The Structure of Elongated Viral Capsids

There are many viruses whose genetic material is protected by a closed elongated protein shell. Unlike spherical viruses, the structure and construction principles of these elongated capsids are not fully known. In this article, we have developed a general geometrical model to describe the structure of prolate or bacilliform capsids. We show that only a limited set of tubular architectures can be built closed by hemispherical icosahedral caps. In particular, the length and number of proteins adopt a very special set of discrete values dictated by the axial symmetry (fivefold, threefold, or twofold) and the triangulation number of the caps. The results are supported by experimental observations and simulations of simplified physical models. This work brings about a general classification of elongated viruses that will help to predict their structure, and to design viral cages with tailored geometrical properties for biomedical and nanotechnological applications.     

A novel method to map and compare protein-protein interactions in spherical viral capsids

Viral capsids are composed of multiple copies of one or a few chemically distinct capsid proteins and are mostly stabilized by inter subunit protein-protein interactions. There have been efforts to identify and analyze these protein-protein interactions, in terms of their extent and similarity, between the subunit interfaces related by quasi- and icosahedral symmetry. Here, we describe a new method to map quaternary interactions in spherical virus capsids onto polar angle space with respect to the icosahedral symmetry axes using azimuthal orthographic diagrams. This approach enables one to map the nonredundant interactions in a spherical virus capsid, irrespective of its size or triangulation number (T), onto the reference icosahedral asymmetric unit space. The resultant diagrams represent characteristic fingerprints of quaternary interactions of the respective capsids. Hence, they can be used as road maps of the protein-protein interactions to visualize the distribution and the density of the interactions. In addition, unlike the previous studies, the fingerprints of different capsids, when represented in a matrix form, can be compared with one another to quantitatively evaluate the similarity (S-score) in the subunit environments and the associated protein-protein interactions. The S-score selectively distinguishes the similarity, or lack of it, in the locations of the quaternary interactions as opposed to other well-known structural similarity metrics (e.g., RMSD, TM-score). Application of this method on a subset of T = 1 and T = 3 capsids suggests that S-score values range between 1 and 0.6 for capsids that belong to the same virus family/genus; 0.6-0.3 for capsids from different families with the same T-number and similar subunit fold; and <0.3 for comparisons of the dissimilar capsids that display different quaternary architectures (T-numbers). Finally, the sequence conserved interface residues within a virus family, whose spatial locations were also conserved have been hypothesized as the essential residues for self-assembly of the member virus capsids.     

Affine extensions of the icosahedral group with applications to the three-dimensional organisation of simple viruses

Since the seminal work of Caspar and Klug on the structure of the protein containers that encapsulate and hence protect the viral genome, it has been recognised that icosahedral symmetry is crucial for the structural organisation of viruses. In particular, icosahedral symmetry has been invoked in order to predict the surface structures of viral capsids in terms of tessellations or tilings that schematically encode the locations of the protein subunits in the capsids. Whilst this approach is capable of predicting the relative locations of the proteins in the capsids, information on their tertiary structures and the organisation of the viral genome within the capsid are inaccessible. We develop here a mathematical framework based on affine extensions of the icosahedral group that allows us to describe those aspects of the three-dimensional structure of simple viruses. This approach complements Caspar-Klug theory and provides details on virus structure that have not been accessible with previous methods, implying that icosahedral symmetry is more important for virus architecture than previously appreciated.      

Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures.

Assembly of bacteriophage P22 procapsids requires the participation of approximately 300 molecules of scaffolding protein in addition to the 420 coat protein subunits. In the absence of the scaffolding, the P22 coat protein can assemble both wild-type-size and smaller size closed capsids. Both sizes of procapsid assembled in the absence of the scaffolding protein have been studied by electron cryomicroscopy. These structural studies show that the larger capsids have T = 7 icosahedral lattices and appear the same as wild-type procapsids. The smaller capsids possess T = 4 icosahedral symmetry. The two procapsids consist of very similar penton and hexon clusters, except for an increased curvature present in the T = 4 hexon. In particular, the pronounced skewing of the hexons is conserved in both sizes of capsid. The T = 7 procapsid has a local non-icosahedral twofold axis in the center of the hexon and thus contains four unique quasi-equivalent coat protein conformations that are the same as those in the T = 4 procapsid. Models of how the scaffolding protein may direct these four coat subunit types into a T = 7 rather than a T = 4 procapsid are presented.     

