INTRODUCTION

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EM (abbreviations are listed at the end of this section) has long been a primary tool for classifying viruses and exploring their structures. The last decade has also seen a burst of activity in the use of EM for the elucidation of virus structures. This has resulted from two advances in technique. Firstly, cryo-EM has allowed the preservation of fragile specimens in the EM (1, 109). Secondly, the development of efficient algorithms for processing micrographs to produce 3D structures of icosahedral particles has allowed this higher-quality data to be used (9, 14, 42, 80, 86, 89, 91, 93, 130, 133, 210). These two developments have made 3D structural information accessible for a broad range of viruses at the same time that high-resolution X-ray diffraction studies have revealed atomic detail about a more limited range. These two approaches are complementary and together are bringing a new excitement to the field of virus structure.

The most widely used approach for the reconstruction of icosahedral structures begins with the method of common lines, which was developed by Crowther in the early 1970s (86, 89). The original application of the method relied upon estimating the orientation of each particle by eye and then using the symmetry relationships which are present in the projection of any icosahedral object to refine these orientations. The data from the resulting set of views are then combined to produce a 3D reconstruction by means of the projection theorem (43). Several 3D structures of negatively stained viruses were solved, and these results helped clarify the principles of quasi-equivalence that were being explored at the time (88, 89, 94, 95, 119, 120, 170, 216). The use of data from negatively stained specimens limited the efficacy of the method. The distortions of the structure caused by drying, flattening, nonuniform staining, and radiation damage resulted in a loss of the icosahedral symmetry upon which the reconstruction method depended. The applicability of the method was further constrained because many interesting structures such as enveloped viruses were destroyed by interaction with the stain. Finally, even ideal conditions of negative staining revealed only the distribution of the heavy metal stain embedding the specimen rather than the density of the specimen itself. The development of cryo-EM changed this situation (1). By maintaining a layer of vitrified water around the specimen, relying on defocus rather than heavy metal stains to generate contrast, and performing microscopy under low-dose conditions at near-liquid-nitrogen temperatures, this method was able to produce data of unprecedented quality. In particular, the distortions and artifacts which had limited the use of 3D reconstruction of icosahedral particles previously were eliminated in data collected by cryo-EM. The limitation then became the processing of the data. Relatively few characteristic views were recognizable by eye because a cryo-electron micrograph shows the entire density of the particle in projection. This was not the case for negatively stained samples, where uneven staining and the fact that only the outline of the specimen is observed resulted in a simpler image. Cryo-electron micrographs revealed higher-resolution data, but they did so with relatively low contrast (1). This further complicated the task of recognizing and refining views. A reformulation of the common lines method was necessary to allow this to be done reliably and automatically for these noisy, low-contrast, and complex images. The past decade has seen the successful development of such methods (9, 14, 42, 80, 93, 130, 133, 210), a resulting flowering of the use of the approach, and an explosion in the number and quality of the results (see Table 1).

The value of the results produced by 3D reconstruction of viruses from cryo-EM must be considered in terms of its contribution to our understanding of viral structural biology. The morphology of the virus is only the first result gleaned from viewing a 3D reconstruction (Fig. 1). For a highly symmetric particle such as a virus, morphology reveals not only the shape of the virion but also the organization and shape of the components. As discussed below, the combination of an understanding of icosahedral symmetry with even a low-resolution density map can often allow one to infer the stoichiometry of the polypeptides comprising the virion. At a somewhat higher resolution (20 to 30 Å), one can visualize systematic changes in subunit conformation that allow the formation of the capsid. This may reveal fundamental relationships among the structures of members of a viral family such as the papovaviruses (14, 15, 19) or unexpected relationships such as that between a bacteriophage and the reoviruses (48). Interactions between the components of the virus which provide a glimpse into the process of virus assembly (e.g., see references 48, 101, 108, 130, 131, 245, 249, and 353) may also be revealed and generate ideas which can be tested by observations of intermediates in the assembly process (48, 100, 166, 213, 245, 294, 311). Together, the information provided by these methods is generating a deeper understanding of capsid assembly (294).

View larger version (131128K): [in this window] [in a new window]   FIG. 1.   Gallery of representative icosahedral viruses studied by us using cryo-EM and 3D image reconstruction methods. The monomer of bacteriorhodopsin, a 26-kDa membrane protein which contains seven  helices oriented perpendicular to the membrane plane, is shown for comparison at the lower right of the right-hand page (extracellular surface faces upward). All shaded-surface virus structures are viewed along a twofold axis of symmetry. Table 1 presents more-detailed information about these and other 3D reconstructions of icosahedral viruses. TBE, tick-borne encephalitis recombinant subviral particle; Nv, Nudaurelia capensis  virus; Nv, Nudaurelia capensis  virus; Ty Retro, yeast retrotransposon Ty1 VLP; SpV4, Spiroplasma virus type 4; DHBc, duck hepatitis B capsid; B19, human parvovirus B19.

An important tool that provides a link between the reconstruction and the biochemistry and that facilitates its interpretation is the use of specific labels. Antibodies are an important example of such labels that have been used to identify components in a reconstruction (e.g., see references 242, 286, and 313). Localization of antibody binding sites also gives important functional information concerning the mechanism of neutralization (285, 287-289, 297) and receptor binding (242, 286). The conclusions reached in these studies can be supported by difference imaging, which takes advantage of the tools of expression and reconstitution to create particles of defined composition (312, 362, 365). The resolution of antibody labeling techniques is higher than expected from the resolution of the reconstruction because the complex can be modeled by fitting in the known structure of an Fab (73, 156, 161, 162, 235, 240, 243, 288, 289, 297, 336, 337). The promise of higher-resolution localization is realized in recent papers in which a combination of site-directed mutagenesis and undecagold labeling has been used to localize a particular residue with high precision in HepBc (364) and in which antibody Fab labeling has been used to localize the six-residue putative immunodominant loop of HepBc (78). Finally, the use of peptide-based difference mapping has been successfully exploited in the hepatitis virus system to locate the N terminus of HepBc (79) and to locate the binding site of peptides that block hepatitis virion assembly (41).

