A general method to quantify quasi-equivalence in icosahedral viruses

A quantitative, atom-based, method is described for comparing protein subunit interfaces in icosahedral virus capsids with quasi-equivalent surface lattices. An integrated, normalized value (between 0 and 1) based on equivalent residue contacts (Q-score) is computed for every pair of subunit interactions and scores that are significantly above zero readily identify interfaces that are quasi-equivalent to each other. The method was applied to all quasi-equivalent capsid structures (T=3, 4, 7 and 13) in the Protein Data Bank and the Q-scores were interpreted in terms of their structural underpinnings. The analysis allowed classification of T=3 structures into three groups with architectures that resemble different polyhedra with icosahedral symmetry. The preference of subunits to form dimers in the T=4 human Hepatitis B virus capsid (HBV) was clearly reflected in high Q-scores of quasi-equivalent dimers. Interesting differences between the classical T=7 capsid and polyoma-like capsids were also identified. Application of the method to the outer-shell of the T=13 Blue tongue virus core (BTVC) highlighted the modest distortion between the interfaces of the general trimers and the strict trimers of VP7 subunits. Furthermore, the method identified the quasi 2-fold symmetry in the inner capsids of the BTV and reovirus cores. The results show that the Q-scores of various quasi-symmetries represent a "fingerprint" for a particular virus capsid architecture allowing particle classification into groups based on their underlying structural and geometric features.     

Specific Encapsidation of Nodavirus RNAs Is Mediated through the C Terminus of Capsid Precursor Protein Alpha

Flock house virus (FHV) is a small icosahedral insect virus with a bipartite, messenger-sense RNA genome. Its T=3 icosahedral capsid is initially assembled from 180 subunits of a single type of coat protein, capsid precursor protein alpha (407 amino acids). Following assembly, the precursor particles undergo a maturation step in which the alpha subunits autocatalytically cleave between Asn363 and Ala364. This cleavage generates mature coat proteins beta (363 residues) and gamma (44 residues) and is required for acquisition of virion infectivity. The X-ray structure of mature FHV shows that gamma peptides located at the fivefold axes of the virion form a pentameric helical bundle, and it has been suggested that this bundle plays a role in release of viral RNA during FHV uncoating. To provide experimental support for this hypothesis, we generated mutant coat proteins that carried deletions in the gamma region of precursor protein alpha. Surprisingly, we found that these mutations interfered with specific recognition and packaging of viral RNA during assembly. The resulting particles contained large amounts of cellular RNAs and varying amounts of the viral RNAs. Single-site amino acid substitution mutants showed that three phenylalanines located at positions 402, 405, and 407 of coat precursor protein alpha were critically important for specific recognition of the FHV genome. Thus, in addition to its hypothesized role in uncoating and RNA delivery, the C-terminal region of coat protein alpha plays a significant role in recognition of FHV RNA during assembly. A possible link between these two functions is discussed.     

Structural peptides of a nonenveloped virus are involved in assembly and membrane translocation

The capsid of infectious bursal disease virus (IBDV), a nonenveloped virus of the family Birnaviridae, has a T=13l icosahedral shell constituted by a single protein, VP2, and several disordered peptides, all derived from the precursor pVP2. In this study, we show that two of the peptides, pep11 and pep46, control virus assembly and cell entry. Deletion of pep11 or even simple substitution of most of its residues blocks the capsid morphogenesis. Removal of pep46 also prevents capsid assembly but leads to the formation of subviral particles formed by unprocessed VP2 species. Fitting with the VP2 atomic model into three-dimensional reconstructions of these particles demonstrates that the presence of uncleaved pep46 causes a steric hindrance at the vertices, blocking fivefold axis formation. Mutagenesis of the pVP2 maturation sites confirms that C terminus processing is necessary for VP2 to acquire the correct icosahedral architecture. All peptides present on virions are accessible to proteases or biochemical labeling. One of them, pep46, is shown to induce large structural rearrangements in liposomes and to destabilize target membranes, demonstrating its implication in cell entry.     

