Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The structure of the Bluetongue virus (BTV) core and its outer layer VP7 has been solved by X-ray crystallography, but the assembly intermediates that lead to the inner scaffolding VP3 layer have not been defined. In this report, we addressed two key questions: (a) the role of VP3 amino terminus in core assembly and its interaction with the transcription complex (TC) components; and (b) the assembly intermediates involved in the construction of the VP3 shell. To do this, deletion mutants in the amino terminal and decamer-decamer interacting region of VP3 (DeltaDD) were generated, expressed in insect cells using baculovirus expression systems, and their ability to assemble into core-like particles (CLPs) and to incorporate the components of TC were investigated. Deletion of the N-terminal 5 (Delta5N) or 10 (Delta10N) amino acids did not affect the ability to assemble into CLPs in the presence of VP7 although the cores assembled using the 10 residue mutant (Delta10N) deletion were very unstable. Removal of five residues also did not effect incorporation of the internal VP1 RNA polymerase and VP4 mRNA capping enzyme proteins of the TC. Removal of the VP3-VP3 interacting domain (DeltaDD) led to failure to assemble into CLPs yet retained interaction with VP1 and VP4. In solution, purified DeltaDD mutant protein readily multimerized into dimers, pentamers, and decamers, suggesting that these oligomers are the authentic assembly intermediates of the subcore. However, unlike wild-type VP3 protein, the dimerization domain-deleted assembly intermediates were found to have lost RNA binding ability. Our study emphasizes the requirement of the N-terminus of VP3 for binding and encapsidation of the TC components, and defines the role of the dimerization domain in subcore assembly and RNA binding.
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PMID:Mapping the assembly pathway of Bluetongue virus scaffolding protein VP3. 1520 24

Bluetongue virus is a large and structurally complex virus composed of three concentric capsid layers that surround 10 segments of a double-stranded RNA genome. X-ray crystallographic analysis of the particles without the outer capsid layer has provided atomic structural details of VP3 and VP7, which form the inner two layers. However, limited structural information is available on the other five proteins in the virion-two of which are important for receptor recognition, hemagglutination, and membrane interaction-are in the outer layer, and the others, important for endogenous transcriptase activity are internal. Here we report the electron cryomicroscopy (cryo-EM) reconstruction of the mature particle, which shows that the outer layer has a unique non-T = 13 icosahedral organization consisting of two distinct triskelion and globular motifs interacting extensively with the underlying T = 13 layer. Comparative cryo-EM analysis of the recombinant corelike particles has shown that VP1 (viral polymerase) and VP4 (capping enzyme) together form a flower-shaped structure attached to the underside of VP3, directly beneath the fivefold axis. The structural data have been substantiated by biochemical studies demonstrating the interactions between the individual outer and inner capsid proteins.
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PMID:Interactions between the inner and outer capsids of bluetongue virus. 1525 77

X-ray and electron microscopy analysis of Bluetongue virus (BTV), the type species of the Orbivirus genus within the family Reoviridae, have revealed various aspects of the organisation and structure of the proteins that form the viral capsid. Orbiviruses have a segmented dsRNA genome, which imposes constraints on their structure and life cycle. The atomic structure of the BTV core particle, the key viral component which transcribes the viral mRNA within the cell cytoplasm, revealed the architecture and assembly of the major core proteins VP7 and VP3. In addition, these studies formed the basis for a plausible model for the organisation of the dsRNA viral genome and the arrangement of the viral transcriptase complex (composed of the RNA-dependent RNA polymerase, the viral capping enzyme and RNA helicase) that resides within the core particle. Electron cryo-microscopy of the viral particle has shown how the two viral proteins VP2 and VP5 are arranged to form the outer capsid, with distinct packing arrangements between them and the core protein VP7. By comparison of the outer capsid proteins of orbiviruses with those of other nonturreted members of the family Reoviridae, we are able to propose a more detailed model of these structures and possible mechanisms for cell entry. Further structural results are also discussed including the atomic structure of an N-terminal domain of nonstructural protein NS2, a protein involved in virus genome assembly and morphogenesis.
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PMID:Structural studies on orbivirus proteins and particles. 1690 1

Bluetongue virus (BTV) is a double-stranded (ds) RNA virus, classified within the genus Orbivirus, family Reoviridae, which causes bluetongue (BT), an infectious, non-contagious disease of ruminants. The virus exists as 24 distinct serotypes, which are currently identified by virus isolation and serum neutralisation assays. The most variable outer capsid protein VP2 (encoded by genome segment 2), is the primary determinant of BTV serotype. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays, based on amplification of segment 2, have been developed for identification of the five European BTV types (BTV-1, BTV-2, BTV-4, BTV-9 and BTV-16). Primer pairs were designed that are specific for each BTV serotype. The resulting RT-PCR assay was both sensitive and specific, providing BTV typing within 24 h. Perfect agreement was recorded between the RT-PCR and virus neutralisation assays. The primers for each serotype could successfully amplify the BTV isolates of that serotype from different regions and showed no cross-amplification of the most closely related BTV serotypes. RT-PCR primers were also developed for the discrimination of field and vaccine strains of BTV serotypes currently circulating in Europe. The primer pairs which could amplify field and vaccine strains of BTV-1, BTV-2, BTV-4 and BTV-9 were validated with several isolates of each serotype from various geographic origins around the world and their type specificity was again tested with the most closely related serotypes. Overall, these RT-PCR assays provide a rapid and reliable method for the identification and differentiation of field and vaccine strains of different BTV types. The primers used in this study are listed on the website of the Institute for Animal Health, Pirbright.
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PMID:Development of reverse transcriptase-polymerase chain reaction-based assays and sequencing for typing European strains of bluetongue virus and differential diagnosis of field and vaccine strains. 2042 85