An increasingly important approach in structural biology is that of "divide and conquer" (16). Complex biological systems which are intractable when studied by a single technique yield to a combined approach. The structures of a number of isolated viral capsid proteins have been solved to high resolution by X-ray diffraction (e.g., see references 74, 254, 255, 324, and 347). Image reconstruction from electron micrographs cannot equal the resolution attained by X-ray diffraction (yet see references 42 and 80), but it provides a context for diffraction results by being able to orient the atomic structure of the subunit into the complex 3D structure of the virion (64, 145, 162, 166, 233, 261). Finally, cryo-EM followed by image reconstruction can be performed with heterogeneous populations of particles. This allows one to explore biochemical treatments which have known effects on virus composition or infectivity but which do not cause a transition of the entire population of particles to a defined state (132, 325). The ability to accommodate flexibility makes cryo-EM and 3D image reconstruction (cryoreconstruction) an ideal tool for exploring changes in virus structure.

This review focuses on the results of 3D reconstruction of icosahedrally symmetric viruses from cryo-electron micrographs. We begin with a brief introduction to the standard terminology so that the reader will share our language for describing the icosahedral structures presented. We then discuss the preparation and microscopy of vitrified specimens and describe 3D reconstruction by the common lines technique as it has been recently implemented for viral structures. More-detailed discussions of the processing methods have been presented elsewhere (9, 133, 210). We then address the question of interpretation of the reconstruction, describing the tools and pointing out some of the pitfalls to be avoided. We continue with a description of some of the portions of virus life cycles illustrated by these results. This section owes much to our colleagues who have very generously allowed us to present their work here. Their willingness to share results and ideas has made the development of this field rapid and enjoyable. We conclude with a comprehensive list of relevant details and literature citations for the bulk of image reconstruction work on icosahedral viruses and a brief discussion of the prospects for extending the technique to higher resolution and the examination of more-complex viral and nonviral systems.

Throughout this review, icosahedral structures are described in language which assumes their regularity and symmetry. This description is based on the theory of quasi-equivalence introduced by Caspar and Klug (53). There are several excellent reviews of icosahedral organization and its expression at the atomic level as determined by X-ray diffraction techniques (148, 149, 176, 202, 260). We will not enter into such a detailed discussion of these results but rather confine ourselves to their description and relevance for the interpretation of 3D reconstructions.

The basic problem addressed by Caspar and Klug (53) in their elegant paper was the understanding of virus structure as the consequence of the self-directed interaction of a large number of chemically identical units. Self-assembly requires specificity of bonding between the units of the structure. One way to accomplish this is to allow all subunits to form identical bonds with their neighbors. A crystal of water or sucrose is formed in this way, as are protein assemblies such as the octahedral assembly of pyruvate dehydrogenase (98). Since a virus structure is optimized for the propagation of its genome, it is advantageous to make a large shell with a small amount of information devoted to structural components. One can imagine that a virus with octahedral symmetry could enclose its genome only if it were formed from very large subunits. This would require that it devote a large fraction of its genome to coding for these structural proteins. Forming a shell from a larger number of small, identically bonded subunits would provide a more parsimonious solution both to the problem of encapsidating a large amount of genetic information and to that of self-assembly.

An icosahedron (Fig. 2, left) is an isometric structure with 12 pentagonal vertices and 20 triangular faces. Any icosahedron has a defined set of exact symmetry elements: 6 fivefold axes through the 12 vertices, 10 threefold axes through the 20 triangular faces, and 15 twofold axes through the edges. The related structure, the pentagonal dodecahedron (Fig. 2, right), shares the same symmetry elements but has a complementary morphology: 6 fivefold axes through the pentagonal faces, 10 threefold axes through the vertices, and 15 twofold axes through the edges. Both of these ideal geometric structures have icosahedral symmetry, as do spherical viruses, whose shapes lie between these two extremes.

View larger version (65K): [in this window] [in a new window]   FIG. 2.   An icosahedron (left) and dodecahedron (right) with symmetry axes and the asymmetric unit used by microscopists. The numbers (2, 3, and 5) indicate the positions of some of the symmetry axes. The white triangle defines the asymmetric unit which is bounded by the lines joining adjacent fivefold and threefold positions.

The positions of the symmetry elements are the landmarks used to describe any icosahedral structure. Application of the icosahedral symmetry elements to a subunit which does not lie on a symmetry axis causes it to be repeated 60 times in the complete structure. This means that the complete structure can be generated by taking 1/60th, called the asymmetric unit, and operating on it with the symmetry elements. The choice of the unit cell is arbitrary. In icosahedral reconstruction, the asymmetric unit is usually defined as being the wedge-shaped volume which extends from the icosahedron's center along edges formed by a threefold axis and two adjacent fivefold axes (14, 130, 133). In most high-resolution X-ray structures of viruses, the conventional asymmetric unit is bounded by a fivefold axis and two adjacent threefold axes (350). Another way of understanding the effect of the symmetry elements which is particularly appropriate to image reconstruction is to realize that a single, nonaxial view of an icosahedral object is equivalent to 59 other views generated by the symmetry axes.

The simplest icosahedral structure is one in which 60 identical subunits interact identically. Caspar and Klug (53) pointed out that, when more than 60 subunits interact to form a closed shell, as appeared to be true for many spherical viruses, all subunits cannot have identical environments. This lack of equivalence between the subunits reopened the question of self-assembly of the subunits. Quasi-equivalence is a solution to this problem (53). Their hypothesis was that shells with more than 60 subunits would be formed from chemically identical subunits with slight but regular changes in the bonding. Hence, a virus comprising 180 subunits would contain three types of bonding and three distinct environments for the subunits. The three types of subunits would no longer be equivalent but rather quasi-equivalent because their environments were similar but not identical.