Large-scale,pH-dependent,quaternary structure changes in an RNA virus capsid are reversible in the absence of subunit autoproteolysis

The assembly and maturation of the coat protein of a T=4, nonenveloped, single-stranded RNA virus, Nudaurelia capensis omega virus (N omega V), was examined by using a recombinant baculovirus expression system. At pH 7.6, the coat protein assembles into a stable particle called the procapsid, which is 450 A in diameter and porous. Lowering the pH to 5.0 leads to a concerted reorganization of the subunits into a 410-A-diameter particle called the capsid, which has no obvious pores. This conformational change is rapid but reversible until slow, autoproteolytic cleavage occurs in at least 15% of the subunits at the lower pH. In this report, we show that expression of subunits with replacement of Asn-570, which is at the cleavage site, with Thr results in assembly of particles with expected morphology but that are cleavage defective. The conformational change from procapsid to capsid is reversible in N570T mutant virus-like particles, in contrast to wild-type particles, which are locked into the capsid conformation after cleavage of the coat protein. The reexpanded procapsids display slightly different properties than the original procapsid, suggesting hysteretic effects. Because of the stability of the procapsid under near-neutral conditions and the reversible properties of the cleavage-defective mutant, N omega V provides an excellent model for the study of pH-induced conformational changes in macromolecular assemblies. Here, we identify the relationship between cleavage and the conformational change and propose a pH-dependent helix-coil transition that may be responsible for the structural rearrangement in N omega V.     

Refined structure of southern bean mosaic virus at 2.9 A resolution

The T = 3 capsid of southern bean mosaic virus is analyzed in detail. The beta-sheets of the beta-barrel folding motif that form the subunits show a high degree of twist, generated by several beta-bulges. Only 34 water molecules were identified in association with the three quasi-equivalent subunits, most of them on the external viral surface. Subunit contacts related by quasi-3-fold axes are similar, are dominated by polar interactions and have almost identical calcium binding sites. There is no metal ion on the quasi-3-fold axis, as previously reported. Subunits related by quasi-2-fold and icosahedral 2-fold axes have different contacts but nevertheless display almost identical interactions between the antiparallel helices alpha A. A dipole-dipole type interaction between these helices may produce an energetically stable hinge that allows two types of dimers in a T = 3 assembly. The temperature factor distribution, the hydrogen-bonding pattern, and the contacts across the icosahedral 2-fold axes suggest that one of the dimer types is present in the intact virion and probably also in solution; the other is produced only during capsid assembly. Interactions along the 5-fold axes are mainly polar and possibly form an ion channel. The beta-sheet structures of the three subunits can be superimposed with considerable precision. Significant relative distortions between quasi-equivalent subunits occur mainly in helices and loops. The two dimeric forms and the subunit distortions are the consequence of the non-equivalent subunit environments in the capsid.     

Three-dimensional structure of baculovirus-expressed Norwalk virus capsids.

The three-dimensional structure of the baculovirus-expressed Norwalk virus capsid has been determined to a resolution of 2.2 nm using electron cryomicroscopy and computer image processing techniques. The empty capsid, 38.0 nm in diameter, exhibits T = 3 icosahedral symmetry and is composed of 90 dimers of the capsid protein. The striking features of the capsid structure are arch-like capsomeres, at the local and strict 2-fold axes, formed by dimers of the capsid protein and large hollows at the icosahedral 5- and 3-fold axes. Despite its distinctive architecture, the Norwalk virus capsid has several similarities with the structures of T = 3 single-stranded RNA (ssRNA) viruses. The structure of the protein subunit appears to be modular with three distinct domains: the distal globular domain (P2) that appears bilobed, a central stem domain (P1), and a lower shell domain (S). The distal domains of the 2-fold related subunits interact with each other to form the top of the arch. The lower domains of the adjacent subunits associate tightly to form a continuous shell between the radii of 11.0 and 15.0 nm. No significant mass density is observed below the radius of 11.0 mm. It is suspected that the hinge peptide in the adjoining region between the central domain and the shell domain may facilitate the subunits adapting to various quasi-equivalent environments. Architectural similarities between the Norwalk virus capsid and the other ssRNA viruses have suggested a possible domain organization along the primary sequence of the Norwalk virus capsid protein. It is suggested that the N-terminal 250 residues constitute the lower shell domain (S) with an eight-strand beta-barrel structure and that the C-terminal residues beyond 250 constitute the protruding (P1+P2) domains. A lack of an N-terminal basic region and the ability of the Norwalk virus capsid protein to form empty T = 3 shells suggest that the assembly pathway and the RNA packing mechanisms may be different from those proposed for tomato bushy stunt virus and southern bean mosaic virus but similar to that in tymoviruses and comoviruses.     

