A lattice of VP7 trimers forms the surface of the icosahedral bluetongue virus (BTV) core. To investigate the role of VP7 oligomerization in core assembly, a series of residues for substitution were predicted based on crystal structures of BTV type 10 VP7 molecule targeting the monomer-monomer contacts within the trimer. Seven site-specific substitution mutations of VP7 have been created using cDNA clones and were employed to produce seven recombinant baculoviruses. The effects of these mutations on VP7 solubility, ability to trimerize and formation of core-like particles (CLPs) in the presence of the scaffolding VP3 protein, were investigated. Of the seven VP7 mutants examined, three severely affected the stability of CLP, while two other mutants had lesser effect on CLP stability. Only one mutant had no apparent effect on the formation of the stable capsid. One mutant in which the conserved tyrosine at residue 271 (lower domain helix 6) was replaced by arginine formed insoluble aggregates, implying an effect in the folding of the molecule despite the prediction that such a change would be accommodated. All six soluble VP7 mutants were purified, and their ability to trimerize was examined. All mutants, including those that did not form stable CLPs, assembled into stable trimers, implying that single substitution may not be sufficient to perturb the complex monomer-monomer contacts, although subtle changes within the VP7 trimer could destabilize the core. The study highlights some of the key residues that are crucial for BTV core assembly and illustrates how the structure of VP7 in isolation underrepresents the dynamic nature of the assembly process at the biological level.Orbiviruses, members of the Reoviridae family, possess a large (86 nm in diameter) nonenveloped virus particle, encapsidating 10 segments of double-stranded RNA genome (31,32,35). Orbivirus virions are composed of a number of discrete proteins arranged in a specific but nonequimolar ratio. Overall, these viruses are icosahedral, with two protein layers that have radically different geometries, and provide a complex subject to study, both in terms of protein-protein interactions, and protein-RNA interactions. Bluetongue virus (BTV), the prototype orbivirus, consists of seven structural proteins (VP1 to VP7), four of which are major (VP2, VP3, VP5, and VP7) and include proteins that interact with cellular receptors and others that form the underlying framework of the virion (30, 31, 32). The three minor proteins (VP1, VP4, and VP6), which are present in low molar ratios within the virion, have RNA transcriptase-and RNA-modifying properties (33). In its mature form the virus exhibits no transcriptase activity until it is activated upon infection with the modification of the outer capsid to create channels in the core architecture that allow metabolites to enter the capsid and the viral mRNA species to be formed and extruded (10,12,13,29).A considerable amount of data has recently been accumulated on the transcriptionally active BTV core architect...
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.Orbiviruses (within the family Reoviridae), are vectored to vertebrate species by arthropods (e.g., gnats, mosquitoes and ticks) and are able to replicate in both hosts. Bluetongue virus (BTV) is the prototype virus of the genus and is transmitted by gnats (Culicoides species), causing diseases of economic importance in ruminants in many parts of the world. Vector-virus interactions play a crucial role in vector-borne disease epidemiology. The spread of Culicoides species from BTV-endemic to non-BTV (or related African horsesickness virus, AHSV, and epizootic hemorrhagic disease virus EHDV, of deer) regions of the world in the past highlights the concern that these viruses are a threat to regions of the world that are presently free from them.The initiation of a virus infection involves virus binding to ligands on the cell surface prior to cell entry by a number of mechanisms (depending on the virus). Like many other viruses, BTV appears to utilize a protein molecule(s) of mammalian cells as a receptor (20); however, it is also possible that alternative receptors may be utilized in different tissues and in different species and as accessory molecules.BTV has a genome composed of 10 segments of doublestranded RNA packaged within a double icosahedral capsid. The outer capsi...
Based on the crystal structure of the VP7 major core protein of bluetongue virus serotype 10 (BTV-10) and that of the top domain of the VP7 protein of African horsesickness virus serotype 4 (AHSV-4), chimeras and site-directed mutants of the proteins were constructed and the products analyzed with respect to their properties and functions. Chimeras with the central upper domain of BTV-10 VP7 replaced by that of AHSV-4 VP7 (construct BAB) formed trimers, as did the converse construct (ABA). Further, both proteins exhibited the expected conformational epitopes of the constituent sequences. Using BAB VP7 it was demonstrated that residues of the upper domain of AHSV-4 VP7 contribute to the observed insolubility of the protein. By contrast, ABA VP7 protein was as soluble as wild-type BTV-10 VP7. Replacement of selected amino acid residues in the top domain (e.g., A167 by R; F209 by T) improved the solubility of BAB VP7. Since the trimeric BAB and ABA VP7 proteins did not form core-like particles (CLPs) when coexpressed with BTV VP3, it was concluded that trimerization of chimeric VP7 is not sufficient for CLP formation. When the N-terminal region of the ABA protein (aa 1-120) was replaced by the respective sequences of BTV VP7 (construct BBA), the protein aggregated and did not form CLPs with coexpressed BTV VP3, most likely due to disruption of the required contacts between the N- and C-terminal regions of the bottom domain, leading to incorrect folding of the chimera.
An indirect enzyme-linked immunosorbent assay was used for the screening of horse sera from The Gambia for antibodies against African horse sickness virus (AHSV). The AHSV antigen used for coating was semipurified according to the method of Manning and Chen (Curr. Microbiol. 4:381, 1980); control mock-infected Vero cell antigen was treated in the same manner. A total of 459 horse serum samples were assayed at a single dilution (1:10), and their reactivities were compared with those of reference positive anti-AHSV and reference negative horse sera. A total of 81% of the horse serum samples clearly contained antibodies against AHSV; this consisted of 18% (of the total number of serum samples) strongly positive, 46.5% moderately positive, and 16.5% weakly but still clearly positive. Such results suggest a high prevalence of AHSV in the regions from whence the samples originated. Reports from investigations in other countries in this area of West Africa have also shown a high prevalence for anti-AHSV antibodies in equids. The question is raised as to how the animals became seropositive and whether the observations represent an increased resistance of horses living in a region in which AHS is enzootic.
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