C terminus of infectious bursal disease virus major capsid protein VP2 is involved in definition of the T number for capsid assembly

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus. The IBDV capsid is formed by two major structural proteins, VP2 and VP3, which assemble to form a T=13 markedly nonspherical capsid. During viral infection, VP2 is initially synthesized as a precursor, called VPX, whose C end is proteolytically processed to the mature form during capsid assembly. We have computed three-dimensional maps of IBDV capsid and virus-like particles built up by VP2 alone by using electron cryomicroscopy and image-processing techniques. The IBDV single-shelled capsid is characterized by the presence of 260 protruding trimers on the outer surface. Five classes of trimers can be distinguished according to their different local environments. When VP2 is expressed alone in insect cells, dodecahedral particles form spontaneously; these may be assembled into larger, fragile icosahedral capsids built up by 12 dodecahedral capsids. Each dodecahedral capsid is an empty T=1 shell composed of 20 trimeric clusters of VP2. Structural comparison between IBDV capsids and capsids consisting of VP2 alone allowed the determination of the major capsid protein locations and the interactions between them. Whereas VP2 forms the outer protruding trimers, VP3 is found as trimers on the inner surface and may be responsible for stabilizing functions. Since elimination of the C-terminal region of VPX is correlated with the assembly of T=1 capsids, this domain might be involved (either alone or in cooperation with VP3) in the induction of different conformations of VP2 during capsid morphogenesis.     

Three-dimensional structure of infectious bursal disease virus determined by electron cryomicroscopy.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae group, is a commercially important pathogen of chickens. From electron micrographs of frozen, hydrated, unstained specimens, we have computed a three-dimensional map of IBDV at about 2 nm resolution. The map shows that the structure of the virus is based on a T=13 lattice and that the subunits are predominantly trimer clustered. The subunits close to the fivefold symmetry axes are at a larger radius than those close to the two- or threefold axes, giving the capsid a markedly nonspherical shape. The trimer units on the outer surface protrude from a continuous shell of density. On the inner surface, the trimers appear as Y-shaped units, but the set of units surrounding the fivefold axes appears to be missing. It is likely that the outer trimers correspond to the protein VP2, carrying the dominant neutralizing epitope, and the inner trimers correspond to protein VP3, which has a basic carboxy-terminal tail expected to interact with the packaged RNA.     

Intermolecular interactions in a two-layered viral capsid that requires a complex symmetry mismatch

The surface of the bluetongue virus core forms a T=13 quasiequivalent icosahedral protein shell with 260 trimers of a single gene product: VP7 protein. Underneath is a smooth layer, made up of VP3 protein, which appears to guide and nucleate the assembly of VP7 trimers. The contacts between the two shells are extensive but nonspecific, and construction of the T=13 icosahedral shell requires polymorphism in the association of the VP7 subunits, each of which has two domains that contribute to trimer formation. We used structural and relative sequence information to guide an investigation of how such a complex structure is achieved during virus assembly and what residues are required to form a stable capsid. Fifteen single or multiple site-specific substitution mutations were introduced into the helical domain of VP7, which is closely associated with the VP3 layer, and the effects on capsid assembly were analyzed. Our data show that both the position and the nature of single residues are critical for the attachment of VP7 to VP3 and that formation of a stable VP7 lattice is not the automatic consequence of trimer formation.     

Structure of Double-Shelled Rice Dwarf Virus

Rice dwarf virus (RDV), a member of the Reoviridae family, is a double-stranded RNA virus. Infection of rice plants with RDV reduces crop production significantly and can pose a major economic threat to Southeast Asia. A 25-Å three-dimensional structure of the 700-Å-diameter RDV capsid has been determined by 400-kV electron cryomicroscopy and computer reconstruction. The structure revealed two distinctive icosahedral shells: a T=13l outer icosahedral shell composed of 260 trimeric clusters of P8 (46 kDa) and an inner T=1 icosahedral shell of 60 dimers of P3 (114 kDa). Sequence and structural comparisons were made between the RDV outer shell trimer and the two crystal conformations (REF and HEX) of the VP7 trimer of bluetongue virus, an animal analog of RDV. The low-resolution structural match of the RDV outer shell trimer to the HEX conformation of VP7 trimer has led to the proposal that P8 consists of an upper domain of β-sandwich motif and a lower domain of α helices. The less well fit REF conformation of VP7 to the RDV trimer may be due to the differences between VP7 and P8 in the sequence of the hinge region that connects the two domains. The additional mass density and the absence of a known signaling peptide on the surface of the RDV outer shell trimer may be responsible for the different interactions between plants and animal reoviruses.     