Dynamics and Stability in Maturation of a T = 4 Virus

Nudaurelia capensis ω virus is a T = 4, icosahedral virus with a bipartite, positive-sense RNA genome. Expression of the coat protein gene in a baculovirus system was previously shown to result in the formation of procapsids when purified at pH 7.6. Procapsids are round, porous particles (480 Å diameter) and have T = 4 quasi-symmetry. Reduction of pH from 7.6 to 5.0 resulted in virus-like particles (VLP_5.0) that are morphologically identical with authentic virions, with an icosahedral-shaped capsid and a maximum dimension of 410 Å. VLP_5.0 undergoes a maturation cleavage between residues N570 and F571, creating the covalently independent γ peptide (residues 571-641) that remains associated with the particle. This cleavage also occurs in authentic virions, and in each case, it renders the morphological change irreversible (i.e., capsids do not expand when the pH is raised back to 7.6). However, a non-cleavable mutant, N570T, undergoes the transition reversibly (NT_7.6 ↔ NT_5.0). We used electron cryo-microscopy and three-dimensional image reconstruction to study the icosahedral structures of NT_7.6, NT_5.0, and VLP_5.0 at about 8, 6, and 6 Å resolution, respectively. We employed the 2. 8-Å X-ray model of the mature virus, determined at pH 7.0 (XR_7.0), to establish (1) how and why procapsid and capsid structures differ, (2) why lowering pH drives the transition, and (3) why the non-cleaving NT_5.0 is reversible. We show that procapsid assembly minimizes the differences in quaternary interactions in the particle. The two classes of 2-fold contacts in the T = 4 surface lattice are virtually identical, both mediated by similarly positioned but dynamic γ peptides. Furthermore, quasi and icosahedral 3-fold interactions are indistinguishable. Maturation results from neutralizing the repulsive negative charge at subunit interfaces with significant differentiation of quaternary interactions (one 2-fold becomes flat, mediated by a γ peptide, while the other is bent with the γ peptide disordered) and dramatic stabilization of the particle. The γ peptide at the flat contact remains dynamic when cleavage cannot occur (NT_5.0) but becomes totally immobilized by noncovalent interactions after cleavage (VLP_5.0).     

Maturation of papillomavirus capsids

The papillomavirus capsid is a nonenveloped icosahedral shell formed by the viral major structural protein, L1. It is known that disulfide bonds between neighboring L1 molecules help to stabilize the capsid. However, the kinetics of inter-L1 disulfide bond formation during particle morphogenesis have not previously been examined. We have recently described a system for producing high-titer papillomavirus-based gene transfer vectors (also known as pseudoviruses) in mammalian cells. Here we show that papillomavirus capsids produced using this system undergo a maturation process in which the formation of inter-L1 disulfide bonds drives condensation and stabilization of the capsid. Fully mature capsids exhibit improved regularity and resistance to proteolytic digestion. Although capsid maturation for other virus types has been reported to occur in seconds or minutes, papillomavirus capsid maturation requires overnight incubation. Maturation of the capsids of human papillomavirus types 16 and 18 proceeds through an ordered accumulation of dimeric and trimeric L1 species, whereas the capsid of bovine papillomavirus type 1 matures into more extensively cross-linked forms. The presence of encapsidated DNA or the minor capsid protein, L2, did not have major effects on the kinetics or extent of capsid maturation. Immature capsids and capsids formed from L1 mutants with impaired disulfide bond formation are infectious but physically fragile. Consequently, capsid maturation is essential for efficient purification of papillomavirus-based gene transfer vectors. Despite their obvious morphological differences, mature and immature capsids are similarly neutralizable by various L1- and L2-specific antibodies.     

The last C-terminal residue of VP3, glutamic acid 257, controls capsid assembly of infectious bursal disease virus

Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH(2)-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process.     

The crystal structure of the human hepatitis B virus capsid.

Hepatitis B is a small enveloped DNA virus that poses a major hazard to human health. The crystal structure of the T = 4 capsid has been solved at 3.3 A resolution, revealing a largely helical protein fold that is unusual for icosahedral viruses. The monomer fold is stabilized by a hydrophobic core that is highly conserved among human viral variants. Association of two amphipathic alpha-helical hairpins results in formation of a dimer with a four-helix bundle as the major central feature. The capsid is assembled from dimers via interactions involving a highly conserved region near the C terminus of the truncated protein used for crystallization. The major immunodominant region lies at the tips of the alpha-helical hairpins that form spikes on the capsid surface.     

