Ion channels in icosahedral virus: a comparative analysis of the structures and binding sites at their fivefold axes.

An analysis of the crystallographically determined structures of the icosahedral protein coats of Tomato Bushy Stunt Virus, Southern Bean Mosaic Virus, Satellite Tobacco Necrosis Virus, Human Rhinovirus 14 and Mengovirus around their fivefold axes is presented. Accessibilities surfaces, electrostatic energy profile calculations, ion-protein interaction energy calculations, free energy perturbation methods and comparisons with structures of chelating agents are used in this study. It is concluded that the structures built around the viral fivefold axes would be adequate for ion binding and transport. Relative ion preferences are derived for the binding sites, using free energy perturbation methods, which are consistent with the experimental data when available. In the cases where crystallographic studies determined the existence of ions on the fivefold axes, our results indicate that they would correspond to ions in crystallization or purification buffers. The environment of the fivefold axes are rich in polar residues in all icosahedral viral structures whose atomic coordinates are available, including some that are not being analyzed in detail in this work. The fivefold channel-like structures have most of the basic properties expected for real ion channels including a funnel at the entrance, a polar internal environment with frequent alternation of acidic and basic residues, ion binding sites, the capability to induce ion dehydration and ion transit from the external viral surface to the binding sites.

Ab initio phase determination for viruses with high symmetry: a feasibility study.

Conditions that would permit the complete structure determination of spherical viruses that have high internal symmetry were examined starting only from an initial spherical shell model. Problems were considered that might arise due to the following. 1. Creation of centric phases due to the simple shell model and its position in the unit cell. The centric symmetry can generally be broken on averaging an initial electron density map based on observed structure amplitudes, provided that the internal molecular symmetry is sufficiently non-parallel to the crystallographic symmetry. 2. Choice of the average model shell radius. Some incorrect radii led to the Babinet opposite solution (electron density is negative instead of positive). Phases derived from other models with incorrect radii failed to converge to the correct solution. 3. Error in structure amplitude measurements. 4. Lack of a complete data set. 5. Error in positioning the initial spherical-shell model within the crystal unit cell. It was found that an error of 1.6 A caused noticeable phasing error at a resolution greater than 20 A.

Three-dimensional structure of Theiler murine encephalomyelitis virus (BeAn strain).

Depending on the strain, Theiler murine encephalomyelitis virus (TMEV) may cause acute encephalitis or chronic demyelinating disease, which is associated with viral persistence in mice. Persistent central nervous system infection and demyelination by the less-virulent TMEV has provided a useful animal model for the human demyelinating disease multiple sclerosis. The less-virulent BeAn strain of TMEV was crystallized and its atomic structure was determined by x-ray crystallography. The alpha-carbon coordinates of the closely related Mengo virus were used to calculate the initial phases to 3.5 A resolution and the interpretable electron density map was produced by 10 cycles of 30-fold noncrystallographic molecular replacement averaging. The structure revealed a high degree of overall structural similarity to Mengo virus as well as substantial differences in the surface loops. These structural changes might be correlated with TMEV host-specific recognition, pH-related stability, and neurovirulence.

The locked rotation function.

It frequently occurs that a biological assembly in a crystallographic asymmetric unit has more than one noncrystallographic symmetry operator. For instance, a tetramer might have the point group 222 or a spherical virus will have the point group 532. A self-rotation function searches for the direction and angle of rotation of the individual noncrystallographic symmetry operations, while a cross-rotation function searches for the relationship of a structure in one unit cell with similar structures in another cell. The power of the rotation function can be greatly enhanced by searching for all noncrystallographic symmetry operators simultaneously. The procedure described previously [Rossmann, Ford, Watson & Banaszak (1972). J. Mol. Biol. 64, 237-249] has been generalized. The increased power of this 'locked' rotation function permits a good determination of the orientation of an icosahedral virus in the presence of less than 1% of the possible diffraction data to 7 A resolution. In addition, the peak-to-noise ratio is substantially improved.

Functional aspects of the capsid structure of Mengo virus.

The three-dimensional structure of the Mengo virus capsid has been determined at a resolution of 3.0 A. This achievement is discussed in an historical context, and the general features of picornavirus capsid design are presented. The dynamic functional aspects of the Mengo virus capsid--namely its ability to interact with specific receptors on host cells, to dissociate and release the viral genomic RNA into the cellular cytoplasm, to assemble with progeny RNA molecules and form new virions, and to alter its external surface in order to evade neutralization by circulating antibodies--are discussed. Comparisons with other picornaviruses whose capsid structures have also been elucidated (poliovirus serotype 1 and 3, human rhinovirus types 14 and 1A, and foot-and-mouth disease virus type O) illustrate both the similarities and the distinctive features of capsid design found within this family of mammalian viruses.

Structural refinement and analysis of Mengo virus.

