Alteration of amino acid 101 within capsid protein VP-1 changes the pathogenicity of Theiler's murine encephalomyelitis virus.

Chronic Theiler's murine encephalomyelitis virus infection of susceptible mice is an animal model for human demyelinating diseases. Previously we described an altered and diminished pattern of central nervous system disease in immunocompetent SJL/J mice infected with a variant virus. This variant virus H7A6-2 was selected with a neutralizing mAb recognizing the capsid protein VP-1 of Theiler's virus. Here we characterize the variant virus by ELISA and neutralization assays and by sequencing selected regions of the viral RNA genome and relate the alteration to disease. The variant virus contains one single point mutation within a neutralizing epitope of VP-1. This nucleotide change lead to an amino acid replacement at amino acid 101 of VP-1, a threonine (wild type) to an isoleucine (variant). Model building based on sequence alignments and the known structure of the related Mengo virus indicates that the altered amino acid is located in an exposed loop on the surface of the virus at the periphery of a site that has been proposed to be the receptor binding site. The results of ELISA, neutralization assay, and direct RNA sequencing provide for the first time an opportunity to precisely map an important structural determinant of neurovirulence.

Three-dimensional structure of poliovirus at 2.9 A resolution.

The three-dimensional structure of poliovirus has been determined at 2.9 A resolution by x-ray crystallographic methods. Each of the three major capsid proteins (VP1, VP2, and VP3) contains a "core" consisting of an eight-stranded antiparallel beta barrel with two flanking helices. The arrangement of beta strands and helices is structurally similar and topologically identical to the folding pattern of the capsid proteins of several icosahedral plant viruses. In each of the major capsid proteins, the "connecting loops" and NH2- and COOH-terminal extensions are structurally dissimilar. The packing of the subunit "cores" to form the virion shell is reminiscent of the packing in the T = 3 plant viruses, but is significantly different in detail. Differences in the orientations of the subunits cause dissimilar contacts at protein-protein interfaces, and are also responsible for two major surface features of the poliovirion: prominent peaks at the fivefold and threefold axes of the particle. The positions and interactions of the NH2- and COOH-terminal strands of the capsid proteins have important implications for virion assembly. Several of the "connecting loops" and COOH-terminal strands form prominent radial projections which are the antigenic sites of the virion.

Structures of poliovirus complexes with anti-viral drugs: implications for viral stability and drug design.

BACKGROUND: Picornaviruses, such as the structurally related polioviruses and rhinoviruses, are important human pathogens which have been the target of major drug development efforts. Receptor-mediated uncoating and thermal inactivation of poliovirus and rhinovirus are inhibited by agents that bind to each virus by inserting into a pocket in the beta barrel of the viral capsid protein, VP1. This pocket, which is normally empty in human rhinovirus-14 (HRV14), is occupied by an unknown natural ligand in poliovirus. Structural studies of HRV14-drug complexes have shown that drug binding causes large, localized changes in the conformation of VP1. RESULTS: We report the crystal structures of six complexes between poliovirus and capsid-binding, antiviral drugs, including complexes of four different drugs with the Sabin vaccine strain of type 3 poliovirus, and complexes of one of these drugs with two other poliovirus strains that contain sequence differences in the drug-binding site. In each complex, the changes in capsid structure associated with drug binding are limited to minor adjustments in the conformations of a few side chains lining the binding site. CONCLUSIONS: The minor structural changes caused by drug binding suggest a model of drug action in which it is the conformational changes prevented by the bound drug, rather than obvious conformational changes induced by drug binding, which exert the biological effect. Our results, along with additional structures of rhinovirus-drug complexes, suggest possible improvements in drug design, and provide important clues about the nature of the conformational changes that are involved in the uncoating process.

The structure of the substrate-free form of MurB, an essential enzyme for the synthesis of bacterial cell walls.

BACKGROUND: The repeating disaccharide and pentapeptide units of the bacterial peptidoglycan layer are connected by a lactyl ether bridge biosynthesized from UDP-N-acetylglucosamine and phosphoenolpyruvate in sequential enol ether transfer and reduction steps catalyzed by MurA and MurB respectively. Knowledge of the structure and mechanism of the MurB enzyme will permit analysis of this unusual enol ether reduction reaction and may facilitate the design of inhibitors as candidate next-generation antimicrobial agents. RESULTS: The crystal structure of UDP-N-acetylenolpyruvylglucosamine reductase, MurB, has been solved at 3.0 A and compared with our previously reported structure of MurB complexed with its substrate enolpyruvyl-UDP-N- acetylglucosamine. Comparison of the liganded structure of MurB with this unliganded form reveals that the binding of substrate induces a substantial movement of domain 3 (residues 219-319) of the enzyme and a significant rearrangement of a loop within this domain. These ligand induced changes disrupt a stacking interaction between two tyrosines (Tyr190 and Tyr254) which lie at the side of the channel leading to the active site of the free enzyme. CONCLUSIONS: The conformational change induced by enolpyruvyl-UDP-N- acetylglucosamine binding to MurB results in the closure of the substrate-binding channel over the substrate. Tyr190 swings over the channel opening and establishes a hydrogen bond with an oxygen of the alpha-phosphate of the sugar nucleotide substrate which is critical to substrate binding.