Triangulation number is a geometric and abstract concept that does not necessarily correspond to the structural components of an individual virus. It refers to the organization of the geometric figure (Fig. 2). One can visualize the formation of an isometric shell by beginning with a flat, hexagonal net. In the original net, all internal bonds are identical in environment. To curve the net and generate a closed structure, one converts some of the hexagons to pentagons. This concept of quasiequivalence can be naturally expressed by the triangulation number for an ideal icosahedron. This can be seen from a consideration of the building of larger icosahedra from a hexagonal net (Fig. 3). The simplest icosahedron has only equivalent units: all bonds are identical and all vertices have identical bonding. The larger icosahedra have hexagons between the pentagons and can be visualized as replacing the original triangular faces with larger ones formed from equilateral triangles. The number of triangles replacing the original one is the triangulation number. More precisely, the triangulation number is given by the following relationship, T = h² + hk + k², where h and k are positive integers which define the position of the fivefold vertex on the original hexagonal net. The different triangulation numbers have very different organizations of bonding (Fig. 4).

View larger version (45K): [in this window] [in a new window]   FIG. 3.   Geometric principles of constructing icosahedral lattices of defined T (triangulation) number. (A) An array of hexamers, represented as a flat sheet of hexagons, is the basis for generating icosahedra (178). A closed icosahedral shell that conforms to the principles of quasi-equivalent symmetry contains 60T subunits organized as hexamer and pentamer units (53). Hexamers are initially considered planar (hexagons in the flat sheet), and pentamers are considered convex and introduce curvature in the sheet of hexamers when they are inserted in place of specific hexamers. A closed shell is generated by inserting 12 pentamers at appropriate positions in the hexamer net as specified by (h,k) lattice points that mark the centers of the original hexagons in the sheet. The model of a particular quasi-equivalent lattice is constructed as follows. Generate one face of an icosahedron by defining an equilateral triangle in the net. The first side of the triangle is a line connecting the origin point of the net (h,k = 0,0) to any (h,k) point. This process will lead to a lattice of T number given by the relation T=h2 + hk + k2. The remaining two sides of the triangle are formed by connecting the (h,k) point to the appropriate points needed to form an equilateral triangle, as illustrated for the nonenantiomeric T=3 (B) and T=4 (C) lattices and the enantiomeric T=7l,d (D) and T=13l,d (E) lattices. A planar sheet of 20 such triangles is formed, and the sheet is then folded up to form a closed icosahedron as depicted for T=3 and T=4 lattices (B and C). In this way, each of the hexamers at the (h,k) lattice points that define the corners of the triangles are replaced by pentamers and each triangular face contains 3T subunits. (F) Examples of viruses with different T-lattice symmetries. Not all viruses conform to the simple rules of quasi-symmetry as stated above. For example, all T=7 papovaviruses such as polyomavirus have capsids built of 360 subunits arranged as 72 pentameric capsomers (29, 252, 293). A further useful relation is that the number of capsomers is given by 10T+2. Adapted from reference 178 with the permission of the author and the publisher.

View larger version (36K): [in this window] [in a new window]   FIG. 4.   The environments of subunits in three different triangulation numbers shown by an arrangement of the heads of the members of one of our groups. Only a single environment is required in the T=1 arrangement, while three and four environments are present in T=3 and T=4, respectively. Notice that a larger head (subunit) is necessary to fill the same-sized asymmetric unit for the lower triangulation numbers. The positions of the icosahedral twofold (2), fivefold (5), and threefold (3) and the quasi-sixfold (6) axes are indicated.

The remnants of the hexagons, now rendered nonequivalent by their construction into an icosahedron, remain hexavalent and are seen as the positions of local or quasi-sixfold axes in the larger icosahedra. In general, the nonfivefold lattice points of the icosahedral net must correspond to the positions of hexavalent structural units. These positions often correspond to the positions of capsomers, which are apparent structural units on the surface of some viruses.

The number of different environments occupied by a subunit increases as the triangulation number increases. The original theory of quasi-equivalence stipulated that the number of different environments should equal the triangulation number; therefore, a T=4 virus would have four different subunit environments and 60T or 240 subunits (120, 130, 230). The correspondence between T number and the number of different subunit environments is not always strictly maintained. In agreement with the theory of quasi-equivalence, a T=1 (h=1,k=0) icosahedral virus such as satellite tobacco necrosis virus has only one type of environment for its subunit and a total of 60 subunits (203). Similarly, the T=3 (h=1,k=1) tomato bushy stunt virus has three separate environments for a total of 180 subunits (150). In contrast to this, the T=7 papovaviruses are composed of 72 pentamers and have six subunit environments and yet only 360 subunits (h=1,k=2 for the T=7dextro or right-handed organization as in SV40) (4, 201). The same arrangement is seen in three papillomaviruses (29, 188). However, not all T=7 viruses violate quasi-equivalence, since bacteriophage P2 (101) and CaMV (65) also display triangulation number T=7 but have 12 pentameric and 60 hexameric capsomers for a total of 420 subunits with seven distinct environments. When the correspondence is direct, determination of the triangulation number provides a count of the number of subunits; otherwise, it remains a descriptive tool. The nominal "T=2" arrangement of the fungal virus capsids (63) and the intermediates of the dsRNA viruses 6 and BTV (48, 144) can be viewed as a T=1 arrangement of pairs of chemically identical subunits.