The structure of Mengo encephalomyelitis virus was refined at 3 A resolution with a final R-factor of 0.221 and a root-mean-square deviation from idealized bond lengths of 0.019 A for 10 A to 3 A data with F greater than or equal to 3 sigma(F). The Hendrickson-Konnert refinement was restrained by the phases derived from the molecular replacement averaging procedure and constrained by the icosahedral symmetry of the virus. The virus consists of 60 protomers each having three major subunits, VP1, VP2 and VP3, along with one smaller internal protein, VP4. The three major subunits form similar eight-stranded beta-barrel structures. Alterations in the original sequence were found at position 45 in VP1 (Arg to Ala) and at position 58 in VP3 (Met to Val). The residues in loops I and II of VP1 (82 to 102), the "FMDV loop" in VP1 (205 to 213), the flexible loop of VP3 in the putative receptor attachment site (175 to 185) as well as the terminal regions 260 to 268 in VP1, 253 to 256 in VP2 and 13 to 15 in VP4 were built or modified in regions of weak density. The variation in temperature factors at the end of the refinement is over a wide range (from 2 to 80 A2), with the disordered outer and inner regions showing high mobility. Four cis proline residues, 105 in VP1, 85 and 152 in VP2 and 59 in VP3, have been identified. The disulfide bridge Cys86 to Cys88 in VP3 has been characterized. One phosphate ion and 233 water positions were included in the refinement. It is suggested that this phosphate is associated with the receptor attachment site. There are two major hydrogen-bonding networks involving solvent atoms; one involving only the subunits of a protomer, and the other connecting the protomers in a pentamer. The distribution of atom types around the icosahedral symmetry axes shows that the 5-fold channel is more hydrophobic than that along the 3-fold axis and that there are more charged residues around the 2-fold axis. The analysis of contacts between the different subunits supports the assignment of the protomeric unit. The five protomers that form the pentameric unit are held together by interactions involving the smaller VP4 protein and the amino termini of VP1 and VP3. The pentamers are associated by means of the amino-terminal region of the VP2 subunits, the beta F strand of the VP3 subunits, the C terminus of the VP4 subunits and the electrostatic helical (alpha A) interactions of VP2 subunits across the icosahedral 2-fold axes. The superposition of the corresponding subunits of Mengo virus, human rhinovirus 14 and southern bean mosaic virus has provided an improved sequence alignment. The largest structural similarity is between the VP3 subunits of Mengo virus and rhinovirus, while the least similarity is between the VP1 subunits. The various specialized insertions in the different subunits can be associated with specific functional requirements.

Structure determination of Mengo virus.

The structure of Mengo virus was determined to 3.0 A resolution using human rhinovirus 14 as an initial phasing model at 8.0 A resolution. Oscillation diffraction photographs were collected at the Cornell High Energy Synchrotron Source using orthorhombic Mengo virus crystals. The crystal space group was P2(1)2(1)2(1), a = 441.4, b = 427.3 and c = 421.9 A, with one icosahedral particle per asymmetric unit, giving 60-fold noncrystallographic redundancy. The orientations of the four viral particles in the unit cell were determined with a rotation function. Their positions relative to the crystallographic symmetry axes were found by a combination of Patterson-function analysis and a subsequent R-factor search using human rhinovirus 14 atomic coordinates as a model. The initial phases to 8.0 A resolution were then computed by placing human rhinovirus 14 particles in the orientations and positions of Mengo virus particles. These phases were improved by ten cycles of real-space molecular replacement averaging. Phases between 8.0 and 3.0 A resolution were obtained by molecular replacement phase extension. One or two reciprocal-space lattice points were used for each extension followed by two cycles of averaging.

Conservation of the putative receptor attachment site in picornaviruses.

A deep canyon or pit on the surfaces of human rhinovirus 14 and Mengo virus, respectively, has been proposed as a putative receptor binding site. Amino acids lining the surface of the canyon or pit have been identified and show greater conservation than other surface residues.

The atomic structure of Mengo virus at 3.0 A resolution.

The structure of Mengo virus, a representative member of the cardio picornaviruses, is substantially different from the structures of rhino- and polioviruses. The structure of Mengo virus was solved with the use of human rhinovirus 14 as an 8 A resolution structural approximation. Phase information was then extended to 3 A resolution by use of the icosahedral symmetry. This procedure gives promise that many other virus structures also can be determined without the use of the isomorphous replacement technique. Although the organization of the major capsid proteins VP1, VP2, and VP3 of Mengo virus is essentially the same as in rhino- and polioviruses, large insertions and deletions, mostly in VP1, radically alter the surface features. In particular, the putative receptor binding "canyon" of human rhinovirus 14 becomes a deep "pit" in Mengo virus because of polypeptide insertions in VP1 that fill part of the canyon. The minor capsid peptide, VP4, is completely internal in Mengo virus, but its association with the other capsid proteins is substantially different from that in rhino- or poliovirus. However, its carboxyl terminus is located at a position similar to that in human rhinovirus 14 and poliovirus, suggesting the same autocatalytic cleavage of VP0 to VP4 and VP2 takes place during assembly in all these picornaviruses.

Structure of the Mengo virion. VII. Crystallization and preliminary X-ray diffraction analysis.

Crystals of Mengo virions have been grown reproducibly and analyzed by X-ray diffraction. These crystals diffract to a resolution of 7.0 A. The unit cell exhibits cubic symmetry with a = 422 A. The space group is P23, with four virus particles situated on crystallographic threefold axes. Picornavirions from three of the four recognized genera (Study Group on Picornaviridae, Intervirology 10, 165-180, 1978) have now been examined at low resolution by X-ray diffraction: poliovirus type 1 (J. T. Finch and A. Klug, Nature (London) 183, 1709-1714, 1959; J. M. Hogle, J. Mol. Biol. 160, 663-668, 1982); human rhinovirus 14 (J. W. Erickson, E. A. Frankenberger, M. G. Rossmann, G. S. Fout, K. C. Medappa, and R. R. Rueckert, Proc. Natl. Acad. Sci. USA 80, 931-934, 1983); and Mengo virus.