Structure of the pseudorabies virus capsid: comparison with herpes simplex virus type 1 and differential binding of essential minor proteins.

The structure of pseudorabies virus (PRV) capsids isolated from the nucleus of infected cells and from PRV virions was determined by cryo-electron microscopy (cryo-EM) and compared to herpes simplex virus type 1 (HSV-1) capsids. PRV capsid structures closely resemble those of HSV-1, including distribution of the capsid vertex specific component (CVSC) of HSV-1, which is a heterodimer of the pUL17 and pUL25 proteins. Occupancy of CVSC on all PRV capsids is near 100%, compared to ~50% reported for HSV-1 C-capsids and 25% or less that we measure for HSV-1 A- and B-capsids. A PRV mutant lacking pUL25 does not produce C-capsids and lacks visible CVSC density in the cryo-EM-based reconstruction. A reconstruction of PRV capsids in which green fluorescent protein was fused within the N-terminus of pUL25 confirmed previous studies with a similar HSV-1 capsid mutant localizing pUL25 to the CVSC density region that is distal to the penton. However, comparison of the CVSC density in a 9-Å-resolution PRV C-capsid map with the available crystal structure of HSV-1 pUL25 failed to find a satisfactory fit, suggesting either a different fold for PRV pUL25 or a capsid-bound conformation for pUL25 that does not match the X-ray model determined from protein crystallized in solution. The PRV capsid imaged within virions closely resembles C-capsids with the addition of weak but significant density shrouding the pentons that we attribute to tegument proteins. Our results demonstrate significant structure conservation between the PRV and HSV capsids.

Development of the dodecahedral penton particle from adenovirus 3 for therapeutic application.

The subviral dodecahedral particle of adenovirus 3, which assembles spontaneously in insect cells expressing the viral penton base protein, shows promise as a vector for drug delivery. Its ability to gain cell entry has been demonstrated and recent structural analysis has outlined details of the interfaces between penton bases and the importance of proteolysis of the penton base N terminus for assembly, providing a basis for understanding particle assembly and stability. Here, work in manipulating the assembly status of the dodecahedron by changing buffer conditions and subsequent success in passively encapsidating a marker molecule is described. This represents an important stage towards development of the dodecahedral particle for use as a delivery vehicle capable of targeting therapeutic molecules to specific cell types.

Human kinetochore-associated kinesin CENP-E visualized at 17 A resolution bound to microtubules.

The highly dynamic process of cell division is effected, in part, by molecular motors that generate the forces necessary for its enactment. Several members of the kinesin superfamily of motor proteins are implicated in mitosis, such as CENP-E, which plays essential roles in cell division, including association with the kinetochore to stabilize attachment of chromosomes to microtubules prior to and during their separation. Neither the functional assembly state of CENP-E nor its direction of motion along the polar microtubule are certain. To determine the mode of interaction between CENP-E and microtubules, we have used cryo-electron microscopy to visualize CENP-E motor domains complexed with microtubules and calculated a density map of the complex to 17 A resolution by combining helical and single-particle reconstruction methods. The interface between the motor domain and microtubules was modeled by docking atomic-resolution models of the subunits into the cryoEM density map. Our results support a plus end motion for CENP-E, consistent with features of the crystallographic structure. Despite considerable functional differences from the monomeric transporter kinesin KIF1A and the oppositely directed ncd kinesin, CENP-E appears to share many features of the intermolecular interactions, suggesting that differences in motor function are governed by small variations in the loops at the microtubule interface.

Bacteriophage T5 structure reveals similarities with HK97 and T4 suggesting evolutionary relationships.