Poliovirus: new insights from an old paradigm.

A combination of structural and genetic studies of poliovirus suggests that the final stages of viral assembly lock the virus in a metastable structure primed to undergo the receptor-catalyzed conformational changes required for cell entry. Future studies promise to provide detailed insights into the conformational dynamics of the virion during its life cycle.

Structural basis of the oligomerization of hepatitis delta antigen.

BACKGROUND: The hepatitis D virus (HDV) is a small satellite virus of hepatitis B virus (HBV). Coinfection with HBV and HDV causes severe liver disease in humans. The small 195 amino-acid form of the hepatitis delta antigen (HDAg) functions as a trans activator of HDV replication. A larger form of the protein containing a 19 amino acid C-terminal extension inhibits viral replication. Both of these functions are mediated in part by a stretch of amino acids predicted to form a coiled coil (residues 13-48) that is common to both forms. It is believed that HDAg forms dimers and higher ordered structures through this coiled-coil region. RESULTS: The high-resolution crystal structure of a synthetic peptide corresponding to residues 12 to 60 of HDAg has been solved. The peptide forms an antiparallel coiled coil, with hydrophobic residues near the termini of each peptide forming an extensive hydrophobic core with residues C-terminal to the coiled-coil domain in the dimer protein. The structure shows how HDAg forms dimers, but also shows the dimers forming an octamer that forms a 50 A ring lined with basic sidechains. This is confirmed by cross-linking studies of full-length recombinant small HDAg. CONCLUSIONS: HDAg dimerizes through an antiparallel coiled coil. Dimers then associate further to form octamers through residues in the coiled-coil domain and residues C-terminal to this region. Our findings suggest that the structure of HDAg represents a previously unseen organization of a nucleocapsid protein and raise the possibility that the N terminus may play a role in binding the viral RNA.

Structure and assembly of turnip crinkle virus. I. X-ray crystallographic structure analysis at 3.2 A resolution.

The structure of turnip crinkle virus has been determined at 3.2 A resolution, using the electron density of tomato bushy stunt virus as a starting point for phase refinement by non-crystallographic symmetry. The structures are very closely related, especially in the subunit arm and S domain, where only small insertions and deletions and small co-ordinate shifts relate one chain to another. The P domains, although quite similar in fold, are oriented somewhat differently with respect to the S domains. Understanding of the structure of turnip crinkle virus has been important for analyzing its assembly, as described in an accompanying paper.

Ab initio phasing of high-symmetry macromolecular complexes: successful phasing of authentic poliovirus data to 3.0 A resolution.

A genetic algorithm-based computational method for the ab initio phasing of diffraction data from crystals of symmetric macromolecular structures, such as icosahedral viruses, has been implemented and applied to authentic data from the P1/Mahoney strain of poliovirus. Using only single-wavelength native diffraction data, the method is shown to be able to generate correct phases, and thus electron density, to 3.0 A resolution. Beginning with no advance knowledge of the shape of the virus and only approximate knowledge of its size, the method uses a genetic algorithm to determine coarse, low-resolution (here, 20.5 A) models of the virus that obey the known non-crystallographic symmetry (NCS) constraints. The best scoring of these models are subjected to refinement and NCS-averaging, with subsequent phase extension to high resolution (3.0 A). Initial difficulties in phase extension were overcome by measuring and including all low-resolution terms in the transform. With the low-resolution data included, the method was successful in generating essentially correct phases and electron density to 6.0 A in every one of ten trials from different models identified by the genetic algorithm. Retrospective analysis revealed that these correct high-resolution solutions converged from a range of significantly different low-resolution phase sets (average differences of 59.7 degrees below 24 A). This method represents an efficient way to determine phases for icosahedral viruses, and has the advantage of producing phases free from model bias. It is expected that the method can be extended to other protein systems with high NCS.

Stabilization of poliovirus by capsid-binding antiviral drugs is due to entropic effects.