Lytic replication of Kaposi's sarcoma-associated herpesvirus results in the formation of multiple capsid species: isolation and molecular characterization of A, B, and C capsids from a gammaherpesvirus

Despite the discovery of Epstein-Barr virus more than 35 years ago, a thorough understanding of gammaherpesvirus capsid composition and structure has remained elusive. We approached this problem by purifying capsids from Kaposi's sarcoma-associated herpesvirus (KSHV), the only other known human gammaherpesvirus. The results from our biochemical and imaging analyses demonstrate that KSHV capsids possess a typical herpesvirus icosahedral capsid shell composed of four structural proteins. The hexameric and pentameric capsomers are composed of the major capsid protein (MCP) encoded by open reading frame 25. The heterotrimeric complexes, forming the capsid floor between the hexons and pentons, are each composed of one molecule of ORF62 and two molecules of ORF26. Each of these proteins has significant amino acid sequence homology to capsid proteins in alpha- and betaherpesviruses. In contrast, the fourth protein, ORF65, lacks significant sequence homology to its structural counterparts from the other subfamilies. Nevertheless, this small, basic, and highly antigenic protein decorates the surface of the capsids, as does, for example, the even smaller basic capsid protein VP26 of herpes simplex virus type 1. We have also found that, as with the alpha- and betaherpesviruses, lytic replication of KSHV leads to the formation of at least three capsid species, A, B, and C, with masses of approximately 200, 230, and 300 MDa, respectively. A capsids are empty, B capsids contain an inner array of a fifth structural protein, ORF17.5, and C capsids contain the viral genome.     

A comparative study of viral capsids and bacterial compartments reveals an enriched understanding of shell dynamics

In this work, we carry out a comparative study of the homo 360‐mer structures of viral capsids and bacterial compartments. Different from viral 360‐mers that all are arranged on a skewed right‐handed icosahedral lattice with a triangulation number T of 7, the new 360‐mer structure of AaLS‐13, an engineered bacterial compartment, offers a novel open conformation that has a unique unskewed lattice arrangement with a triangulation number T of 1 and large keyhole‐shaped pores in the shell. By comparing their differences, we are able to predict a closed conformation of AaLS‐13 that has the same lattice arrangement as existing viral capsid structures and in which all the keyhole‐shaped pores are shut. We find that there is a smooth transition pathway between the open and closed conformations. There exists another close conformation but with an opposite, left handedness, which, however, is not kinetically accessible from the open conformation. Our finding thus provides a clue why existing 360‐mer capsid structures all share the same right handedness. We further show that the conformation transition between the open and closed forms aligns extremely well with the intrinsic dynamics of the system as revealed from normal mode analysis, indicating that conformation transition can be fully driven by thermal fluctuations. The significance of this work is that it provides a better understanding of shell dynamics of both viral capsids and bacterial compartments, paving a way for future study of pore dynamics and the selective permeability of these systems.     