Bluetongue (BT) is an important viral disease of ruminants that is transmitted by hematophagous Culicoides midges. We examined the seasonal patterns of abundance and infection of Culicoides sonorensis at four dairy farms in the northern Central Valley of California to develop estimates of risk for bluetongue virus (BTV) transmission to cattle at each farm. These four farms were selected because of their similar meteorological conditions but varying levels of vector abundance and BTV infection of cattle. C. sonorensis midges were collected weekly at each farm during the seasonal transmission period, using three different trapping methods: traps baited with either carbon dioxide (CO(2)) alone or traps with CO(2) and UV light, and by direct aspiration of midges from sentinel cattle. Analysis of BTV-infected midges using group and serotype-specific quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) assays confirmed that BTV serotypes 10, 11, 13 and 17 are all present in the region, but that midge infection rates and the number of BTV serotypes circulating differed markedly among the individual farms. Furthermore, more serotypes of BTV were present in midges than in sentinel cattle at individual farms where BTV circulated, and the virus was detected at each farm in midges prior to detection in cattle. BTV infection rates were remarkably lower among female C. sonorensis midges collected by CO(2) traps with UV light than among midges collected by either animal-baited aspirations or in CO(2) traps without light. A subsample of female midges examined from each collection method showed no overall differences in the proportion of female midges that had previously fed on a host. Findings from this study confirm the importance of using sensitive surveillance methods for both midge collection and virus detection in epidemiological studies of BTV infection, which is especially critical if the data are to be used for development of mathematical models to predict the occurrence of BTV infection of livestock.
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PMID:The combination of abundance and infection rates of Culicoides sonorensis estimates risk of subsequent bluetongue virus infection of sentinel cattle on California dairy farms. 2228 Nov 50

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.
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PMID:Establishment of an entirely plasmid-based reverse genetics system for Bluetongue virus. 2640 55

Epizootic haemorrhagic disease virus (EHDV) is an insect-transmitted pathogen which causes high mortality in deer populations and may also cause high morbidity in cattle. EHDV belongs to the Orbivirus genus and is closely related to the prototype Bluetongue virus (BTV). To date seven distinct serotypes have been recognized. However, a live-attenuated vaccine is commercially available against only one serotype namely EHDV-2, which has been responsible for multiple outbreaks in North America, Canada, Asia and Australia. Here we expressed four major capsid proteins (VP2, VP3, VP5 and VP7) of EHDV-1 using baculovirus multiple gene expression systems and demonstrated that three-layered VLPs were assembled mimicking the authentic EHDV particles but lacking the viral genomic RNA segments and the transcriptase complex (TC). Antibodies generated with VLPs not only neutralized EHDV-1 infection in cell culture but also showed cross neutralizing reactivity against two other serotypes, EHDV-2 and EHDV-6. For proof of concept, we demonstrated that EHDV-2 VLPs could be generated rapidly by expressing the EHDV-2 variable outer capsid proteins (VP2, VP5) together with EHDV-1 VP3 and VP7, the two inner capsid proteins, which are highly conserved among the 7 serotypes. Data presented in this study validate the VLPs as a potential vaccine and demonstrate that a vaccine could be developed rapidly in the event of an outbreak of a new serotype.
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PMID:Generation of virus-like particles for emerging epizootic haemorrhagic disease virus: Towards the development of safe vaccine candidates. 2680 95

Bluetongue is a major disease of economic importance that affects ruminants worldwide. It is transmitted by species of Culicoides midges (Diptera: Ceratopogonidae). The Inner Mongolia Autonomous Region is one of the main pastoral areas for farmed sheep in Mainland China and, because of its large area, represents an ideal candidate region for the study of Bluetongue virus (BTV) distribution and prevalence characteristics. The present study conducted a detailed investigation into the spatial patterns of BTV transmission in sheep in the Inner Mongolia Autonomous Region, and assessed the inter-relationships between meteorological factors, land cover and the transmission of the virus was conducted. Reverse-transcriptase polymerase chain reaction (RT-PCR) was used for the determination of BTV infection in the surveyed animals. Between June 2013 and February 2015, 6199 sheep were subjected to virus detection and 2199 sheep (35.47%) were determined to be positive for BTV. Subsequently, a maximum entropy model (MaxEnt) was used to investigate the relationship between land cover, meteorological factors and the prevalence of BTV infection. Jackknife analysis revealed that the mean monthly temperature, rainfall and average wind speed were associated with the occurrence of BTV infection and that BTV infection positivity was significantly higher among animals from districts with a high percentage of grassland and forest area. Our findings indicate that meteorological factors and land cover may be important variables affecting transmission of BTV and should be taken into account in the development of future surveillance programmes for BTV.
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PMID:Meteorological conditions and land cover as predictors for the prevalence of Bluetongue virus in the Inner Mongolia Autonomous Region of Mainland China. 2823 39

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