The 2.6-Angstrom structure of infectious bursal disease virus-derived T=1 particles reveals new stabilizing elements of the virus capsid

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus that causes a highly contagious disease in young chickens leading to significant economic losses in the poultry industry. The VP2 protein, the only structural component of the IBDV icosahedral capsid, spontaneously assembles into T=1 subviral particles (SVP) when individually expressed as a chimeric gene. We have determined the crystal structure of the T=1 SVP to 2.60 A resolution. Our results show that the 20 trimeric VP2 clusters forming the T=1 shell are further stabilized by calcium ions located at the threefold icosahedral axes. The structure also reveals a new unexpected domain swapping that mediates interactions between adjacent trimers: a short helical segment located close to the end of the long C-terminal arm of VP2 is projected toward the threefold axis of a neighboring VP2 trimer, leading to a complex network of interactions that increases the stability of the T=1 particles. Analysis of crystal packing shows that the exposed capsid residues, His253 and Thr284, determinants of IBDV virulence and the adaptation of the virus to grow in cell culture, are involved in particle-particle interactions.     

Three-dimensional structure of vaccinia virus-produced human papillomavirus type 1 capsids.

The capsid proteins of papillomavirus self-assemble to form empty capsids or virus-like particles that appear quite similar to naturally occurring virions by conventional electron microscopy. To characterize such virus-like particles more fully, cryoelectron microscopy and image analysis techniques were used to generate three-dimensional reconstructions of capsids produced by vaccinia virus recombinants (V capsids) that expressed human papillomavirus type 1 L1 protein only or both L1 and L2 proteins. All V capsids had 72 pentameric capsomers arranged on a T = 7 icosahedral lattice. Each particle (approximately 60 nm in diameter) consisted of an approximately 2-nm-thick shell of protein with a radius of 22 nm with capsomers that extend approximately 6 nm from the shell. At a resolution of 3.5 nm, both V capsid structures appear identical to the capsid structure of native human papillomavirus type 1 (T. S. Baker, W. W. Newcomb, N. H. Olson, L. M. Cowsert, C. Olson, and J. C. Brown, Biophys. J. 60:1445-1456, 1991), thus implying that expressed and native capsids are structurally equivalent.     

Structure Determination of Feline Calicivirus Virus-Like Particles in the Context of a Pseudo-Octahedral Arrangement

The vesivirus feline calicivirus (FCV) is a positive strand RNA virus encapsidated by an icosahedral T=3 shell formed by the viral VP1 protein. Upon its expression in the insect cell - baculovirus system in the context of vaccine development, two types of virus-like particles (VLPs) were formed, a majority built of 60 subunits (T=1) and a minority probably built of 180 subunits (T=3). The structure of the small particles was determined by x-ray crystallography at 0.8 nm resolution helped by cryo-electron microscopy in order to understand their formation. Cubic crystals belonged to space group P2₁3. Their self-rotation function showed the presence of an octahedral pseudo-symmetry similar to the one described previously by Agerbandje and co-workers for human parvovirus VLPs. The crystal structure could be solved starting from the published VP1 structure in the context of the T=3 viral capsid. In contrast to viral capsids, where the capsomers are interlocked by the exchange of the N-terminal arm (NTA) domain, this domain is disordered in the T=1 capsid of the VLPs. Furthermore it is prone to proteolytic cleavage. The relative orientation of P (protrusion) and S (shell) domains is alerted so as to fit VP1 to the smaller T=1 particle whereas the intermolecular contacts around 2-fold, 3-fold and 5-fold axes are conserved. By consequence the surface of the VLP is very similar compared to the viral capsid and suggests a similar antigenicity. The knowledge of the structure of the VLPs will help to improve their stability, in respect to a use for vaccination.     

Structures of bovine and human papillomaviruses. Analysis by cryoelectron microscopy and three-dimensional image reconstruction.