Evolutionary relationships between viruses may be obscure by protein sequence but unmasked by structure. Analysis of bacteriophage T5 by cryo-electron microscopy and protein sequence analysis reveals analogies with HK97 and T4 that suggest a mosaic of such connections. The T5 capsid is consistent with the HK97 capsid protein fold but has a different geometry, incorporating three additional hexamers on each icosahedral facet. Similarly to HK97, the T5 major capsid protein has an N-terminal extension, or Delta-domain that is missing in the mature capsid, and by analogy with HK97, may function as an assembly or scaffold domain. This Delta-domain is predicted to be largely coiled-coil, as for that of HK97, but is approximately 70% longer correlating with the larger capsid. Thus, capsid architecture appears likely to be specified by the Delta-domain. Unlike HK97, the T5 capsid binds a decoration protein in the center of each hexamer similarly to the "hoc" protein of phage T4, suggesting a common role for these molecules. The tail-tube has unusual trimeric symmetry that may aid in the unique two-stage DNA-ejection process, and joins the tail-tip at a disk where tail fibers attach. This intriguing mix of characteristics embodied by phage T5 offers insights into virus assembly, subunit function, and the evolutionary connections between related viruses.

Structure of the dodecahedral penton particle from human adenovirus type 3.

The sub-viral dodecahedral particle of human adenovirus type 3, composed of the viral penton base and fiber proteins, shares an important characteristic of the entire virus: it can attach to cells and penetrate them. Structure determination of the fiberless dodecahedron by cryo-electron microscopy to 9 Angstroms resolution reveals tightly bound pentamer subunits, with only minimal interfaces between penton bases stabilizing the fragile dodecahedron. The internal cavity of the dodecahedron is approximately 80 Angstroms in diameter, and the interior surface is accessible to solvent through perforations of approximately 20 Angstroms diameter between the pentamer towers. We observe weak density beneath pentamers that we attribute to a penton base peptide including residues 38-48. The intact amino-terminal domain appears to interfere with pentamer-pentamer interactions and its absence by mutation or proteolysis is essential for dodecamer assembly. Differences between the 9 Angstroms dodecahedron structure and the adenovirus serotype 2 (Ad2) crystallographic model correlate closely with differences in sequence. The 3D structure of the dodecahedron including fibers at 16 Angstroms resolution reveals extra density on the top of the penton base that can be attributed to the fiber N terminus. The fiber itself exhibits striations that correlate with features of the atomic structure of the partial Ad2 fiber and that represent a repeat motif present in the amino acid sequence. These new observations offer important insights into particle assembly and stability, as well as the practicality of using the dodecahedron in targeted drug delivery. The structural work provides a sound basis for manipulating the properties of this particle and thereby enhancing its value for such therapeutic use.

Epitope diversity of hepatitis B virus capsids: quasi-equivalent variations in spike epitopes and binding of different antibodies to the same epitope.

To investigate the range of antigenic variation of HBV capsids, we have characterized the epitopes for two anti-capsid antibodies by cryo-electron microscopy and image reconstruction of Fab-labeled capsids to approximately 10A resolution followed by molecular modeling. Both antibodies engage residues on the protruding spikes but their epitopes and binding orientations differ. Steric interference effects limit maximum binding to approximately 50% average occupancy in each case. However, the occupancies of the two copies of a given epitope that are present on a single spike differ, reflecting subtle distinctions in structure and hence, binding affinity, arising from quasi-equivalence. The epitope for mAb88 is conformational but continuous, consisting of a loop-helix motif (residues 77-87) on one of the two polypeptide chains in the spike. In contrast, the epitope for mAb842, like most conformational epitopes, is discontinuous, consisting of a loop on one polypeptide chain (residues 74-78) combined with a loop-helix element (residues 78-83) on the other. The epitope of mAb842 is essentially identical with that previously mapped for mAb F11A4, although the binding orientations of the two monoclonal antibodies (mAbs) differ, as do their affinities measured by surface plasmon resonance. From the number of monoclonals (six) whose binding had to be characterized to give the first duplicate epitope, we estimate the total number of core antigen (cAg) epitopes to be of the order of 20. Given that different antibodies may share the same epitope, the potential number of distinct anti-cAg clones should be considerably higher. The observation that the large majority of cAg epitopes are conformational reflects the relative dimensions of a Fab (large) and the small size and close packing of the motifs that are exposed and accessible on the capsid surface.