Combined EM/X-ray imaging yields a quasi-atomic model of the adenovirus-related bacteriophage PRD1 and shows key capsid and membrane interactions

BACKGROUND: The dsDNA bacteriophage PRD1 has a membrane inside its icosahedral capsid. While its large size (66 MDa) hinders the study of the complete virion at atomic resolution, a 1.65-A crystallographic structure of its major coat protein, P3, is available. Cryo-electron microscopy (cryo-EM) and three-dimensional reconstruction have shown the capsid at 20-28 A resolution. Striking architectural similarities between PRD1 and the mammalian adenovirus indicate a common ancestor. RESULTS: The P3 atomic structure has been fitted into improved cryo-EM reconstructions for three types of PRD1 particles: the wild-type virion, a packaging mutant without DNA, and a P3-shell lacking the membrane and the vertices. Establishing the absolute EM scale was crucial for an accurate match. The resulting "quasi-atomic" models of the capsid define the residues involved in the major P3 interactions, within the quasi-equivalent interfaces and with the membrane, and show how these are altered upon DNA packaging. CONCLUSIONS: The new cryo-EM reconstructions reveal the structure of the PRD1 vertex and the concentric packing of DNA. The capsid is essentially unchanged upon DNA packaging, with alterations limited to those P3 residues involved in membrane contacts. These are restricted to a few of the N termini along the icosahedral edges in the empty particle; DNA packaging leads to a 4-fold increase in the number of contacts, including almost all copies of the N terminus and the loop between the two beta barrels. Analysis of the P3 residues in each quasi-equivalent interface suggests two sites for minor proteins in the capsid edges, analogous to those in adenovirus.     

Finding and using local symmetry in identifying lower domain movements in hexon subunits of the herpes simplex virus type 1 B capsid

A characteristic of virus assembly is the use of symmetry to construct a complex capsid from a limited number of different proteins. Many spherical viruses display not only icosahedral symmetry, but also local symmetries, which further increase the redundancy of their structural proteins. We have developed a computational procedure for evaluating the quality of these local symmetries that allows us to probe the extent of local structural variations among subunits. This type of analysis can also provide orientation parameters for carrying out non-icosahedral averaging of quasi-equivalent subunits during three-dimensional structural determination. We have used this procedure to analyze the three types of hexon (P, E and C) in the 8.5 A resolution map of the herpes simplex virus type 1 (HSV-1) B capsid, determined by electron cryomicroscopy. The comparison of the three hexons showed that they have good overall 6-fold symmetry and are almost identical throughout most of their lengths. The largest difference among the three lies near the inner surface in a region of about 34 A in thickness. In this region, the P hexon displays slightly lower 6-fold symmetry than the C and E hexons. More detailed analysis showed that parts of two of the P hexon subunits are displaced counterclockwise with respect to their expected 6-fold positions. The most highly displaced subunit interacts with a subunit from an adjacent P hexon (P'). Using the local 6-fold symmetry axis of the P hexon as a rotation axis, we examined the geometrical relationships among the local symmetry axes of the surrounding capsomeres. Deviations from exact symmetry are also found among these local symmetry axes. The relevance of these findings to the process of capsid assembly is considered.     

Crystallization and preliminary crystallographic analysis of San Miguel sea lion virus: an animal calicivirus

The Caliciviridae is a family of nonenveloped, icosahedral, positive-sense single-stranded RNA viruses. This family of viruses consists of both animal and human pathogens. Adapting human caliciviruses to cell culture has not been successful, whereas some animal caliciviruses, including San Miguel sea lion virus, have been successfully propagated in vitro. Here we report the crystallization of San Miguel sea lion virus serotype 4 (SMSV4) and the preliminary X-ray crystallographic analysis of the crystals. SMSV4 have been crystallized using the hanging-drop method. These crystals diffracted to approximately 3A resolution using a synchrotron radiation source. A single crystal under cryo-conditions yielded a complete set of diffraction data. Data processing of the diffraction patterns showed that SMSV crystals belong to I23 space group with cell dimensions a=b=c=457 A. The crystallographic asymmetric unit includes five icosahedral asymmetric units, each consisting of three capsid protein subunits. In the space group I23, given the icosahedral symmetry and the size of the virus particle, the location of the particle is constrained to be at the point where the crystallographic 2- and 3-fold axes intersect. The orientation of the virus particle in the unit cell was ascertained by self-rotation function calculations.     