Cryo-EM in combination with image reconstruction provides a direct, objective way to determine triangulation numbers of spherical viruses and also allows direct determination of the number of subunits in the virion when individual units are resolved. For example, the trimeric nature of the capsomers that lie on the T=25 net of adenovirus is apparent in the reconstruction (296), and the number of subunits and their environments are clearly identified. Reconstructions also provide information concerning the alteration of subunit conformations and their interactions in the structure. In adenovirus, these changes appear to occur at the sites of interaction with the minor structural proteins of the virus (135, 296, 298) and so provide a view of the quasi-equivalence in practice.

Classically, the projected images of negatively stained viruses have been used for the determination of triangulation numbers (53). Two approaches have been employed. The first is the inference of capsomer positions and their placement on an icosahedral net, often by comparison with a model of the triangulated structure (121, 188, 189). This method is not only technically difficult but fraught with difficulties due to the vagaries of negative staining and the mental gymnastics associated with inferring a 3D structure from two-dimensional views of particles of unknown relative orientation. It has, however, produced the correct triangulation number when applied carefully to structures that respond well to negative staining (188). The second approach is to combine an estimate of the subunit size with the radius of the virus to infer the number of subunits and a triangulation number (i.e., 60 [T=1], 180 [T=3], 240 [T=4], ... ). This approach is very straightforward, but it often produces the wrong answer. Microscopists who are tempted to utilize this second method should realize that their conclusions may be challenged by the more objective 3D reconstruction methods described here. Naturally, either method depends upon but does not validate the assumption of icosahedral symmetry (134).

The methods of preparing viruses and many other types of biological macromolecules for cryo-EM studies are well established (1, 71, 109, 218). A vitrified specimen of a spherical virus is typically prepared in the following way (Fig. 5). A 2- to 5-µl aliquot of a purified, 0.05- to 5-mg/ml suspension of virus is applied to an EM grid coated with a holey carbon support film. The grid is secured with a pair of forceps in a guillotine-like device and suspended over a bath of cryogen slush (usually ethane or propane) maintained near its freezing point by a reservoir of liquid nitrogen. The grid is then blotted nearly dry by pressing a piece of filter paper directly against it, the guillotine is released, and the grid is rapidly plunged into the cryogen.

View larger version (56K): [in this window] [in a new window]   FIG. 5.   The steps in a typical preparation of a specimen for cryo-EM are shown. A holey carbon film (A) is prepared by the evaporation of carbon onto a grid bearing a holey plastic film and the removal of the plastic by exposure of the grid to the fumes of ethyl acetate. This film contains holes with diameters between 1 and 5 µm. The specimen is applied to the film at concentrations between 50 µg/ml and 5 mg/ml (B). The grid may then be floated on a drop of water or low-ionic-strength buffer to remove excess salt. The grid is then placed in a pair of forceps which are locked into a guillotine-like device (C) and blotted with filter paper to produce a very thin aqueous film (1,000 to 2,000 Å) across the holes of the grid (D). Immediately after blotting (and before the aqueous film has had time to dry), the plunger is released to allow the forceps to drop into a bath of ethane slush held in a container of liquid nitrogen. The efficient cooling afforded by the ethane slush causes vitrification of the sample. It is then either stored under liquid nitrogen or placed in a liquid-nitrogen-cooled specimen holder for viewing in the microscope.

Vitrification of the water rather than crystallization (i.e., ice formation) occurs if the sample is thin enough (~0.2 µm or less), so that cooling occurs very rapidly. Estimates vary, but there is agreement that the rate of cooling exceeds 10⁴ °C/s (see discussion in reference 31). An efficient cryogen such as propane or ethane slush rather than liquid nitrogen must be used because the Leidenfrost effect (the creation of gas upon contact with the specimen) would otherwise slow heat transfer so that crystalline ice would be formed (110).

The concentration of the sample is critical and is usually higher than that needed for negative staining. Application of the sample to a continuous carbon film followed by a brief period of absorption (1 to 2 s) before negative staining can be used to determine the appropriate range of concentration. Prolonged absorption before staining will concentrate the sample on the grid so that it appears more concentrated than it will by cryo-EM.

The thickness of the water layer primarily depends on blotting time, wetting properties of the support, and the humidity near the sample. Excessive drying rapidly thins the specimen, often causes particles to migrate toward the periphery of the holes in the substrate, and can lead to drastically increased solute concentrations as well as dehydration, all of which alters the specimen's environment and perhaps structure (211, 212, 330). Alternatively, inadequate drying leaves a sample in which particles are superimposed or embedded in a water layer that is too thick for the electron beam to penetrate. Some workers prepare their samples in cold rooms (174) or in 37°C rooms at 100% humidity (211, 212) to avoid the effects of drying on sensitive specimens. However, the use of humidity-controlled glove boxes (174), double-blotting techniques (310, 334), or a simple temperature-controlled mist device (75) may preserve the state of the specimen with less stress on the investigator.

All subsequent steps, including the recording of images in the microscope, are carried out with the sample below 160°C to avoid the devitrification which occurs at ~140°C (113, 198, 200). These include transfer of the grid from the cryogen into liquid nitrogen (where it may be stored indefinitely) and then into a cooled cryospecimen holder which is rapidly (and carefully) inserted into the EM. All of the transfer steps must be performed rapidly to avoid warming of the specimen and contamination by the condensation of water vapor. Such condensation can be minimized by continuously bathing the cold sample in dry nitrogen gas during transfer to the microscope vacuum, which is an option on some cryotransfer systems. Excess water vapor that gets into the microscope severely overloads the high-vacuum system and reduces its useful lifetime. This has become increasingly critical with the use of sensitive FEG sources. The beginner should become comfortable with all steps of transfer with cryoholder and specimen at room temperature before preparing a vitrified specimen.