Rotavirus spike structure and polypeptide composition.

Negatively stained preparations of rotavirus imaged with a low dose of electrons provide sufficient contrast to reveal surface projections or spikes. The number of spikes found projecting from different particles indicates that not all 60 peripentonal sites are occupied. Treatment at pH 11.2 with 250 mM ammonium hydroxide specifically removes the spikes, yielding smooth double-shelled particles of the same diameter as that of the native virus. Protein analysis confirms that the released spikes are composed of polypeptide VP4 (or its two cleavage products VP5* and VP8*) and that the smooth particle retains the other major outer shell protein VP7. Spikeless particles can be decorated by a monoclonal antibody specific for the major immunodominant neutralizing domain of VP7, implying that removal of the spikes does not denature the VP7 that is retained on the surface of the smooth particle.     

Three-dimensional structure of the inner core of rice dwarf virus

Rice dwarf virus (RDV) is a double-shelled icosahedral virus.Using electron cryomicroscopy and computer reconstruction techniques, we have determined a 3.3 nm resolution three-dimensional (3D) structure of the inner shell capsid without the outer shell and viral RNA. The results show that the inner shell is a thin, densely packed, smooth structure, which provides a scaffold for the full virus. A total of 120 copies of the major inner shell capsid protein P3 forms 60 dimers arranged in a T=1 icosahedral lattice. A close examination on the subunit packing of the T=1 inner core P3 with that of the T=13l outer shell P8 indicated that P8 trimers connect with P3 through completely non-equivalent, yet highly specific, intermolecular interactions.     

Three-dimensional structure of the inner core of rice dwarf virus

Rice dwarf virus (RDV) is a double-shelled icosahedral virus. Using electron cryomicro-scopy and computer reconstruction techniques, we have determined a 3.3 nm resolution three-dimensional (3D) structure of the inner shell capsid without the outer shell and viral RNA. The results show that the inner shell is a thin, densely packed, smooth structure, which provides a scaffold for the full virus. A total of 120 copies of the major inner shell capsid protein P3 forms 60 dimers arranged in a T=1 icosahedral lattice. A close examination on the subunit packing of the T=1 inner core P3 with that of the T=13/ outer shell P8 indicated that P8 trimers connect with P3 through completely non-equivalent, yet highly specific, intermolecular interactions.     

Atomic structure of the major capsid protein of rotavirus: implications for the architecture of the virion

The structural protein VP6 of rotavirus, an important pathogen responsible for severe gastroenteritis in children, forms the middle layer in the triple-layered viral capsid. Here we present the crystal structure of VP6 determined to 2 A resolution and describe its interactions with other capsid proteins by fitting the atomic model into electron cryomicroscopic reconstructions of viral particles. VP6, which forms a tight trimer, has two distinct domains: a distal beta-barrel domain and a proximal alpha-helical domain, which interact with the outer and inner layer of the virion, respectively. The overall fold is similar to that of protein VP7 from bluetongue virus, with the subunits wrapping about a central 3-fold axis. A distinguishing feature of the VP6 trimer is a central Zn(2+) ion located on the 3-fold molecular axis. The crude atomic model of the middle layer derived from the fit shows that quasi-equivalence is only partially obeyed by VP6 in the T = 13 middle layer and suggests a model for the assembly of the 260 VP6 trimers onto the T = 1 viral inner layer.     

Electron cryo-microscopy and image reconstruction of adeno-associated virus type 2 empty capsids

Adeno-associated virus type 2 empty capsids are composed of three proteins, VP1, VP2 and VP3, which have relative molecular masses of 87, 72 and 62 kDa, respectively, and differ in their N-terminal amino acid sequences. They have a likely molar ratio of 1:1:8 and occupy symmetrical equivalent positions in an icosahedrally arranged protein shell. We have investigated empty capsids of adeno-associated virus type 2 by electron cryo-microscopy and icosahedral image reconstruction. The three-dimensional map at 1.05 nm resolution showed sets of three elongated spikes surrounding the three-fold symmetry axes and narrow empty channels at the five-fold axes. The inside of the capsid superimposed with the previously determined structure of the canine parvovirus (Q. Xie and M.S. Chapman, 1996, J. Mol. Biol., 264, 497–520), whereas the outer surface showed clear discrepancies. Globular structures at the inner surface of the capsid at the two-fold symmetry axes were identified as possible positions for the N-terminal extensions of VP1 and VP2.     