Spectral signal-to-noise ratio and resolution assessment of 3D reconstructions.

Measuring the quality of three-dimensional (3D) reconstructed biological macromolecules by transmission electron microscopy is still an open problem. In this article, we extend the applicability of the spectral signal-to-noise ratio (SSNR) to the evaluation of 3D volumes reconstructed with any reconstruction algorithm. The basis of the method is to measure the consistency between the data and a corresponding set of reprojections computed for the reconstructed 3D map. The idiosyncrasies of the reconstruction algorithm are taken explicitly into account by performing a noise-only reconstruction. This results in the definition of a 3D SSNR which provides an objective indicator of the quality of the 3D reconstruction. Furthermore, the information to build the SSNR can be used to produce a volumetric SSNR (VSSNR). Our method overcomes the need to divide the data set in two. It also provides a direct measure of the performance of the reconstruction algorithm itself; this latter information is typically not available with the standard resolution methods which are primarily focused on reproducibility alone.

Diversity of core antigen epitopes of hepatitis B virus.

Core antigen (cAg), the viral capsid, is one of the three major clinical antigens of hepatitis B virus. cAg has been described as presenting either one or two conformational epitopes involving the "immunodominant loop." We have investigated cAg antigenicity by cryo-electron microscopy at approximately 11-A resolution of capsids labeled with monoclonal Fabs, combined with molecular modeling, and describe here two conformational epitopes. Both Fabs bind to the dimeric external spikes, and each epitope has contributions from the loops on both subunits, explaining their discontinuous nature: however, their binding aspects and epitopes differ markedly. To date, four cAg epitopes have been characterized: all are distinct. Although only two regions of the capsid surface are accessible to antibodies, local clustering of the limited number of exposed peptide loops generates a potentially extensive set of discontinuous epitopes. This diversity has not been evident from competition experiments because of steric interference effects. These observations suggest an explanation for the distinction between cAg and e-antigen (an unassembled form of capsid protein) and an approach to immunodiagnosis, exploiting the diversity of cAg epitopes.

Characterization of a conformational epitope on hepatitis B virus core antigen and quasiequivalent variations in antibody binding.

We have characterized a conformational epitope on capsids of hepatitis B virus (HBV) by cryo-electron microscopy and three-dimensional image reconstruction of Fab-labeled capsids to approximately 10-A resolution, combined with molecular modeling. The epitope straddles the interface between two adjacent subunits and is discontinuous, consisting of five peptides-two on one subunit and three on its neighbor. Together, the two icosahedral forms of the HBV capsid-T=3 and T=4 particles-present seven quasiequivalent variants of the epitope. Of these, only three bind this Fab. Occupancy ranges from approximately 100 to approximately 0%, reflecting conformational variations in the epitope and steric blocking effects. In the former, small shifts of the component peptides have large effects on binding affinity. This approach appears to hold general promise for elucidating conformational epitopes of HBV and other viruses, including those of neutralizing and diagnostic significance.

Virus maturation involving large subunit rotations and local refolding.

Large-scale conformational changes transform viral precursors into infectious virions. The structure of bacteriophage HK97 capsid, Head-II, was recently solved by crystallography, revealing a catenated cross-linked topology. We have visualized its precursor, Prohead-II, by cryoelectron microscopy and modeled the conformational change by appropriately adapting Head-II. Rigid-body rotations ( approximately 40 degrees) cause switching to an entirely different set of interactions; in addition, two motifs undergo refolding. These changes stabilize the capsid by increasing the surface area buried at interfaces and bringing the cross-link-forming residues, initially approximately 40 angstroms apart, close together. The inner surface of Prohead-II is negatively charged, suggesting that the transition is triggered electrostatically by DNA packaging.