As with other techniques in microscopy, various alternative procedures have been developed for each of the steps described above. Usually the specimen is prepared on a holey carbon film, which sometimes is glow discharged to enhance the spreading of the specimen. Alternatively, continuous carbon films, carbon-coated plastic films, or even bare grids (1) have been successfully used as supports for different viruses. The age of the carbon film often determines the quality of the specimen spreading: old support films can be recarboned to produce usable grids (335a). Sometimes, specimens prepared on holey films do not appear in the holes because they strongly adhere to the support film. If this occurs, the properties of the support must be altered. If hydrophobic support films are required, grids may be glow discharged in an atmosphere of amyl amine (112). However, support films that are too hydrophobic usually exhibit regions of thin "ice" surrounded by very thick "ice." An alternative is to apply a dilute solution of lipids (0.1% [wt/vol] phosphatidylcholine or phosphatidylethanolamine in H₂O) to the grid before the specimen. This results in a vitrified layer of water sandwiched between lipid monolayers (325). A number of different techniques and apparatuses that are capable of producing uniformly thin, vitrified samples have been developed. These procedures include the double blot (310), carbon sandwich (302, 303), jet spray (111, 114), pressurized liquid nitrogen (109), and many others.

Another concern of the microscopist is that many aqueous samples of biological specimens contain high concentrations of buffer salts (>100 mM) or solutes such as glycerol (5 to 10%). Their presence can lead to phase separation upon cooling and diminish the contrast in the image. This alteration of the specimen environment can lead to structural changes. Such solutes may sometimes be removed without disrupting the virus on the grid by floating the grid on a drop of distilled water or an appropriate, low-concentration buffer for a fraction of a minute, in much the same way as is common with the preparation of negatively stained samples. This washing technique works even when holey carbon films are used, since it appears that the sample adheres to the air-water interface (325). A somewhat more concentrated sample (3 to 5×) should be used when this washing approach is employed.

Success in obtaining a vitrified sample suitable for cryo-EM thus depends on many factors, the most important of which are dictated by the properties of the virus (pI, enveloped or not, etc.), solution (pH, ionic strength, etc.), and support film and certainly the experience (and persistence) of the microscopist. A number of studies have demonstrated that the dimensions and integrity of most viruses and other fragile macromolecular assemblies are well preserved by cryopreparation techniques (1, 26, 130, 211, 228). This provides strong evidence that the specimens are fully embedded in a vitreous layer; those that are not become distorted and compressed (197).

Aqueous specimens examined by cryo-EM must be maintained at or below the devitrification temperature (~140°C) to prevent conversion of water in the sample to a crystalline state. A number of different designs for liquid nitrogen-cooled, cryospecimen holders have been described elsewhere (105, 109, 151, 152, 155, 301). Side-entry cryoholders have improved enormously over the past few years, but they remain less stable than conventional, room temperature holders.

Cryoholders are subject to greater instabilities due to the temperature gradient between the microscope at room temperature and the specimen and as a result of boiling of the coolant which transmits vibrations to the specimen. Current stage designs have minimized these problems with a satisfying improvement in resolution. Nevertheless, the maximum instrumental resolving power of most modern microscopes (~0.7 to 2 Å) cannot yet be achieved with currently available cold holders which promise stability in the 2- to 4-Å range. The development of a microscope which incorporates a liquid-helium-cooled, specimen stage and a superconducting objective lens has provided a means to achieve even better resolving power (127, 128, 196, 354, 355), as has the development of practical top- and side-entry stages for liquid helium work. These stages have shown their worth in the increased resolution obtained with crystalline specimens (52, 153, 154, 190) and should provide similar advantages for single particle specimens such as viruses.

Even when the specimen holder is rapidly transferred to the microscope, water vapor is carried in by the rod of the holder. The low temperature of the specimen unfortunately makes it an efficient sink for contaminants. The consequent high rate of specimen contamination forces the cryomicroscopist to work quickly or use an auxiliary anticontamination device. Several devices such as the blade-type anticontaminators, which closely sandwich the specimen, have been constructed and are commercially available (113, 164, 173). These devices significantly reduce the level of contamination so that cryo-EM can now routinely be carried out for a period of several hours with an individual specimen grid. One drawback to the use of the auxiliary anticontaminator is that it often restricts the range of tilt normally allowed on a goniometer stage. Fortunately, this does not pose severe constraints in most work with icosahedral viruses because they tend to be randomly oriented in the vitreous sample layer, and it is usually not necessary to record different views by tilting in order to obtain 3D information. When virions show a strong preference for one or a few orientations as occurs with reovirus and adenovirus due to their surface projections, a tilt of 10°, which can be accommodated by the anticontaminator, is usually enough to give an adequate sampling of orientations (108, 296). Should higher tilts be necessary, the anticontaminator can be retracted after about an hour when most of the water vapor from the specimen holder has been trapped. The structure of adenovirus was determined to 35-Å resolution by combining images of 29 particles (296). The fibers which project from the fivefold axes cause the virus to tend to lie in orientations which are away from the surface of the water layer. This orientation preference was overcome by tilting the stage by 10° during microscopy. This was sufficient in combination with the rotational disorder of the virions in the water layer to allow the collection of a complete data set (296).

After allowing some time (>15 min) for the specimen stage to stabilize after insertion into the microscope, the EM grid is searched at very low magnification (<×2,000 to ×3,000) and at a very low irradiation level (<0.05 e/Ų/s) to locate a suitable specimen area for photography. At low magnification it is possible to assess the relative thickness of the vitrified sample (Fig. 6). The switch from high- to low-magnification operation is conveniently selected in many microscopes by the push of a single button. Some cryomicroscopists prefer to view the "false," low-magnification image that is formed by highly defocusing the pattern formed in diffraction mode. The advantages of this technique are that the "diffraction" image exhibits very high contrast, and realignment of the microscope is usually not required. Many newer microscopes have a three-state system which allows switching among low magnification for searching, high magnification for focusing, and intermediate magnification for recording images. Areas on the grid that appear black are too thick for the electron beam to penetrate; areas that appear bright are likely to be dry. Sometimes, regions of the specimen appear to be vitrified but are actually dehydrated regions in which the buffer salt has crystallized and appears like an "ice" layer. Truly vitrified specimens of optimal thickness are usually characterized by a cloudy appearance. One can verify the vitrified nature of a particular specimen area at high magnification by the occurrence of bubbling at the sites of particles within the area during the first few seconds of irradiation.

