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.     

Phage P22 procapsids equilibrate with free coat protein subunits

Assembly of bacteriophage P22 procapsids has long served as a model for assembly of spherical viruses. Historically, assembly of viruses has been viewed as a non-equilibrium process. Recently alternative models have been developed that treat spherical virus assembly as an equilibrium process. Here we have investigated whether P22 procapsid assembly reactions achieve equilibrium or are irreversibly trapped. To assemble a procapsid-like particle in vitro, pure coat protein monomers are mixed with scaffolding protein. We show that free subunits can exchange with assembled structures, indicating that assembly is a reversible, equilibrium process. When empty procapsid shells (procapsids with the scaffolding protein stripped out) were diluted so that the concentration was below the dissociation constant ( approximately 5 microM) for coat protein monomers, free monomers were detected. The released monomers were assembly-competent; when NaCl was added to metastable partial capsids that were aged for an extended period, the released coat subunits were able to rapidly re-distribute from the partial capsids and form whole procapsids. Lastly, radioactive monomeric coat subunits were able to exchange with the subunits from empty procapsid shells. The data presented illustrate that coat protein monomers are able to dissociate from procapsids in an active state, that assembly of procapsids is consistent with reactions at equilibrium and that the reaction follows the law of mass action.     

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.     

Electrostatic effects in a large enzyme complex: subunit interactions and electrostatic potential field of the icosahedral beta 60 capsid of heavy riboflavin synthase

The role of the electrostatic interactions in the stability of the icosahedral beta 60 capsid of heavy riboflavin synthase from Bacillus subtilis has been investigated using an approach based on the theory of Kirkwood and Tanford. The pH dependence of the electrostatic subunit interactions agrees well with experimental data. The electrostatic subunit interaction energy has a pronounced minimum at pH 8.2 for both the ligated and ligand-free capsid. The latter is characterized by a reduction of the magnitude and the pH range of the electrostatic attraction. It is found that only 8 charged groups, which form one cluster and two ion pairs, provide a significant contribution to the capsid stability. The analysis has shown that the aggregation/disaggregation equilibrium seems to be regulated by electrostatic interactions between beta-subunits forming dimers, which connect the relatively stable pentamers in the beta-60 capsid. The release of the ligand causes a reduction of the electrostatic attraction of the dimers, which may induce disaggregation of the capsid. The electrostatic potential field due to the titratable groups and alpha-helix macrodipoles has been calculated on the basis of the Coulomb relation. Two different values of the dielectric constant have been used for the protein and the surrounding solvent, respectively. The electrostatic potential shows a radially polar distribution with a positive pole at the inner capsid wall and a negative pole outside the capsid. An interesting feature of the electrostatic field is the formation of positive potential "channels" that coincide with the channels constituted by the pentameric and trimeric beta-subunit aggregates. It is supposed that the electrostatic potential field plays a role in enzyme-substrate recognition.     

A Pirhemocyton-like parasite of the Blenny, Blennius pholis L. (Teleostei; Blenniidae) and its relationship to Immanoplasma Neumann, 1909

A light and electron microscopic study was made of a Pirhemocyton-like infection of the red blood cells of B. pholis, an intertidal fish. Electron micrographs show that polygonal particles occur in the cytoplasm of infected cells; these particles resemble the supposed icosahedral virus of Pirhemocyton. Inclusion bodies associated with the infection also resemble those seen in Pirhemocyton.Immanoplasma Neumann, 1909, was re-examined from blood films taken from three infected Scyliorhinus canicula collected in 1970. A marked similarity was noted between the inclusion bodies of the Blenny infection, Pirhemocyton and the Immanoplasma body. The nature of the numerous particles in the infected red blood cells of S. canicula is not known, therefore, Immanoplasma is only tentatively included in the group of known and suspected icosahedral cytoplasmic deoxyriboviruses.     

Structure of an insect virus at 3.0 A resolution

We report the first atomic resolution structure of an insect virus determined by single crystal X-ray diffraction. Black beetle virus has a bipartite RNA genome encapsulated in a single particle. The capsid contains 180 protomers arranged on a T = 3 surface lattice. The quaternary organization of the protomers is similar to that observed in the T = 3 plant virus structures. The protomers consist of a basic, crystallographically disordered amino terminus (64 residues), a beta-barrel as seen in other animal and plant virus subunits, an outer protrusion composed predominantly of beta-sheet and formed by three large insertions between strands of the barrel, and a carboxy terminal domain composed of two distorted helices lying inside the shell. The outer surfaces of quasi-threefold related protomers form trigonal pyramidyl protrusions. A cleavage site, located 44 residues from the carboxy terminus, lies within the central cavity of the protein shell. The structural motif observed in BBV (a shell composed of 180 eight-stranded antiparallel beta-barrels) is common to all nonsatellite spherical viruses whose structures have so far been solved. This highly conserved shell architecture suggests a common origin for the coat protein of spherical viruses, while the primitive genome structure of BBV suggests that this insect virus represents an early stage in the evolution of spherical viruses from cellular genes.     