The cellular receptor to human rhinovirus 2 binds around the 5-fold axis and not in the canyon: a structural view.

Human rhinovirus serotype 2 (HRV2) belongs to the minor group of HRVs that bind to members of the LDL-receptor family including the very low density lipoprotein (VLDL)-receptor (VLDL-R). We have determined the structures of the complex between HRV2 and soluble fragments of the VLDL-R to 15 A resolution by cryo-electron microscopy. The receptor fragments, which include the first three ligand-binding repeats of the VLDL-R (V1-3), bind to the small star-shaped dome on the icosahedral 5-fold axis. This is in sharp contrast to the major group of HRVs where the receptor site for ICAM-1 is located at the base of a depression around each 5-fold axis. Homology models of the three domains of V1-3 were used to explore the virus-receptor interaction. The footprint of VLDL-R on the viral surface covers the BC- and HI-loops on VP1.

Novel fold and capsid-binding properties of the lambda-phage display platform protein gpD.

The crystal structure of gpD, the capsid-stabilizing protein of bacteriophage lambda, was solved at 1.1 A resolution. Data were obtained from twinned crystals in space group P21 and refined with anisotropic temperature factors to an R-factor of 0.098 (Rfree = 0. 132). GpD (109 residues) has a novel fold with an unusually low content of regular secondary structure. Noncrystallographic trimers with substantial intersubunit interfaces were observed. The C-termini are well ordered and located on one side of the trimer, relatively far from its three-fold axis. The N-termini are disordered up to Ser 15, which is close to the three-fold axis and on the same side as the C-termini. A density map of the icosahedral viral capsid at 15 A resolution, obtained by cryo-electron microscopy and image reconstruction, reveals gpD trimers, seemingly indistinguishable from the ones seen in the crystals, at all three-fold sites. The map further reveals that the side of the trimer that binds to the capsid is the side on which both termini reside. Despite this orientation of the gpD trimer, fusion proteins connected by linker peptides to either terminus bind to the capsid, allowing protein and peptide display.

Maturation dynamics of a viral capsid: visualization of transitional intermediate states.

Typical of DNA bacteriophages and herpesviruses, HK97 assembles in two stages: polymerization and maturation. First, capsid protein polymerizes into closed shells; then, these precursors mature into larger, stabler particles. Maturation is initiated by proteolysis, producing a metastable particle primed for expansion-the major structural transition. We induced expansion in vitro by acidic pH and monitored the resulting changes by time-resolved X-ray diffraction and cryo-electron microscopy. The transition, which is not synchronized over the population, proceeds in a series of stochastically triggered subtransitions. Three distinct intermediates were identified, which are comparable to transitional states in protein folding. The intermediates' structures reveal the molecular events occurring during expansion. Integrated into a movie (see Dynamic Visualization below), they show capsid maturation as a dynamic process.

Molecular tectonic model of virus structural transitions: the putative cell entry states of poliovirus.

Upon interacting with its receptor, poliovirus undergoes conformational changes that are implicated in cell entry, including the externalization of the viral protein VP4 and the N terminus of VP1. We have determined the structures of native virions and of two putative cell entry intermediates, the 135S and 80S particles, at approximately 22-A resolution by cryo-electron microscopy. The 135S and 80S particles are both approximately 4% larger than the virion. Pseudoatomic models were constructed by adjusting the beta-barrel domains of the three capsid proteins VP1, VP2, and VP3 from their known positions in the virion to fit the 135S and 80S reconstructions. Domain movements of up to 9 A were detected, analogous to the shifting of tectonic plates. These movements create gaps between adjacent subunits. The gaps at the sites where VP1, VP2, and VP3 subunits meet are plausible candidates for the emergence of VP4 and the N terminus of VP1. The implications of these observations are discussed for models in which the externalized components form a transmembrane pore through which viral RNA enters the infected cell.