View larger version (133K): [in this window] [in a new window]   FIG. 6.   Low-magnification views of vitrified samples of icosahedral viruses, including Nudaurelia capensis  virus (upper left and upper right), SV40 (lower left), and a mixture of polyomavirus and bromegrass mosaic virus (lower right). The grid square with the N. capensis  virus specimen (upper left) is a particularly poor candidate for cryo-EM, but it nicely exhibits a complete spectrum of ice thicknesses, from much too thick at the top (black region) to completely absent at the bottom (holes with no specimen). Only a few holes in the carbon film at the border of the very thick ice have a suitable thickness and distribution of virus particles. The strongly mottled appearance of the N. capensis  virus sample (top right) is caused by ice contamination that quickly builds up on specimens if a blade-type anticontaminator is not used during microscopy. The SV40 sample is a nearly perfect monodispersed distribution of particles in a uniform layer of ice. The bare circular region presumably resulted when surface tension effects in the thinning water layer excluded the ~500-Å-diameter particles just before vitrification occurred. The mixed polyomavirus and bromegrass mosaic virus sample shows a graded change in specimen thickness, from a thin region near the center of the hole where the small (~300-Å-diameter) bromegrass mosaic virus particles congregate in a single layer to progressively thicker regions marked by a ring of 500-Å-diameter polyomaviruses surrounded by more polyomaviruses and multiple layers of bromegrass mosaic virus. All magnification bars represent 5,000 Å.

Because most viruses smaller than ~1,000 Å are difficult to see directly at very low magnification, a convenient way to assess the particle concentration is to view one area (grid square) at a higher magnification and at a higher level of defocus, thereby sacrificing that specimen region. One then assumes that the particle concentration remains relatively constant in other areas of the grid for vitrified samples of similar thickness. The use of a video-rate TV camera system (e.g., see reference 104) provides a very effective means for scanning cryospecimens at a very low magnification and dose and simultaneously allows the user to easily assess particle distributions for viruses as small as 300 Å in diameter. Alternatively, a charge-coupled device camera can be used to visualize particles at higher magnification (232) and to check imaging conditions by performing a Fourier transform online.

Unstained, vitrified biological specimens are very sensitive to the effects of electron irradiation (Fig. 7). At 170°C, features in the 20- to 40-Å resolution range are unaffected by a total electron dose of ~10 to 20 e/Ų, a value similar to that found for negatively stained specimens (7, 318) but 5 to 10 times greater than that for unstained specimens at room temperature (317). Thus, microscopy must be performed in a way which minimizes exposure of the specimen to the electron beam. This requires the use of minimal irradiation procedures (e.g., see references 8, 317, and 345) which act to limit specimen exposure to the minimum necessary to record a micrograph with sufficient optical density and signal. Most modern microscopes offer some convenient mode of low-dose operation. Dose levels may be measured directly by the use of a Faraday cage (215, 316) or they may be estimated from the standard microscope exposure meter after calibration. This latter procedure is done by measuring the optical densities on an EM film of known sensitivity which has been exposed for different times and developed under carefully controlled conditions (e.g., see reference 8). A careful study of the effect of radiation damage on the cryoreconstruction of the HSV-1 capsid showed that the reconstructions of particles which had received 30 to 40 e/Ų had recognizable but blurred features compared to particles which had received a more standard 7 to 10 e/Ų (84). The high-resolution reconstructions of HepBc which yielded resolutions of better than 10 Å used single images taken at 10 to 16 e/Ų or pairs of images taken at 7 to 10 e/Ų each (42, 80).

View larger version (129K): [in this window] [in a new window]   FIG. 7.   Radiation damage in vitrified SV40 samples. Fine structural details are progressively lost in the SV40 particles as the electron dose increases from 10 to 40 e/Å2 (left series). The most prominent damage first appears as bubbling that occurs in the ice over the carbon support film (right series). Bubbling also occurs within particles suspended over the holes of the support film but usually only after doses of about 50 e/Å2 or more (data not shown).

The relationship between the electron image of the specimen and the specimen itself is, unfortunately, not straightforward. This relationship is described by the contrast transfer function, or CTF, which involves both phase- and amplitude-contrast components. These are characteristic of the particular microscope used, the specimen, and the conditions of imaging. The contrast-enhancing effect of the CTF arises from the spherical aberration present in all electromagnetic lenses and varies as a function of the objective lens focal setting and accelerating voltage (Fig. 8). The function includes both a phase-contrast and an amplitude-contrast component:
where (v) =  ·  · v²(f  0.5 · C_s · ² · ²), v is the spatial frequency, (per angstrom), F_amp is the fraction amplitude contrast,  is the electron wavelength (angstroms), where (0.042 Å for 80-kV electrons and 0.02527 Å for 200-kV electrons), V is the voltage (volts), f is the underfocus (micrometers), and C_s is the spherical aberration of the objective lens of the microscope in millimeters. The CTF is usually attenuated by an envelope function which depends upon the coherence of the beam, drift, and other factors (116, 329, 331). Thus, at any particular defocus setting of the objective lens, phase contrast in the electron image is positive and maximal at only a few specific spatial frequencies; at all other frequencies, contrast is either lower than expected, completely absent, or opposite (inverted or reversed) from what it should be. This allows the microscopist to selectively accentuate image details of a particular size much as an optical-phase microscope accentuates some features at the expense of others. This discussion ignores the effects of inelastic scattering, which can be severe for vitrified samples embedded in thick layers of water (192, 283). Inelastic scattering effects can be modeled by including an envelope function which imposes an exponential decay in the transfer function similar to that resulting from the lack of coherence in the beam (124). The effect is dramatically revealed in thick regions of the specimen; images of particles in these areas appear unsharp due to significant chromatic aberration effects.