When poliovirus attaches to its receptor or is heated in hypotonic buffers, the virion undergoes an irreversible conformational transition from the native 160 S (or N) particle to the 135 S (or A) particle, which is believed to mediate cell entry. The first-order rate constants for the thermally induced transition have been measured as a function of temperature for virus alone and for complexes of the virus with capsid-binding drugs that inhibit the receptor and thermally mediated conversion. Although the drugs have minimum inhibitory concentrations (MIC) that differ by almost three orders of magnitude, the activation energies for the N to A transition for the drug complexes (145 kcal/mol) were indistinguishable from each other or from that of the virus alone. We conclude that the antiviral activity of these drugs derives from a novel mechanism in which drug-binding stabilizes the virions through entropic effects.

A structurally biased combinatorial approach for discovering new anti-picornaviral compounds.

BACKGROUND: Picornaviruses comprise a family of small, non-enveloped RNA viruses. A common feature amongst many picornaviruses is a hydrophobic pocket in the core of VP1, one of the viral capsid proteins. The pocket is normally occupied by a mixture of unidentified, fatty acid-like moieties, which can be competed out by a family of capsid-binding, antiviral compounds. Many members of the Picornaviridae family are pathogenic to both humans and livestock, yet no adequate therapeutics exist despite over a decade's worth of research in the field. To address this challenge, we developed a strategy for rapid identification of capsid-binding anti-picornaviral ligands. The approach we took involved synthesizing structurally biased combinatorial libraries that had been targeted to the VP1 pocket of poliovirus and rhinovirus. The libraries are screened for candidate ligands with a high throughput mass spectrometry assay. RESULTS: Using the mass spectrometry assay, we were able to identify eight compounds from a targeted library of 75 compounds. The antiviral activity of these candidates was assessed by (i) measuring the effect on the kinetics of viral uncoating and (ii) the protective effect of each drug in traditional cell-based assays. All eight of the candidates exhibited antiviral activity, but three of them were particularly effective against poliovirus and rhinovirus. CONCLUSIONS: The results illustrate the utility of combining structure-based design with combinatorial chemistry. The success of our approach suggests that assessment of small, targeted libraries, which query specific chemical properties, may be the best strategy for surveying all of chemical space for ideal anti-picornaviral compounds.

Crystallization and preliminary X-ray crystallographic studies of UDP-N-acetylenolpyruvylglucosamine reductase.

The overexpression and purification of the second enzyme in Escherichia coli peptidoglycan biosynthesis, UDP-N-acetylenolpyruvylglucosamine reductase (MurB), provided sufficient protein to undertake crystallization and X-ray crystallographic studies of the enzyme. MurB crystallizes in 14-20% PEG 8000, 100 mM sodium cacodylate, pH 8.0, and 200 mM calcium acetate in the presence of its substrate UDP-N-acetylglucosamine enolpyruvate. Crystals of MurB belong to the tetragonal space group P4(1)2(1)2 with a = b = 49.6 A, c = 263.2 A, and alpha = beta = gamma = 90 degrees at -160 degrees C and diffract to at least 2.5 A. Screening for heavy atom derivatives has yielded a single site that is reactive with both methylmercury nitrate and Thimerosal.

Role and mechanism of the maturation cleavage of VP0 in poliovirus assembly: structure of the empty capsid assembly intermediate at 2.9 A resolution.

The crystal structure of the P1/Mahoney poliovirus empty capsid has been determined at 2.9 A resolution. The empty capsids differ from mature virions in that they lack the viral RNA and have yet to undergo a stabilizing maturation cleavage of VP0 to yield the mature capsid proteins VP4 and VP2. The outer surface and the bulk of the protein shell are very similar to those of the mature virion. The major differences between the 2 structures are focused in a network formed by the N-terminal extensions of the capsid proteins on the inner surface of the shell. In the empty capsids, the entire N-terminal extension of VP1, as well as portions corresponding to VP4 and the N-terminal extension of VP2, are disordered, and many stabilizing interactions that are present in the mature virion are missing. In the empty capsid, the VP0 scissile bond is located some 20 A away from the positions in the mature virion of the termini generated by VP0 cleavage. The scissile bond is located on the rim of a trefoil-shaped depression in the inner surface of the shell that is highly reminiscent of an RNA binding site in bean pod mottle virus. The structure suggests plausible (and ultimately testable) models for the initiation of encapsidation, for the RNA-dependent autocatalytic cleavage of VP0, and for the role of the cleavage in establishing the ordered N-terminal network and in generating stable virions.

The antigenic structure of poliovirus.