Protruding Features of Viral Capsids Are Clustered on Icosahedral Great Circles

Spherical viruses are remarkably well characterized by the Triangulation (T) number developed by Casper and Klug. The T-number specifies how many viral capsid proteins are required to cover the virus, as well as how they are further subdivided into pentamer and hexamer subunits. The T-number however does not constrain the orientations of these proteins within the subunits or dictate where the proteins should place their protruding features. These protrusions often take the form of loops, spires and helices, and are significant because they aid in stability of the capsid as well as recognition by the host organism. Until now there has be no overall understanding of the placement of protrusions for spherical viruses, other than they have icosahedral symmetry. We constructed a set of gauge points based upon the work affine extensions of Keef and Twarock, which have fixed relative angular locations with which to measure the locations of these features. This work adds a new element to our understanding of the geometric arrangement of spherical viral capsid proteins; chiefly that the locations of protruding features are not found stochastically distributed in an icosahedral manner across the viral surface, but instead these features are found only in specific locations along the 15 icosahedral great circles. We have found that this result holds true as the T number and viral capsids size increases, suggesting an underlying geometric constraint on their locations. This is in spite of the fact that the constraints on the pentamers and hexamer orientations change as a function of T-number, as you need to accommodate more hexamers in the same solid angle between pentamers. The existence of this angular constraint of viral capsids suggests that there is a fitness or energetic benefit to the virus placing its protrusions in this manner. This discovery may have profound impacts on identifying and eliminating viral pathogens, understanding evolutionary constraints as well as bioengineering for capsid drug delivery systems. This result also suggests that in addition to biochemical attachment restrictions, there are additional geometric constraints that should be adhered to when modifying protein capsids.     

Structure of the capsid of Kilham rat virus from small-angle neutron scattering

The structure of empty capsids of Kilham rat virus, an autonomous parvovirus with icosahedral symmetry, was investigated by small-angle neutron scattering. From the forward scatter, the molecular weight was determined to be 4.0 X 10(6), and from the Guinier region, the radius of gyration was found to be 105 A in D2O and 104 A in H2O. On the basis of the capsid molecular weight and the molecular weights and relative abundances of the capsid proteins, we propose that the capsid has a triangulation number of 1. Extended scattering curves and mathematical modeling revealed that the capsid consists of two shells of protein, the inner shell extending from 58 to 91 A in D2O and from 50 to 91 A in H2O and containing 11% of the capsid scattering mass, and the outer shell extending to 121 A in H2O and D2O. The inner shell appears to have a higher content of basic amino acids than the outer shell, based on its lower scattering density in D2O than in H2O. We propose that all three capsid proteins contribute to the inner shell and that this basic region serves DNA binding and partial charge neutralization functions.     

A crystallographic approach to structural transitions in icosahedral viruses

Viruses with icosahedral capsids, which form the largest class of all viruses and contain a number of important human pathogens, can be modelled via suitable icosahedrally invariant finite subsets of icosahedral 3D quasicrystals. We combine concepts from the theory of 3D quasicrystals, and from the theory of structural phase transformations in crystalline solids, to give a framework for the study of the structural transitions occurring in icosahedral viral capsids during maturation or infection. As 3D quasicrystals are in a one-to-one correspondence with suitable subsets of 6D icosahedral Bravais lattices, we study systematically the 6D-analogs of the classical Bain deformations in 3D, characterized by minimal symmetry loss at intermediate configurations, and use this information to infer putative viral-capsid transition paths in 3D via the cut-and-project method used for the construction of quasicrystals. We apply our approach to the Cowpea Chlorotic Mottle virus (CCMV) and show that the putative transition path between the experimentally observed initial and final CCMV structures is most likely to preserve one threefold axis. Our procedure suggests a general method for the investigation and prediction of symmetry constraints on the capsids of icosahedral viruses during structural transitions, and thus provides insights into the mechanisms underlying structural transitions of these pathogens.     

Group theory of icosahedral virus capsid vibrations: A top-down approach

We explore the use of a top-down approach to analyse the dynamics of icosahedral virus capsids and complement the information obtained from bottom-up studies of viral vibrations available in the literature. A normal mode analysis based on protein association energies is used to study the frequency spectrum, in which we reveal a universal plateau of low-frequency modes shared by a large class of Caspar-Klug capsids. These modes break icosahedral symmetry and are potentially relevant to the genome release mechanism. We comment on the role of viral tiling theory in such dynamical considerations.     