Methods for reconstructing density maps of "single" particles from cryoelectron micrographs to subnanometer resolution.

Recently the resolution attainable in density maps calculated from cryo-electron micrographs of free-standing virus capsids has advanced to resolutions below 1 nm. This represents a significant milestone in that resolutions of this order potentially allow direct visualization of individual elements of protein secondary structure (i.e., alpha-helices), in addition to the shapes and connectivity of subdomains. We describe here a computational strategy for structural analyses at this level of detail: its principal innovation is a procedure for correcting the contrast transfer function of the electron microscope. Also important is the practice of combining data from pairs of differently defocused micrographs to improve the signal-to-noise ratio of the images, thereby allowing more precise determinations of the particles' orientations and origins and contributing to higher resolution reconstructions. These procedures proved instrumental in our analysis of the capsid of hepatitis B virus at 9-A resolution (Conway et al., 1997, Nature 386, 91-94). Finally, we discuss the prospects for achieving comparable resolutions for isolated macromolecular complexes with lower symmetry or no symmetry and for further extension of the resolution.

Tetrairidium, a four-atom cluster, is readily visible as a density label in three-dimensional cryo-EM maps of proteins at 10-25 A resolution.

Heavy metal clusters derivatized to bind to designated chemical groups on proteins have great potential as density labels for cryo-electron microscopy. Smaller clusters offer higher resolution and penetrate more easily into sterically restricted sites, but are more difficult to detect. In this context, we have explored the potential of tetrairidium (Ir(4)) as a density label by attaching it via maleimide linkage to the C-terminus of the hepatitis B virus (HBV) capsid protein. Although the clusters are not visible in unprocessed cryo-electron micrographs, they are distinctly visible in three-dimensional density maps calculated from them, even at only partial occupancy. The Ir(4) label was clearly visualized in our maps at 11-14 A resolution of both size variants of the HBV capsid, thus confirming our previous localization of this site with undecagold (Zlotnick, A., Cheng, N., Stahl, S. J., Conway, J. F., Steven, A. C., and Wingfield, P. T., Proc. Natl. Acad. Sci. USA 94, 9556-9561, 1997). Ir(4) penetrated to the interior of intact capsids to label this site on their inner surface, unlike undecagold for which labelling was achieved only with dissociated dimers that were then reassembled into capsids. The Ir(4) cluster remained visible as the resolution of the maps was lowered progressively to approximately 25 A.

Localization of the N terminus of hepatitis B virus capsid protein by peptide-based difference mapping from cryoelectron microscopy.

Recently, cryoelectron microscopy of isolated macromolecular complexes has advanced to resolutions below 10 A, enabling direct visualization of alpha-helical secondary structure. To help correlate such density maps with the amino acid sequences of the component proteins, we advocate peptide-based difference mapping, i. e., insertion of peptides, approximately 10 residues long, at targeted points in the sequence and visualization of these peptides as bulk labels in cryoelectron microscopy-derived difference maps. As proof of principle, we have appended an extraneous octapeptide at the N terminus of hepatitis B virus capsid protein and determined its location on the capsid surface by difference imaging at 11 A resolution. Hepatitis B virus capsids are icosahedral particles, approximately 300 A in diameter, made up of T-shaped dimers (subunit Mr, 16-21 kDa, depending on construct). The stems of the Ts protrude outward as spikes, whereas the crosspieces pack to form the contiguous shell. The two N termini per dimer reside on either side of the spike-stem, at the level at which it enters the shell. This location is consistent with formation of the known intramolecular disulfide bond between the cysteines at positions 61 and -7 (in the residual propeptide) in the "e-antigen" form of the capsid protein and has implications for why this clinically important antigen remains unassembled in vivo.

Shared motifs of the capsid proteins of hepadnaviruses and retroviruses suggest a common evolutionary origin.