We have solved the structure of the Mahoney strain of type 1 and the Sabin (attenuated vaccine) strain of type 3 poliovirus by X-ray crystallographic methods. By providing a three-dimensional framework for the interpretation of a wealth of experimental data, the structures have yielded insight into the architecture and assembly of the virus particle, have provided information regarding the entry of virus into susceptible cells, and defined the sites on the virus particle that are recognized by neutralizing monoclonal antibodies. Thus locating mutations in variants selected for resistance to neutralizing monoclonal antibodies has defined three antigenic sites of the surface of the virion, and provided clues as to the mechanisms by which viruses escape neutralization. Finally, comparison of the structures of the two strains, together with analysis of sequences of many poliovirus strains, have begun to define the structural changes associated with serotypic differences between polioviruses.

Cell-induced conformational change in poliovirus: externalization of the amino terminus of VP1 is responsible for liposome binding.

Upon attachment to susceptible cells, poliovirus and a number of other picornaviruses undergo conformational transitions which result in changes in antigenicity, increased protease sensitivity, the loss of the internal capsid protein VP4, and a loss of the ability to attach to cells. These conformationally altered particles have been characterized by using a number of sequence-specific probes, including two proteases, a panel of antiviral monoclonal antibodies, and a panel of antisera against synthetic peptides which correspond to sequences from the capsid protein VP1. With these probes, cell-altered virus is clearly distinguishable from native and heat-inactivated virions. The probes also demonstrate that the cell-induced conformational change alters the accessibility of several regions of the virus. In particular, the amino terminus of VP1, which is entirely internal in the native virion, becomes externalized. Unlike native and heat-inactivated virus, cell-altered virions are able to attach to liposomes. The exposed amino terminus of VP1 is shown to be responsible for liposome attachment. We propose that during infection the amino terminus of VP1 inserts into endosomal membranes and thus plays a role in the mechanism of cell entry.

Trypsin sensitivity of the Sabin strain of type 1 poliovirus: cleavage sites in virions and related particles.

Treatment of the Sabin strain of type 1 poliovirus with trypsin produced two stable fragments of capsid protein VP1 which remained associated with the virions. Trypsinized virus was fully infectious and was neutralized by type-specific antisera. The susceptible site in the Sabin 1 strain was between the lysine at position 99 and the asparagine at position 100. A similar tryptic cleavage occurred in the Leon and Sabin strains of type 3 poliovirus, probably at the arginine at position 100, but not in the type 1 Mahoney strain, which lacks a basic residue at either position 99 or position 100. Tryptic treatment of heat-treated virus and 14S assembly intermediates produced unique stable fragments which were different from those produced in virions. The implications of our results for future characterization of the surface structures of these particles and structural rearrangements in the poliovirus capsid are discussed.

A pseudo-cell based approach to efficient crystallographic refinement of viruses.

Strategies have been developed for the inexpensive refinement of atomic models of viruses and of other highly symmetric structures. These methods, which have been used in the refinement of several strains of poliovirus, focus on an arbitrary-sized parallelepiped (termed the 'protomer' box) containing a single complete averaged copy of the structural motif which forms the protein capsid, together with the fragments of other symmetry-related copies of the motif which are located in its immediate neighborhood. The Fourier transform of the protomer box provides reference structure factors for stereochemically restrained crystallographic refinement of the atomic model parameters. The phases of the reference structure factors are based on the averaged map, and are not permitted to change during the refinement. It is demonstrated that models refined using the protomer box methods do not differ significantly from models refined by more expensive full-cell calculations.

A genetic algorithm for the ab initio phasing of icosahedral viruses.

Genetic algorithms have been investigated as computational tools for the de novo phasing of low-resolution X-ray diffraction data from crystals of icosahedral viruses. Without advance knowledge of the shape of the virus and only approximate knowledge of its size, the virus can be modeled as the symmetry expansion of a short list of nearly tetrahedrally arranged lattice points which coarsely, but uniformly, sample the icosahedrally unique volume. The number of lattice points depends on an estimate of the non-redundant information content at the working resolution limit. This parameterization permits a simple matrix formulation of the model evaluation calculation, resulting in a highly efficient survey of the space of possible models. Initially, one bit per parameter is sufficient, since the assignment of ones and zeros to the lattice points yields a physically reasonable low-resolution image of the virus. The best candidate solutions identified by the survey are refined to relax the constraints imposed by the coarseness of the modeling, and then trials whose intensity-based statistics are comparatively good in all resolution ranges are chosen. This yields an acceptable starting point for symmetry-based direct phase extension about half the time. Improving efficiency by incorporating the selection criterion directly into the genetic algorithm's fitness function is discussed.