View larger version (16K): [in this window] [in a new window]   FIG. 8.   The CTF for a Philips CM200 FEG at 200 kV is plotted as a function of resolution in angstroms for an underfocus of 2 µm and an underfocus of 1,000 Å and a magnification of ×36,000. The decrease in the amplitude of the function with increased resolution reflects the measured attenuation due to the lack of coherence in the source, specimen movement, and other optical effects. The value of 0.1 at the origin is the amplitude contrast portion of the function.

In practice, cryomicroscopists typically underfocus images by 8,000 to 20,000 Å (0.8 to 2 µm) to enhance specimen features in the 20- to 40-Å size range (Fig. 9). This amount of defocus is quite foreign to conventional biological microscopists who are used to imaging shadowed or stained specimens much closer to focus (3,000- to 5,000-Å underfocus). Sometimes it is advantageous to record two or more images at different focal settings to selectively enhance different features of the cryospecimen. For example, the overall organization of the glycoprotein spikes in the enveloped SFV is best enhanced at an ~3-µm or larger underfocus (Fig. 9), whereas the lipid bilayer is best visualized in images recorded much closer to focus (<1.5 µm). Images from a focal series can also be combined to compute 3D reconstructions as described in "Image analysis and 3D reconstruction" or can be useful for analyzing very noisy data (e.g., see reference 65).

View larger version (100K): [in this window] [in a new window]   FIG. 9.   The effect of defocus is shown for cryo-EM of SFV. The top four images show a defocus series taken on a Philips 400 microscope with a tungsten filament at 80 kV for illumination and underfocus values of 1.5, 3, 6, and 12 µm. Notice that the overall contrast is lower in the closer-to-focus image; however, the very fine details, such as the membrane, are recorded. The further-from-focus images do not show these details but do show better contrast for the large features of the specimen such as the spikes and the outline of the particle. This reflects the fact that there is high attenuation of the transfer function with a tungsten filament and hence only the information in the first peak of the CTF is transmitted efficiently. The bottom two panels show cryo-electron micrographs of the same sample taken on a Philips CM200 FEG under the same defocus conditions for which the CTF is plotted in Fig. 8. Both images show fine details because the illumination from a field emission source is more coherent than the tungsten filament and hence a larger range in resolution is transmitted. Images at different defocus must still be combined because information is lost at the transfer function nodes and attenuated near them. Identical particles in each of the focal series are indicated (arrows).

The first work on cryo-EM studies of viruses was performed with conventional transmission EMs operated at accelerating voltages of 80 or 100 kV. Recent experience shows that the use of FEG illumination is an enormous advantage for cryo-EM (72, 210). The major source of contrast in an electron micrograph of an unstained specimen is phase contrast. The higher coherence of a FEG source makes the phase contrast in the image much stronger. Practically, focusing and aberration correction become much simpler, and the coherence-dominated falloff of intensity with resolution is greatly reduced. The drive toward higher resolution has required the correction of the contrast transfer effects in the data. This is best done by combining images at different defocus levels (see below). This can be done much more reliably with FEG data because a greater number of Thon rings are visible in optical or computed diffraction patterns of images (116, 304). Higher-voltage instruments are also being employed. The potential advantages of the use of much higher voltages include potentially higher resolution and beam penetration and reduced problems with specimen charging. Higher voltage is particularly important for obtaining higher resolution for larger viruses (>1,000-Å diameter) (360) due to the curvature of the Ewald sphere at lower voltages (210). Finally, several groups have used spot scan imaging for the collection of virus data. This method was originally developed for two-dimensional crystalline specimens, for which it has been shown to decrease the apparent beam-induced drift seen in conventional flood illumination (102, 103). Although a carefully controlled study has not yet been published, several groups have used spot scan images for reconstructions (214, 234, 306, 358, 361-363).

The icosahedral image reconstruction method produces a 3D structure by interpreting the separate images of different particles as distinct views of the same structure. Once the cryo-electron micrographs are obtained, the determination of the structure requires three fundamental steps: (i) determining an initial orientation and center for each particle, (ii) refining these parameters by comparison of common data among different views, and (iii) combining the data from a sufficient number of unique views with their relative orientations to produce the final 3D structure (Fig. 10). This section gives an overview of the processing methods and factors that affect the quality of the reconstruction; the details of this procedure are discussed more extensively elsewhere (9, 133, 210). The methods are implemented in a number of continually evolving software packages (9, 92, 93, 194) which are available from the individual authors. One hallmark of the field is the willingness to share code and ideas among different groups, and consequently the different packages use similar approaches. They all had their origin in the original implementation of common lines (86).

View larger version (65K): [in this window] [in a new window]   FIG. 10.   Schematic diagram of the 3D image reconstruction process from digitization of the micrograph to the dissemination of the structural results. Though some steps such as the boxing out of individual particle images lend themselves to automation (e.g., see references 33 and 306), many steps including the determination of particle origins and orientations must be repeated and involve some trial-and-error decision making (e.g., to determine which data should or should not be included). The scheme depicted here, which uses both cross-common lines (133) and model-based (9) approaches to determine and refine particle origin and orientation parameters, is but one of many suitable schemes.

Other reconstruction methods have been used for cryo-electron micrographs of icosahedral particles, including the ROSE (reconstruction by optimized series expansion) method (326, 327), the COMET (constrained maximum entropy tomography) method (282), and angular reconstitution (297) as implemented in the IMAGIC software package (322). In the latter two papers, the reconstruction was performed by using orientations derived from the common lines approach (282, 297).