The structure of the dimeric C-terminal domain of the HIV-1 capsid protein (CA), recently determined by X-ray crystallography (Gamble et al. (1997)), has a notable resemblance to the structure of the hepatitis B virus (HBV) capsid protein (Cp) dimer, previously determined by cryo-electron microscopy (Conway et al. (1997), Böttcher et al. (1997)). In both proteins, dimerization is effected by formation of a four-helix bundle, whereby each subunit contributes a helix-loop-helix and most of the interaction between subunits is mediated by one pair of helices. These are the first two observations of a motif that is common to the capsid proteins of two enveloped viruses and quite distinct from the eight-stranded anti-parallel beta-barrel found in most other virus capsid proteins solved to date (Harrison et al. (1996)). Motivated by the structural resemblance, we have examined retroviral and HBV capsid protein sequences and found weak but significant similarities between them. These similarities further support an evolutionary relationship between these two virus families of great medical importance -- the hepadnaviruses (e.g. HBV) and retroviruses (e.g. HIV).

Hepatitis B virus capsid: localization of the putative immunodominant loop (residues 78 to 83) on the capsid surface, and implications for the distinction between c and e-antigens.

Hepatitis B virus capsid protein comprises a 149 residue "assembly" domain that polymerizes into icosahedral particles, and a 34 residue RNA-binding "protamine" domain. Recently, the capsid structure has been studied to resolutions below 10 A by cryo-electron microscopy, revealing much of its alpha-helical substructure and that it appears to have a novel fold for a capsid protein; however, the resolution is still too low for chain-tracing by conventional criteria. Aiming to establish a fiducial marker to aid in the process of chain-tracing, we have used cryo-microscopy to pinpoint the binding site of a monoclonal antibody that recognizes the peptide from residues 78 to 83. This epitope resides on the outer rim of the 30 A long spikes that protrude from the capsid shell. These spikes are four-helix bundles formed by the pairing of helix-turn-helix motifs from two subunits; by means of a tilting experiment, we have determined that this bundle is right-handed. Variants of the same protein present two clinically important and non-crossreactive antigens: core antigen (HBcAg), which appears early in infection as assembled capsids; and the sentinel e-antigen (HBeAg), a non-particulate form. Knowledge of the binding site of our anti-HBcAg antibody bears on the molecular basis of the distinction between the two antigens, which appears to reflect conformational differences between the assembled and unassembled states of the capsid protein dimer, in addition to epitope masking in capsids.

Localization of the C terminus of the assembly domain of hepatitis B virus capsid protein: implications for morphogenesis and organization of encapsidated RNA.

The capsid protein of hepatitis B virus, consisting of an "assembly" domain (residues 1-149) and an RNA-binding "protamine" domain (residues 150-183), assembles from dimers into icosahedral capsids of two different sizes. The C terminus of the assembly domain (residues 140-149) functions as a morphogenetic switch, longer C termini favoring a higher proportion of the larger capsids, it also connects the protamine domain to the capsid shell. We now have defined the location of this peptide in capsids assembled in vitro by engineering a mutant assembly domain with a single cysteine at its C terminus (residue 150), labeling it with a gold cluster and visualizing the cluster by cryo-electron microscopy. The labeled protein is unimpaired in its ability to form capsids. Our density map reveals a single undecagold cluster under each fivefold and quasi-sixfold vertex, connected to sites at either end of the undersides of the dimers. Considering the geometry of the vertices, the C termini must be more crowded at the fivefolds. Thus, a bulky C terminus would be expected to favor formation of the larger (T = 4) capsids, which have a greater proportion of quasi-sixfolds. Capsids assembled by expressing the full-length protein in Escherichia coli package bacterial RNAs in amounts equivalent to the viral pregenome. Our density map of these capsids reveals a distinct inner shell of density-the RNA. The RNA is connected to the protein shell via the C-terminal linkers and also makes contact around the dimer axes.