Trypsin enhances rotavirus infectivity by an unknown mechanism. To examine the structural basis of trypsin-enhanced infectivity in rotaviruses, SA11 4F triple-layered particles (TLPs) grown in the absence (nontrypsinized rotavirus [NTR]) or presence (trypsinized rotavirus [TR]) of trypsin were characterized to determine the structure, the protein composition, and the infectivity of the particles before and after trypsin treatment. As expected, VP4 was not cleaved in NTR particles and was cleaved into VP5‫ء‬ and VP8 ‫ء‬ in TR particles. However, surprisingly, while the VP4 spikes were clearly visible and well ordered in the electron cryomicroscopy reconstructions of TR TLPs, they were totally absent in the reconstructions of NTR TLPs. Biochemical analysis with radiolabeled particles indicated that the stoichiometry of the VP4 in NTR particles was the same as that in TR particles and that the VP8 ‫ء‬ portion of NTR, but not TR, particles is susceptible to further proteolysis by trypsin. Taken together, these structural and biochemical data show that the VP4 spikes in the NTR TLPs are icosahedrally disordered and that they are conformationally different. Structural studies on the NTR TLPs after trypsin treatment showed that spike structure could be partially recovered. Following additional trypsin treatment, infectivity was enhanced for both NTR and TR particles, but the infectivity of NTR remained 2 logs lower than that of TR particles. Increased infectivity in these particles corresponded to additional cleavages in VP5 ‫ء‬ , at amino acids 259, 583, and putatively 467, which are conserved in all P serotypes of human and animal group A rotaviruses and also corresponded with a structural change in VP7. These biochemical and structural results show that trypsin cleavage imparts order to VP4 spikes on de novo synthesized virus particles, and these ordered spikes make virus entry into cells more efficient.Rotaviruses are the leading cause of severe gastroenteritis in young children worldwide (16). Structural and biochemical analyses show that rotaviruses are large, icosahedral particles having a complex architecture consisting of three concentric capsid layers surrounding a genome of 11 segments of doublestranded RNA (41, 42). The innermost capsid layer, composed of VP2, encloses the genomic double-stranded RNA. A significant portion of the genomic RNA, particularly that in close contact with the inner surface of the VP2 layer, is icosahedrally ordered (43). Anchored to the inner surface of VP2 at the icosahedral vertices are two proteins, VP1 and VP3, involved in transcription of the genome within the intact particle. The intermediate capsid layer is composed of trimers of VP6 organized on a Tϭ13 (levo) icosahedral lattice (44). The outermost layer in the infectious virus is composed of the major capsid glycoprotein VP7 and the hemagglutinin spike protein VP4 (16). VP7 is present as 780 molecules grouped as 260 trimers at the local and strict threefold axes of a Tϭ13 left-handed icosahedral lattice (44, 51). The VP7 ...

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...

Rotaviruses are the leading cause of severe infantile gastroenteritis worldwide. These viruses are large, complex icosahedral particles consisting of three concentric capsid layers enclosing a genome of eleven segments of double-stranded RNA (dsRNA). The amino terminus of the innermost capsid protein VP2 possesses a nonspecific single-stranded RNA and dsRNA binding activity, and the amino terminus is also essential for the incorporation of the polymerase enzyme VP1 and guanylyltransferase VP3 into the core of the virion. Biochemical and structural studies have suggested that VP2, and especially the amino terminus, appears to act as a scaffold for proper assembly of the components of the viral core. To locate the amino terminus of VP2 within the core, we have used electron cryomicroscopy and image reconstruction to determine the three-dimensional structures of recombinant virus-like particles that contain either full-length or amino-terminal-deleted forms of VP2 coexpressed with the intermediate capsid protein VP6. A comparison of these structures indicates two significant changes along the inner surface of VP2 in the structure lacking the amino terminus: a loss of mass adjacent to the fivefold axes and a redistribution of mass along the fivefold axes. Examination of the VP2 layer suggests that the proteins are arranged as dimers of 120 quasi-equivalent molecules, with each dimer extending between neighboring fivefold axes. Our results indicate that the amino termini of both quasi-equivalent VP2 molecules are located near the icosahedral vertices.

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.

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