Structures of T=1 and T=3 particles of cucumber necrosis virus: evidence of internal scaffolding

Cucumber necrosis virus (CNV) is a member of the genus Tombusvirus, of which tomato bushy stunt virus (TBSV) is the type member. The capsid protein for this group of viruses is composed of three major domains: the R domain, which interacts with the RNA genome: the S domain, which forms the tight capsid shell: and the protruding P domain, which extends approximately 40 Angstrom from the surface. Here, we present the cryo-transmission electron microscopy structures of both the T=1 and T=3 capsids to a resolution of approximately 12 Angstrom. The T=3 capsid is essentially identical with that of TBSV, and the T=1 particles are well described by the A subunit pentons from TBSV. Perhaps most notable is the fact that the T=3 particles have an articulated internal structure with two major internal shells, while the internal core of the T=1 particle is essentially disordered. These internal shells of the T=3 capsid agree extremely well in both dimension and character with published neutron-scattering results. This structure, combined with mutagenesis results in the accompanying article, suggests that the R domain forms an internal icosahedral scaffold that may play a role in T=3 capsid assembly. In addition, the N-terminal region has been shown to be involved in chloroplast targeting. Therefore, this region apparently has remarkably diverse functions that may be distributed unevenly among the quasi-equivalent A, B, and C subunits.     

Cell-penetrating peptides derived from viral capsid proteins

Cell-penetrating peptides (CPP) can translocate across the cell membrane and have been extensively studied for the delivery of proteins, nucleic acids, and therapeutics in mammalian cells. However, characterizations of CPP in plants have only recently been initiated. We showed that the intact virion and a recombinant capsid protein (CaP) from a plant-infecting nonenveloped icosahedral RNA virus, Brome mosaic virus (BMV), can penetrate the membranes of plant protoplasts but are trapped by the extracellular matrix. Furthermore, a 22-residue peptide derived from the N-terminal region of the CaP (CPNT) can enter barley protoplasts and cells of intact barley and Arabidopsis roots. An inhibitor of the macropinocytosis reduced CPNT entry, while treatment with NiCl(2) changed the cellular localization of CPNT. CPNT increased uptake of the green flourescent protein (GFP) into the cell when covalently fused to GFP or when present in trans of GFP. The BMV CPNT overlaps with the sequence known to bind BMV RNA, and it can deliver BMV RNAs into cells, resulting in viral replication, as well as deliver double-stranded RNAs that can induce gene silencing.     

3D domain swapping modulates the stability of members of an icosahedral virus group

BACKGROUND: Rice yellow mottle virus (RYMV) is a major pathogen that dramatically reduces rice production in many African countries. RYMV belongs to the genus sobemovirus, one group of plant viruses with icosahedral capsids and single-stranded, positive-sense RNA genomes. RESULTS: The structure of RYMV was determined and refined to 2.8 A resolution by X-ray crystallography. The capsid contains 180 copies of the coat protein subunit arranged with T = 3 icosahedral symmetry. Each subunit adopts a jelly-roll beta sandwich fold. The RYMV capsid structure is similar to those of other sobemoviruses. When compared with these viruses, however, the betaA arm of the RYMV C subunit, which is a molecular switch that regulates quasi-equivalent subunit interactions, is swapped with the 2-fold-related betaA arm to a similar, noncovalent bonding environment. This exchange of identical structural elements across a symmetry axis is categorized as 3D domain swapping and produces long-range interactions throughout the icosahedral surface lattice. Biochemical analysis supports the notion that 3D domain swapping increases the stability of RYMV. CONCLUSIONS: The quasi-equivalent interactions between the RYMV proteins are regulated by the N-terminal ordered residues of the betaA arm, which functions as a molecular switch. Comparative analysis suggests that this molecular switch can also modulate the stability of the viral capsids.     

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.     

Comparison of the structures of three circoviruses: chicken anemia virus, porcine circovirus type 2, and beak and feather disease virus

Circoviruses are small, nonenveloped icosahedral animal viruses characterized by circular single-stranded DNA genomes. Their genomes are the smallest possessed by animal viruses. Infections with circoviruses, which can lead to economically important diseases, frequently result in virus-induced damage to lymphoid tissue and immunosuppression. Within the family Circoviridae, different genera are distinguished by differences in genomic organization. Thus, Chicken anemia virus is in the genus Gyrovirus, while porcine circoviruses and Beak and feather disease virus belong to the genus CIRCOVIRUS: Little is known about the structures of circoviruses. Accordingly, we investigated the structures of these three viruses with a view to determining whether they are related. Three-dimensional maps computed from electron micrographs showed that all three viruses have a T=1 organization with capsids formed from 60 subunits. Porcine circovirus type 2 and beak and feather disease virus show similar capsid structures with flat pentameric morphological units, whereas chicken anemia virus has stikingly different protruding pentagonal trumpet-shaped units. It thus appears that the structures of viruses in the same genus are related but that those of viruses in different genera are unrelated.