Three-dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconstruction

The structure of rhesus rotavirus was examined by cryoelectron microscopy and image analysis. Three-dimensional reconstructions of infectious virions were computed at 26- and 37-A resolution from electron micrographs recorded at two different levels of defocus. The major features revealed by the reconstructions are (a) both outer and inner capsids are constructed with T = 13l icosahedral lattice symmetry; (b) 60 spikelike projections, attributed to VP4, extend at least 100 A from the outer capsid surface; (c) the outer capsid, attributed primarily to VP7, has a smoothly rippled surface at a mean radius of 377 A and is perforated by 132 aqueous holes ranging from 40-65 A in diameter; (d) the inner capsid has a "bristled" outer surface composed of 260 trimeric-shaped columns of density, attributed to VP6, which merge with a smooth, spherical shell of density at a lower, mean radius of 299 A, and which is perforated by holes in register with those in the outer capsid; (e) a "core" region contains a third, nonspherical shell of density at a mean radius of 225 A that encapsidates the double-stranded RNA genome; and (f) the space between the outer and inner capsids forms an open aqueous network that may provide pathways for the diffusion of ions and small regulatory molecules as well as the extrusion of RNA. The assignment of different viral structural proteins to specific features of the reconstruction has been tentatively made on the basis of excluded volume estimates and previous biochemical characterizations of rotavirus.     

3D reconstruction and capsid protein characterization of grass carp reovirus

Grass carp reovirus (GCRV) is a relatively new virus first isolated in China and is a member of the Aquareovirus genus of the Reoviridae family. Recent report of genomic sequencing showed that GCRV shared high degree of homology with mammalian reovirus (MRV). As a step of our effort to understand the structural basis of GCRV pathogenesis, we determined the three-dimensional (3D) structure of GCRV capsid at 17 Å resolution by electron cryomicroscopy. Each GCRV capsid has a multilayered organization, consisting of an RNAcore, an inner, middle and outer protein layer. The outer layer is made up of 200 trimers that are arranged on an incomplete T=13 icosahedral lattice. A characteristic feature of this layer is the depression resulting from the absence of trimers around the peripentonal positions, revealing the underlying trimers on the middle layer. There are 120 subunits in the inner layer arranged with T=1 symmetry. These structural features are common to other members of the Reoviridae. Moreover, SDS-PAGE analysis showed that GCRV virions contain seven structural proteins (VP1-VP7). These structural proteins have a high degree of sequence homology to MRV, consistent with the structural similarities observed in our study. The high structural similarities of isolated GCRV and MRV suggest that future structural studies focusing on GCRV entering into and replicating within its host cell are necessary in order to fully understand the structural basis of GCRV pathogenesis.     

Two forms of VP7 are involved in assembly of SA11 rotavirus in endoplasmic reticulum

Two pools of the glycoprotein VP7 were detected in the endoplasmic reticulum (ER) of SA11 rotavirus-infected cells. One portion of the newly synthesized protein with VP3 composed the virus outer capsid, while the rest remained associated with the membrane. The two populations could be separated biochemically by fluorocarbon extraction or by immunological methods which used two classes of antibodies. A monoclonal antibody with neutralizing activity recognized VP7 only as displayed on intact virus particles, while a polyclonal antiserum precipitated predominantly the unassembled ER form of the protein and precipitated virus-assembled VP7 poorly. Virus-associated VP7 was localized by immunofluorescence to small punctate structures, presumably corresponding to accumulated virus particles, and to regions of the ER surrounding viroplasmic inclusions, whereas the membrane-associated molecules were distributed in an arborizing reticular pattern throughout the ER. VP3 and the nonstructural glycoprotein NCVP5 displayed a localization similar to that of virus-associated VP7. Intracellular virus particles were isolated from infected cells to determine the kinetics of assembly of VP7 and of the other structural proteins into virions. It was found that incorporation of the inner capsid proteins into single-shelled particles occurred rapidly, while VP7 and VP3 appeared in mature double-shelled particles with a lag time of 10 to 15 min. In addition, the alpha-mannosidase processing kinetics of virus-associated VP7 oligosaccharides showed a 15-min lag compared with that of the membrane-associated form, suggesting that the latter is the precursor to virion VP7. This lag may represent the time required for virus budding and outer capsid assembly.     

Role and mechanism of the maturation cleavage of VP0 in poliovirus assembly: structure of the empty capsid assembly intermediate at 2.9 A resolution.