The structures of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 1 (HPV-1) were determined at 2.5 nm resolution by cryoelectron microscopy and three dimensional image reconstruction techniques. As expected, the reconstructions showed that both viruses consist of a T = 7 icosahedral capsid (approximately 60 nm in diameter) which surrounds a nucleohistone core. The capsid morphologies of the two viruses are nearly indistinguishable. Each capsid consists of a shell layer (approximately 2 nm thick) of nearly continuous density from which capsomers project radially to a maximum height of approximately 5.8 nm. The five-coordinate (pentavalent) and six-coordinate (hexavalent) capsomers both exhibit distinct five-fold axial symmetry as was observed for SV40 and polyoma viruses. Thus, both genera (papilloma and polyoma) of the papovavirus family have now been shown to have the characteristic "all-pentamer" capsid construction. BPV-1 and HPV-1 capsomers consist of a thick (8.6 nm diameter) trunk that broadens distally to form a regular five-pointed, star-shaped head, and proximally to create the shell layer where capsomers associate. A cylindrical channel (approximately 2.8 nm diameter) extends along the axis of each capsomer from the interior of the virus to a point approximately half way to the capsomer surface. Computationally sectioned views of individual capsomers displayed at decreasing radii show that each of the five capsomer subunits (in both pentavalent and hexavalent capsomers) makes a pronounced (30 degrees) left-handed twist just above the outer surface of the capsid shell. Similar views of the reconstructions also clarify the morphology of intercapsomer contacts. For example, they show how hexavalent capsomers coordinate six neighboring capsomers despite the fact that they contain only five subunits. The system of intercapsomer contacts is indistinguishable in BPV-1 and HPV-1, but quite different from that reported for polyoma virus capsids assembled in vitro from the major capsid protein, VP1 (D. M. Salunke, D. L. D. Caspar, and R. L. Garcea. 1989. Biophys. J. 56:887-900). Thus, because both polyoma and papilloma viruses have all-pentamer capsids, it appears that intracapsomer subunit-subunit interactions which stabilize pentameric capsomers are better preserved evolutionarily than those involved in capsomer-capsomer contacts.     

Crystallographic structure of the T=1 particle of brome mosaic virus

T=1 icosahedral particles of amino terminally truncated brome mosaic virus (BMV) protein were created by treatment of the wild-type T=3 virus with 1M CaCl2 and crystallized from sodium malonate. Diffraction data were collected from frozen crystals to beyond 2.9 A resolution and the structure determined by molecular replacement and phase extension. The particles are composed of pentameric capsomeres from the wild-type virions which have reoriented with respect to the original particle pentameric axes by rotations of 37 degrees , and formed tenuous interactions with one another, principally through conformationally altered C-terminal polypeptides. Otherwise, the pentamers are virtually superimposable upon those of the original T=3 BMV particles. The T=1 particles, in the crystals, are not perfect icosahedra, but deviate slightly from exact symmetry, possibly due to packing interactions. This suggests that the T=1 particles are deformable, which is consistent with the loose arrangement of pentamers and latticework of holes that penetrate the surface. Atomic force microscopy showed that the T=3 to T=1 transition could occur by shedding of hexameric capsomeres and restructuring of remaining pentamers accompanied by direct condensation. Knowledge of the structures of the BMV wild-type and T=1 particles now permit us to propose a tentative model for that process. A comparison of the BMV T=1 particles was made with the reassembled T=1 particles produced from the coat protein of trypsin treated alfalfa mosaic virus (AlMV), another bromovirus. There is little resemblance between the two particles. The BMV particle, with a maximum diameter of 195 A, is made from distinctive pentameric capsomeres with large holes along the 3-fold axis, while the AlMV particle, of approximate maximum diameter 220 A, has subunits closely packed around the 3-fold axis, large holes along the 5-fold axis, and few contacts within pentamers. In both particles crucial linkages are made about icosahedral dyads.     

Structure of bluetongue virus particles by cryoelectron microscopy.

The structure of the bluetongue virus (BTV) particle, determined by cryoelectron microscopy and image analysis, reveals a well-ordered outer shell which differs markedly from other known Reoviridae. The inner shell is known to have an icosahedral structure with 260 triangular spikes of VP7 trimers arranged on a T = 13,l lattice. The outer shell is seen to consist of 120 globular regions (possibly VP5), which sit neatly on each of the six-membered rings of VP7 trimers. "Sail"-shaped spikes located above 180 of the VP7 trimers form 60 triskelion-type motifs which cover all but 20 of the VP7 trimers. These spikes are possibly the hemagglutinating protein VP2 which contains a virus neutralization epitope. Thus, VP2 and VP5 together form a continuous layer around the inner shell except for holes on the 5-fold axis.     

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.     

Three-dimensional structure of physalis mottle virus: implications for the viral assembly.

