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. Bluetongue virus (BTV) is an economically important member of the Orbivirus genus in the familyReoviridae. This is a large family and contains important viruses isolated from vertebrates (including humans), invertebrates, and plants. BTV infects mainly ruminants and is transmitted by insect vectors of the Culicoides species.BTV is a large (ϳ850-Å -diameter) and structurally complex virus composed of three concentric capsid layers that surround 10 segments of a double-stranded RNA (dsRNA) genome. The outer layer, composed of VP2 (111 kDa) and VP5 (ϳ59 kDa), is removed during the initial stages of the viral life cycle revealing the transcriptionally competent inner capsid termed the "core" particle. The outer layer of the core particle is composed of 260 trimers of VP7 (ϳ38 kDa) organized on a T ϭ 13 icosahedral lattice. This VP7 layer interacts with the underlying innermost layer made from 120 copies of VP3 (ϳ103 kDa) arranged as 60 dimers on a T ϭ 1 icosahedral lattice (10). Such a unique icosahedral organization indeed appears to be a common feature of the dsRNA viruses (26). The VP3 layer houses the segmented genome as well as three minor structural proteins: VP1, an RNA-dependent RNA polymerase (1); VP4, a methyl transferase (28, 30); and VP6, a helicase (18, 33). The transcriptionally competent core particles have been extensively studied both by electron cryomicroscopy (cryo-EM) and X-ray crystallographic techniques (9-11). Although these studies provided detailed structural description of the VP7 and the VP3 layers, little information was gleaned about the endogenous transcription enzyme complex of the virus.In contrast to the core particles, structural studies on the intact virions are more difficult as a result of the la...

Aquareovirus, a member of the family Reoviridae, is a large virus with multiple capsid layers surrounding a genome composed of 11 segments of double-stranded RNA. Biochemical studies have shown that treatment with the proteolytic agent trypsin significantly alters the infectivity of the virus. The most infectious stage of the virus is produced by a 5-min treatment with trypsin. However, prolonged trypsin treatment almost completely abolishes the infectivity. We have used three-dimensional electron cryomicroscopy to gain insight into the structural basis of protease-induced alterations in infectivity by examining the structural changes in the virion at various time intervals of trypsin treatment. Our data show that after 5 min of trypsinization, projection-like spikes made of VP7 (35 kDa), associated with the underlying trimeric subunits, are completely removed. Concurrent with the removal of VP7, conformational changes are observed in the trimeric subunit composed of putative VP5 (71 kDa). The removal of VP7 and the accompanied structural changes may expose regions in the putative VP5 important for cell entry processes. Prolonged trypsinization not only entirely removes the outer capsid layer, producing the poorly infectious core particle, but also causes significant conformational changes in the turret protein. These changes result in shortening of the turret and narrowing of its central channel. The turret, as in orthoreoviruses, is likely to play a major role in the capping and translocation of mRNA during transcription, and the observed conformational flexibility in the turret protein may have implications in rendering the particle transcriptionally active or inactive.

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...

Mammalian reoviruses undergo acid-dependent proteolytic disassembly within endosomes, resulting in formation of infectious subvirion particles (ISVPs). ISVPs are obligate intermediates in reovirus disassembly that mediate viral penetration into the cytoplasm. The initial biochemical event in the reovirus disassembly pathway is the proteolysis of viral outer-capsid protein 3. Mutant reoviruses selected during persistent infection of murine L929 cells (PI viruses) demonstrate enhanced kinetics of viral disassembly and resistance to inhibitors of endocytic acidification and proteolysis. To identify sequences in 3 that modulate aciddependent and protease-dependent steps in reovirus disassembly, the 3 proteins of wild-type strain type 3 Dearing; PI viruses L/C, PI 2A1, and PI 3-1; and four novel mutant 3 proteins were expressed in insect cells and used to recoat ISVPs. Treatment of recoated ISVPs (rISVPs) with either of the endocytic proteases cathepsin L or cathepsin D demonstrated that an isolated tyrosine-to-histidine mutation at amino acid 354 (Y354H) enhanced 3 proteolysis during viral disassembly. Yields of rISVPs containing Y354H in 3 were substantially greater than those of rISVPs lacking this mutation after growth in cells treated with either acidification inhibitor ammonium chloride or cysteine protease inhibitor E64. Image reconstructions of electron micrographs of virus particles containing wild-type or mutant 3 proteins revealed structural alterations in 3 that correlate with the Y354H mutation. These results indicate that a single mutation in 3 protein alters its susceptibility to proteolysis and provide a structural framework to understand mechanisms of 3 cleavage during reovirus disassembly.

Reovirus virions are nonenveloped icosahedral particles consisting of two concentric protein shells, termed outer capsid and core. Outer-capsid protein 1 is the viral attachment protein and binds carbohydrate molecules on the surface of host cells. Monoclonal antibody (MAb) 4F2, which is specific for outer-capsid protein 3, blocks the binding of 1 protein to sialic acid and inhibits reovirus-induced hemagglutination (HA). To determine whether MAb 4F2 inhibits HA by altering 1-3 interactions or by steric hindrance, we analyzed the effect of 4F2 immunoglobulin G (IgG) and Fab fragments (Fabs) on HA induced by reovirus strain type 3 Dearing (T3D). The concentration of 4F2 IgG sufficient to inhibit T3D-induced HA was 12.5 g per ml, whereas that of Fabs was >200 g per ml. Dynamic light scattering analysis showed that at the concentration of IgG sufficient to inhibit HA, virion-antibody complexes were monodispersed and not aggregated. The affinity of 4F2 Fabs for T3D virions was only threefold less than that of intact IgG, which suggests that differences in HA inhibition titer exhibited by 4F2 IgG and Fabs are not attributable to differences in the affinity of these molecules for T3D virions. We used cryoelectron microscopy and three-dimensional image analysis to visualize T3D virions alone and in complex with either IgG or Fabs of MAb 4F2. IgG and Fabs bind the same site at the distal portion of 3, and binding of IgG and Fabs induces identical conformational changes in outer-capsid proteins 3 and 1. These results suggest that MAb 4F2 inhibits reovirus binding to sialic acid by steric hindrance and provide insight into the conformational flexibility of reovirus outer-capsid proteins.