The crystal structure of the P1/Mahoney poliovirus empty capsid has been determined at 2.9 A resolution. The empty capsids differ from mature virions in that they lack the viral RNA and have yet to undergo a stabilizing maturation cleavage of VP0 to yield the mature capsid proteins VP4 and VP2. The outer surface and the bulk of the protein shell are very similar to those of the mature virion. The major differences between the 2 structures are focused in a network formed by the N-terminal extensions of the capsid proteins on the inner surface of the shell. In the empty capsids, the entire N-terminal extension of VP1, as well as portions corresponding to VP4 and the N-terminal extension of VP2, are disordered, and many stabilizing interactions that are present in the mature virion are missing. In the empty capsid, the VP0 scissile bond is located some 20 A away from the positions in the mature virion of the termini generated by VP0 cleavage. The scissile bond is located on the rim of a trefoil-shaped depression in the inner surface of the shell that is highly reminiscent of an RNA binding site in bean pod mottle virus. The structure suggests plausible (and ultimately testable) models for the initiation of encapsidation, for the RNA-dependent autocatalytic cleavage of VP0, and for the role of the cleavage in establishing the ordered N-terminal network and in generating stable virions.     

Antibodies to rotavirus outer capsid glycoprotein VP7 neutralize infectivity by inhibiting virion decapsidation

The rotavirus capsid is composed of three concentric protein layers. Proteins VP4 and VP7 comprise the outer layer. VP4 forms spikes, is the viral attachment protein, and is cleaved by trypsin into VP8* and VP5*. VP7 is a glycoprotein and the major constituent of the outer protein layer. Both VP4 and VP7 induce neutralizing and protective antibodies. To gain insight into the virus neutralization mechanisms, the effects of neutralizing monoclonal antibodies (MAbs) directed against VP8*, VP5*, and VP7 on the decapsidation process of purified OSU and RRV virions were studied. Changes in virion size were followed in real time by 90 degrees light scattering. The transition from triple-layered particles to double-layered particles induced by controlled low calcium concentrations was completely inhibited by anti-VP7 MAbs but not by anti-VP8* or anti-VP5* MAbs. The inhibitory effect of the MAb directed against VP7 was concentration dependent and was abolished by papain digestion of virus-bound antibody under conditions that generated Fab fragments but not under conditions that generated F(ab')(2) fragments. Electron microscopy showed that RRV virions reacted with an anti-VP7 MAb stayed as triple-layered particles in the presence of excess EDTA. Furthermore, the infectivity of rotavirus neutralized via VP8*, but not that of rotavirus neutralized via VP7, could be recovered by lipofection of neutralized particles into MA-104 cells. These data are consistent with the notion that antibodies directed at VP8* neutralize by inhibiting binding of virus to the cell. They also indicate that antibodies directed at VP7 neutralize by inhibiting virus decapsidation, in a manner that is dependent on the bivalent binding of the antibody.     

Periostracal mineralization in the gastrochaenid bivalve Spengleria

We investigated the spikes on the outer shell surface of the endolithic gastrochaenid bivalve genus Spengleria with a view to understand the mechanism by which they form and evaluate their homology with spikes in other heterodont and palaeoheterodont bivalves. We discovered that spike formation varied in mechanism between different parts of the valve. In the posterior region, spikes form within the translucent layer of the periostracum but separated from the calcareous part of the shell. By contrast those spikes in the anterior and ventral region, despite also forming within the translucent periostracal layer, become incorporated into the outer shell layer. Spikes in the posterior area of Spengleria mytiloides form only on the outer surface of the periostracum and are therefore, not encased in periostracal material. Despite differences in construction between these gastrochaenid spikes and those of other heterodont and palaeoheterodont bivalves, all involve calcification of the inner translucent periostracal layer which may indicate a deeper homology.     

The three-dimensional structure of reovirus obtained by cryo-electron microscopy.

The structures of reovirus serotypes T2J (Jones), T3D (Dearing) and the T3D core particle have been determined by cryo-electron microscopy and image processing. At a resolution of 30 A the two serotypes have similar features. The core is visible within the virus structure. The outer surface of the virus particles contains 120 holes at T = 13.1 local 6-fold axes. The holes penetrate into the virus as far as the surface of the internal core shell. Protrusions extending 4 nm from the virus surface surround each hole on the outside of the virus. At the 5-fold axes on the surface of the virus flat 'penton craters' form covers over the underlying core spikes. The detailed structure of the reovirus shell is very different to that of rotavirus although both have holes at T = 13.1 axes. Little evidence was seen of reovirus fibres extending from the virus surface.