The structure of the T=3 single stranded RNA tymovirus, physalis mottle virus (PhMV), has been determined to 3.8 A resolution. PhMV crystals belong to the rhombohedral space group R 3, with one icosahedral particle in the unit cell leading to 20-fold non-crystallographic redundancy. Polyalanine coordinates of the related turnip yellow mosaic virus (TYMV) with which PhMV coat protein shares 32 % amino acid sequence identity were used for obtaining the initial phases. Extensive phase refinement by real space molecular replacement density averaging resulted in an electron density map that revealed density for most of the side-chains and for the 17 residues ordered in PhMV, but not seen in TYMV, at the N terminus of the A subunits. The core secondary and tertiary structures of the subunits have a topology consistent with the capsid proteins of other T=3 plant viruses. The N-terminal arms of the A subunits, which constitute 12 pentamers at the icosahedral 5-fold axes, have a conformation very different from the conformations observed in B and C subunits that constitute hexameric capsomers with near 6-fold symmetry at the icosahedral 3-fold axes. An analysis of the interfacial contacts between protein subunits indicates that the hexamers are held more strongly than pentamers and hexamer-hexamer contacts are more extensive than pentamer-hexamer contacts. These observations suggest a plausible mechanism for the formation of empty capsids, which might be initiated by a change in the conformation of the N-terminal arm of the A subunits. The structure also provides insights into immunological and mutagenesis results. Comparison of PhMV with the sobemovirus, sesbania mosaic virus reveals striking similarities in the overall tertiary fold of the coat protein although the capsid morphologies of these two viruses are very different.     

Three-dimensional structures of maturable and abortive capsids of equine herpesvirus 1 from cryoelectron microscopy.

Cryoelectron microscopy and three-dimensional computer reconstruction techniques have been used to compare the structures of two types of DNA-free capsids of equine herpesvirus 1 at a resolution of 4.5 nm. "Light" capsids are abortive, whereas "intermediate" capsids are related to maturable intracellular precursors. Their T = 16 icosahedral outer shells, approximately 125 nm in diameter, are indistinguishable and may be described in terms of three layers of density, totalling 15 nm in thickness. The outermost layer consists of protruding portions of both the hexon and the penton capsomers, rising approximately 5 nm above a midlayer of density. The innermost layer, or "floor," is a 4-nm-thick sheet of virtually continuous density except for the orifices of the channels that traverse each capsomer. Hexon protrusions are distinctly hexagonal in shape, and penton protrusions are pentagonal. The structures of the three kinds of hexons (distinguished according to their positions on the surface lattice) are closely similar but differ somewhat in their respective orientations and in the shapes of their channels. The most prominent features of the midlayer are threefold nodules ("triplexes") at the trigonal lattice points. By analogy with other viral capsids, the triplexes may represent trimers of another capsid protein, possibly VP23 (36 kilodaltons [kDa]) or VP26 (12 kDa). Intermediate capsids differ from light capsids, which are empty, in having one or more internal components. In individual images from which the shell structure has been filtered away, these components are seen to have dimensions of 20 to 30 nm but to lack a visible substructure. This material--which is smeared out in the reconstruction, implying that its distribution is not icosahedrally symmetric or necessarily consistent from particle to particle--consists of aggregates of VP22 (46 kDa). From several lines of evidence, we conclude that this protein is located entirely within the capsid shell. These aggregates may be the remnants of morphogenetic cores retained in capsids interrupted in the process of DNA packaging.     

Particle Polymorphism Caused by Deletion of a Peptide Molecular Switch in a Quasiequivalent Icosahedral Virus

The capsid of flock house virus is composed of 180 copies of a single type of coat protein which forms a T=3 icosahedral shell. High-resolution structural analysis has shown that the protein subunits, although chemically identical, form different contacts across the twofold axes of the virus particle. Subunits that are related by icosahedral twofold symmetry form flat contacts, whereas subunits that are related by quasi-twofold symmetry form bent contacts. The flat contacts are due to the presence of ordered genomic RNA and an ordered peptide arm which is inserted in the groove between the subunits and prevents them from forming the dihedral angle observed at the bent quasi-twofold contacts. We hypothesized that by deleting the residues that constitute the ordered peptide arm, formation of flat contacts should be impossible and therefore result in assembly of particles with only bent contacts. Such particles would have T=1 symmetry. To test this hypothesis we generated two deletion mutants in which either 50 or 31 residues were eliminated from the N terminus of the coat protein. We found that in the absence of residues 1 to 50, assembly was completely inhibited, presumably because the mutation removed a cluster of positively charged amino acids required for neutralization of encapsidated RNA. When the deletion was restricted to residues 1 to 31, assembly occurred, but the products were highly heterogeneous. Small bacilliform-like structures and irregular structures as well as wild-type-like T=3 particles were detected. The anticipated T=1 particles, on the other hand, were not observed. We conclude that residues 20 to 30 are not critical for formation of flat protein contacts and formation of T=3 particles. However, the N terminus of the coat protein appears to play an essential role in regulating assembly such that only one product, T=3 particles, is synthesized.     