Structure of the mature P3-virus particle complex of cauliflower mosaic virus revealed by cryo-electron microscopy

The cauliflower mosaic virus (CaMV) has an icosahedral capsid composed of the viral protein P4. The viral product P3 is a multifunctional protein closely associated with the virus particle within host cells. The best-characterized function of P3 is its implication in CaMV plant-to-plant transmission by aphid vectors, involving a P3-virion complex. In this transmission process, the viral protein P2 attaches to virion-bound P3, and creates a molecular bridge between the virus and a putative receptor in the aphid's stylets. Recently, the virion-bound P3 has been suggested to participate in cell-to-cell or long-distance movement of CaMV within the host plant. Thus, as new data accumulate, the importance of the P3-virion complex during the virus life-cycle is becoming more and more evident. To provide a first insight into the knowledge of the transmission process of the virus, we determined the 3D structures of native and P3-decorated virions by cryo-electron microscopy and computer image processing. By difference mapping and biochemical analysis, we show that P3 forms a network around the capsomers and we propose a structural model for the binding of P3 to CaMV capsid in which its C terminus is anchored deeply in the inner shell of the virion, while the N-terminal extremity is facing out of the CaMV capsid, forming dimers by coiled-coil interactions. Our results combined with existing data reinforce the hypothesis that this coiled-coil N-terminal region of P3 could coordinate several functions during the virus life-cycle, such as cell-to-cell movement and aphid-transmission.     

C(alpha) chemical shift tensors in helical peptides by dipolar-modulated chemical shift recoupling NMR

The C chemical shift tensors of proteins contain information on the backbone conformation. We have determined the magnitude and orientation of the C chemical shift tensors of two peptides with -helical torsion angles: the Ala residue in G*AL (=–65.7°, =–40°), and the Val residue in GG*V (=–81.5°, =–50.7°). The magnitude of the tensors was determined from quasi-static powder patterns recoupled under magic-angle spinning, while the orientation of the tensors was extracted from C–H and C–N dipolar modulated powder patterns. The helical Ala C chemical shift tensor has a span of 36 ppm and an asymmetry parameter of 0.89. Its ₁₁ axis is 116° ± 5° from the C–H bond while the ₂₂ axis is 40° ± 5° from the C–N bond. The Val tensor has an anisotropic span of 25 ppm and an asymmetry parameter of 0.33, both much smaller than the values for -sheet Val found recently (Yao and Hong, 2002). The Val ₃₃ axis is tilted by 115° ± 5° from the C–H bond and 98° ± 5° from the C–N bond. These represent the first completely experimentally determined C chemical shift tensors of helical peptides. Using an icosahedral representation, we compared the experimental chemical shift tensors with quantum chemical calculations and found overall good agreement. These solid-state chemical shift tensors confirm the observation from cross-correlated relaxation experiments that the projection of the C chemical shift tensor onto the C–H bond is much smaller in -helices than in -sheets.     

The Capsid Proteins of a Large, Icosahedral dsDNA Virus

Chilo iridescent virus (CIV) is a large (∼ 1850 Å diameter) insect virus with an icosahedral, T = 147 capsid, a double-stranded DNA (dsDNA) genome, and an internal lipid membrane. The structure of CIV was determined to 13 Å resolution by means of cryoelectron microscopy (cryoEM) and three-dimensional image reconstruction. A homology model of P50, the CIV major capsid protein (MCP), was built based on its amino acid sequence and the structure of the homologous Paramecium bursaria chlorella virus 1 Vp54 MCP. This model was fitted into the cryoEM density for each of the 25 trimeric CIV capsomers per icosahedral asymmetric unit. A difference map, in which the fitted CIV MCP capsomers were subtracted from the CIV cryoEM reconstruction, showed that there are at least three different types of minor capsid proteins associated with the capsomers outside the lipid membrane. “Finger” proteins are situated at many, but not all, of the spaces between three adjacent capsomers within each trisymmetron, and “zip” proteins are situated between sets of three adjacent capsomers at the boundary between neighboring trisymmetrons and pentasymmetrons. Based on the results of segmentation and density correlations, there are at least eight finger proteins and three dimeric and two monomeric zip proteins in one asymmetric unit of the CIV capsid. These minor proteins appear to stabilize the virus by acting as intercapsomer cross-links. One transmembrane “anchor” protein per icosahedral asymmetric unit, which extends from beneath one of the capsomers in the pentasymmetron to the internal leaflet of the lipid membrane, may provide additional stabilization for the capsid. These results are consistent with the observations for other large, icosahedral dsDNA viruses that also utilize minor capsid proteins for stabilization and for determining their assembly.