Structural determinants of tissue tropism and in vivo pathogenicity for the parvovirus minute virus of mice

Two strains of the parvovirus minute virus of mice (MVM), the immunosuppressive (MVMi) and the prototype (MVMp) strains, display disparate in vitro tropism and in vivo pathogenicity. We report the crystal structures of MVMp virus-like particles (MVMp(b)) and native wild-type (wt) empty capsids (MVMp(e)), determined and refined to 3.25 and 3.75 A resolution, respectively, and their comparison to the structure of MVMi, also refined to 3.5 A resolution in this study. A comparison of the MVMp(b) and MVMp(e) capsids showed their structures to be the same, providing structural verification that some heterologously expressed parvovirus capsids are indistinguishable from wt capsids produced in host cells. The structures of MVMi and MVMp capsids were almost identical, but local surface conformational differences clustered from symmetry-related capsid proteins at three specific domains: (i) the icosahedral fivefold axis, (ii) the "shoulder" of the protrusion at the icosahedral threefold axis, and (iii) the area surrounding the depression at the icosahedral twofold axis. The latter two domains contain important determinants of MVM in vitro tropism (residues 317 and 321) and forward mutation residues (residues 399, 460, 553, and 558) conferring fibrotropism on MVMi. Furthermore, these structural differences between the MVM strains colocalize with tropism and pathogenicity determinants mapped for other autonomous parvovirus capsids, highlighting the importance of common parvovirus capsid regions in the control of virus-host interactions.     

Crystal structure of the potato leafroll virus coat protein and implications for viral assembly

Luteoviruses, poleroviruses, and enamoviruses are insect-transmitted, agricultural pathogens that infect a wide array of plants, including staple food crops. Previous cryo-electron microscopy studies of virus-like particles show that luteovirid viral capsids are built from a structural coat protein that organizes with T = 3 icosahedral symmetry. Here, we present the crystal structure of a truncated version of the coat protein monomer from potato leafroll virus at 1.80-Å resolution. In the crystal lattice, monomers pack into flat sheets that preserve the two-fold and three-fold axes of icosahedral symmetry and show minimal structural deviations when compared to the full-length subunits of the assembled virus-like particle. These observations have important implications in viral assembly and maturation and suggest that the CP N-terminus and its interactions with RNA play an important role in generating capsid curvature.     

The role of arginine-rich motif and beta-annulus in the assembly and stability of Sesbania mosaic virus capsids

Sesbania mosaic virus (SeMV) capsids are stabilized by protein-protein, protein-RNA and calcium-mediated protein-protein interactions. The N-terminal random domain of SeMV coat protein (CP) controls RNA encapsidation and size of the capsids and has two important motifs, the arginine-rich motif (ARM) and the beta-annulus structure. Here, mutational analysis of the arginine residues present in the ARM to glutamic acid was carried out. Mutation of all the arginine residues in the ARM almost completely abolished RNA encapsidation, although the assembly of T=3 capsids was not affected. A minimum of three arginine residues was found to be essential for RNA encapsidation. The mutant capsids devoid of RNA were less stable to thermal denaturation when compared to wild-type capsids. The results suggest that capsid assembly is entirely mediated by CP-dependent protein-protein inter-subunit interactions and encapsidation of genomic RNA enhances the stability of the capsids. Because of the unique structural ordering of beta-annulus segment at the icosahedral 3-folds, it has been suggested as the switch that determines the pentameric and hexameric clustering of CP subunits essential for T=3 capsid assembly. Surprisingly, mutation of a conserved proline within the segment that forms the beta-annulus to alanine, or deletion of residues 48-53 involved in hydrogen bonding interactions with residues 54-58 of the 3-fold related subunit or deletion of all the residues (48-59) involved in the formation of beta-annulus did not affect capsid assembly. These results suggest that the switch for assembly into T=3 capsids is not the beta-annulus. The ordered beta-annulus observed in the structures of many viruses could be a consequence of assembly to optimize intersubunit interactions.     