An externalized polypeptide partitions between two distinct sites on genome-released poliovirus particles

During cell entry, native poliovirus (160S) converts to a cell-entry intermediate (135S) particle, resulting in the externalization of capsid proteins VP4 and the amino terminus of VP1 (residues 1 to 53). Externalization of these entities is followed by release of the RNA genome (uncoating), leaving an empty (80S) particle. The antigen-binding fragment (Fab) of a monospecific peptide 1 (P1) antibody, which was raised against a peptide corresponding to amino-terminal residues 24 to 40 of VP1, was utilized to track the location of the amino terminus of VP1 in the 135S and 80S states of poliovirus particles via cryogenic electron microscopy (cryo-EM) and three-dimensional image reconstruction. On 135S, P1 Fabs bind to a prominent feature on the external surface known as the “propeller tip.” In contrast, our initial 80S-P1 reconstruction showed P1 Fabs also binding to a second site, at least 50 Å distant, at the icosahedral 2-fold axes. Further analysis showed that the overall population of 80S-P1 particles consisted of three kinds of capsids: those with P1 Fabs bound only at the propeller tips, P1 Fabs bound only at the 2-fold axes, or P1 Fabs simultaneously bound at both positions. Our results indicate that, in 80S particles, a significant fraction of VP1 can deviate from icosahedral symmetry. Hence, this portion of VP1 does not change conformation synchronously when switching from the 135S state. These conclusions are compatible with previous observations of multiple conformations of the 80S state and suggest that movement of the amino terminus of VP1 has a role in uncoating. Similar deviations from icosahedral symmetry may be biologically significant during other viral transitions.     

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.     

Bacteriophage lambda stabilization by auxiliary protein gpD: timing, location, and mechanism of attachment determined by cryo-EM

We report the cryo-EM structure of bacteriophage lambda and the mechanism for stabilizing the 20-A-thick capsid containing the dsDNA genome. The crystal structure of the HK97 bacteriophage capsid fits most of the T = 7 lambda particle density with only minor adjustment. A prominent surface feature at the 3-fold axes corresponds to the cementing protein gpD, which is necessary for stabilization of the capsid shell. Its position coincides with the location of the covalent cross-link formed in the docked HK97 crystal structure, suggesting an evolutionary replacement of this gene product in lambda by autocatalytic chemistry in HK97. The crystal structure of the trimeric gpD, in which the 14 N-terminal residues required for capsid binding are disordered, fits precisely into the corresponding EM density. The N-terminal residues of gpD are well ordered in the cryo-EM density, adding a strand to a beta-sheet formed by the capsid proteins and explaining the mechanism of particle stabilization.     

The structure of pariacoto virus reveals a dodecahedral cage of duplex RNA

The 3.0 A resolution crystal structure of Pariacoto virus (PaV) reveals extensive interactions between portions of the viral RNA genome and the icosahedral capsid. Under the protein shell of the T = 3 quasi equivalent capsid lies a dodecahedral cage composed of RNA duplex that accounts for approximately 35% of the single-stranded RNA genome. The highly basic N-terminal regions (residues 7-54) of the subunits, forming pentamers (A subunits) are clearly visible in the density map and make numerous interactions with the RNA cage. The C-terminal segments (residues 394-401) of the A subunits lie in channels near the quasi three-fold axes. Electron cryo-microscopy and image reconstruction of PaV particles clearly show the dodecahedral RNA cage.     

Mechanical disassembly of single virus particles reveals kinetic intermediates predicted by theory

New experimental approaches are required to detect the elusive transient intermediates predicted by simulations of virus assembly or disassembly. Here, an atomic force microscope (AFM) was used to mechanically induce partial disassembly of single icosahedral T=1 capsids and virions of the minute virus of mice. The kinetic intermediates formed were imaged by AFM. The results revealed that induced disassembly of single minute-virus-of-mice particles is frequently initiated by loss of one of the 20 equivalent capsomers (trimers of capsid protein subunits) leading to a stable, nearly complete particle that does not readily lose further capsomers. With lower frequency, a fairly stable, three-fourths-complete capsid lacking one pentamer of capsomers and a free, stable pentamer were obtained. The intermediates most frequently identified (capsids missing one capsomer, capsids missing one pentamer of capsomers, and free pentamers of capsomers) had been predicted in theoretical studies of reversible capsid assembly based on thermodynamic-kinetic models, molecular dynamics, or oligomerization energies. We conclude that mechanical manipulation and imaging of simple virus particles by AFM can be used to experimentally identify kinetic intermediates predicted by simulations of assembly or disassembly.     

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.