compared to control cells. This work establishes RNAi as a genetic tool for the study of AHSV and offers new possibilities for the analysis of viral genes important for AHSV physiology.
Assuntos
Vírus da Doença Equina Africana/crescimento & desenvolvimento , Vírus da Doença Equina Africana/genética , Antígenos Virais/biossíntese , Inativação Gênica , Interferência de RNA , Proteínas do Core Viral/biossíntese , Animais , Antígenos Virais/genética , Linhagem Celular , Células Cultivadas , Cricetinae , Plasmídeos/genética , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , Proteínas do Core Viral/genética , Ensaio de Placa ViralRESUMO
Biological properties of four recombinant HIV-1 subtype C envelope glycoproteins from viruses with different phenotypic characteristics (CCR5 and/or CXCR4-utilizing) were investigated. The gp160 genes were cloned, expressed in Spodoptera frugiperda insect cells, purified, and their biological characteristics were examined. The conformational and functional integrity of the HIV-1 subtype C rgp160 was intact since they reacted with the A32, C11, IgG1b12, 7B2, and 17b conformational dependant monoclonal antibodies (MAb), sCD4, and patient sera. Baculovirus derived rgp160 can be used for further structural, functional, antigenic, and immunological studies.