Partitivirus structure reveals a 120-subunit, helix-rich capsid with distinctive surface arches formed by quasisymmetric coat-protein dimers

Two distinct partitiviruses, Penicillium stoloniferum viruses S and F, can be isolated from the fungus Penicillium stoloniferum. The bisegmented dsRNA genomes of these viruses are separately packaged in icosahedral capsids containing 120 coat-protein subunits. We used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structure of Penicillium stoloniferum virus S at 7.3 A resolution. The capsid, approximately 350 A in outer diameter, contains 12 pentons, each of which is topped by five arched protrusions. Each of these protrusions is, in turn, formed by a quasisymmetric dimer of coat protein, for a total of 60 such dimers per particle. The density map shows numerous tubular features, characteristic of alpha helices and consistent with secondary structure predictions for the coat protein. This three-dimensional structure of a virus from the family Partitiviridae exhibits both similarities to and differences from the so-called "T = 2" capsids of other dsRNA viruses.     

Encapsulation of a polymer by an icosahedral virus

The coat proteins of many viruses spontaneously form icosahedral capsids around nucleic acids or other polymers. Elucidating the role of the packaged polymer in capsid formation could promote biomedical efforts to block viral replication and enable use of capsids in nanomaterials applications. To this end, we perform Brownian dynamics on a coarse-grained model that describes the dynamics of icosahedral capsid assembly around a flexible polymer. We identify several mechanisms by which the polymer plays an active role in its encapsulation, including cooperative polymer-protein motions. These mechanisms are related to experimentally controllable parameters such as polymer length, protein concentration and solution conditions. Furthermore, the simulations demonstrate that assembly mechanisms are correlated with encapsulation efficiency, and we present a phase diagram that predicts assembly outcomes as a function of experimental parameters. We anticipate that our simulation results will provide a framework for designing in vitro assembly experiments on single-stranded RNA virus capsids.     

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.     

Native hepatitis B virions and capsids visualized by electron cryomicroscopy

Hepatitis B virus (HBV) infects more than 350 million people, of which one million will die every year. The infectious virion is an enveloped capsid containing the viral polymerase and double-stranded DNA genome. The structure of the capsid assembled in vitro from expressed core protein has been studied intensively. However, little is known about the structure and assembly of native capsids present in infected cells, and even less is known about the structure of mature virions. We used electron cryomicroscopy (cryo-EM) and image analysis to examine HBV virions (Dane particles) isolated from patient serum and capsids positive and negative for HBV DNA isolated from the livers of transgenic mice. Both types of capsids assembled as icosahedral particles indistinguishable from previous image reconstructions of capsids. Likewise, the virions contained capsids with either T = 3 or T = 4 icosahedral symmetry. Projections extending from the lipid envelope were attributed to surface glycoproteins. Their packing was unexpectedly nonicosahedral but conformed to an ordered lattice. These structural features distinguish HBV from other enveloped viruses.     

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.     

Structural transitions in Cowpea chlorotic mottle virus (CCMV)

Viral capsids act as molecular containers for the encapsulation of genomic nucleic acid. These protein cages can also be used as constrained reaction vessels for packaging and entrapment of synthetic cargos. The icosahedral Cowpea chlorotic mottle virus (CCMV) is an excellent model for understanding the encapsulation and packaging of both genomic and synthetic materials. High-resolution structural information of the CCMV capsid has been invaluable for evaluating structure-function relationships in the assembled capsid but does not allow insight into the capsid dynamics. The dynamic nature of the CCMV capsid might play an important role in the biological function of the virus. The CCMV capsid undergoes a pH and metal ion dependent reversible structural transition where 60 separate pores in the capsid open or close, exposing the interior of the protein cage to the bulk medium. In addition, the highly basic N-terminal domain of the capsid, which is disordered in the crystal structure, plays a significant role in packaging the viral cargo. Interestingly, in limited proteolysis and mass spectrometry experiments the N-terminal domain is the first part of the subunit to be cleaved, confirming its dynamic nature. Based on our fundamental understanding of the capsid dynamics in CCMV, we have utilized these aspects to direct packaging of a range of synthetic materials including drugs and inorganic nanoparticles.