The bulk of the work in the reconstruction process rests in the determination and refinement of the orientations and origins of the views. Five parameters must be determined for each particle (Fig. 11): , the inclination of the view vector from a selected twofold axis (typically the z axis); , the azimuthal angle relative to the line connecting the adjacent fivefold axes; , the rotation of the projected view relative to the x axis in the scanned image; and x and y, the coordinates of the center of symmetry of the particle where all the symmetry axes cross. Icosahedral symmetry allows one to refer any view to one in the asymmetric unit. Hence, the unique views comprise a spherical triangle bounded by neighboring fivefold axes ( = 90.0°,  = ±31.72°) and an adjacent threefold axis ( = 69.09°,  = 0.0°). Typically, the view parameters must be determined to within about 1° for a low-resolution reconstruction and about 0.25° for a high-resolution reconstruction. This is possible because the projected image of a large object such as a virus changes dramatically with changes in its orientation (Fig. 12).

View larger version (25K): [in this window] [in a new window]   FIG. 11.   Definition of particle view orientation angles. By convention (189), , , and  angles define the orientation in which each icosahedral particle is viewed. The standard setting of the icosahedron places three mutually perpendicular twofold axes of the icosahedron coincident with the x, y, and z axes of a Cartesian coordinate system. The direction of the view vector (thick arrow) is given by  and  (the view direction 80°,15° is illustrated in this diagram), where  is the angle of the vector projected onto the xz plane measured positive from the z axis, and  is the angle of the vector projected onto the xy plane measured positive from the x axis. The rotational orientation of the icosahedron about the vector is given by the angle . Because the symmetry of an icosahedron makes it 60-fold redundant, any view vector can be referenced to a single asymmetric unit (shaded region), which is a spherical triangle bounded by neighboring fivefold axes ( = 90.0°,  = ±31.72°) and an adjacent threefold axis ( = 69.09°,  = 0.0°).

View larger version (118K): [in this window] [in a new window]   FIG. 12.   Change in projected structure with change in view orientation. (Upper panel) Images of a 3D reconstruction of BPV (19), projected on a regular grid at 3° intervals of  and  ( always = 0°) within a half of the icosahedral asymmetric unit (represented by dots in the right half of the inset). This gallery demonstrates how small changes in viewing angle can produce dramatic differences in the projected views of a 600-Å-diameter particle. The magnitude of these differences is correlated with the change in view direction and with the size of the particle. Hence, a 3° change in view direction leads to more-pronounced differences in projection images for a larger particle (>600 Å) or less-pronounced differences for smaller particles (<600 Å). (Lower panel [corresponds to boxed region of inset]). Demonstration that projected views at , and , are enantiomers related by a vertical line of mirror symmetry. Note that when  = 0°, the particle itself is mirror symmetric about a central vertical line (true for all images in leftmost column of upper panel). All equatorial views give rise to mirror-symmetric images. An icosahedron has 15 equators, each of which encircles the icosahedron along a direction that follows adjacent symmetry axes. For example, the equator in the xy plane ( always = 90°;  varies between 180° and +180°) crosses, in order, the following symmetry axes: two-, five-, three-, two-, three-, five-, two-, five-, three-, two-, three-, and fivefold. Any view corresponding to a combination of  and  which lies on this equator will be a mirror-symmetric image with the mirror line parallel to the equator. For example, the bottom row of projected images in the upper panel (which represent some of the views along the xy equator) all exhibit horizontal lines of mirror symmetry. Projection views along strict symmetry axes are additionally unique because they exhibit n mirror lines, where n (= 2, 3, or 5) is the symmetry of the axis in view.

When a symmetry axis is along the direction of view or projection, the image obeys the symmetry of the axis. An axis which is not along the direction of view gives rise to common lines in the transform of the projection. Application of the symmetry operation to the transform of the projection yields a second, identical plane which intersects the first along a line. Application of the inverse of the symmetry element to the original transform generates a third, identical plane with a second line of intersection. These lines in the original transform are designated "common lines" since they are common to the original and symmetry-related planes. The values of the transform in the original plane along them must be identical since they are related by symmetry operations on the original plane (89, 133). Each pair of a symmetry element with its inverse gives rise to such a pair of common lines which lie to either side of the projection of the symmetry axis.

The 60 symmetry elements of the 532 point group symmetry of an icosahedral particle generate 37 pairs of common lines in the Fourier transform of an image of its projection. These 37 pairs include two pairs from each of the 6 fivefold axes, one pair from each of the 10 threefold axes, and one pair from each of the 15 twofold axes. Sixty pairs of common lines exist between the transforms of any two projected particle views. The positions of these lines, along which the values of the transform should be identical for objects with perfect icosahedral symmetry, are determined solely by the direction of the view. The orientation angles corresponding to an observed view can be found by stepping  and  through the asymmetric unit and selecting the values which give the lowest sum of residuals over all 37 pairs of common lines in the transform of the projection (86, 89, 91, 133). These are the view angles for which the observed view most closely matches that of an ideal icosahedron. The agreement of the phases along the 60 symmetry-related lines between pairs of images, which is called the cross-common lines residual, provides a measure of the agreement between the transforms of different projections and, hence, allows screening for consistency in the data set as well as the refinement of the orientations (14, 93, 130, 133).

The resolution of the reconstruction, by this or any other technique, is always limited by the worst steps in the entire process, from specimen preparation through imaging, densitometry, view parameter determination, and final calculation of the map (Fig. 10). For example, if the specimen was destroyed by the beam prior to imaging, nothing can be done to retrieve the information that was lost. Thus, it is important to design the steps of the processing to preserve the information in the images and not to expect to recapture information lost by a previous step.

A prelude to the computer processing of images is the selection of micrographs and screening of them by eye (and sometimes by optical diffraction if there is enough carbon substrate in the image to give a strong transfer function pattern) to identify those of high quality which have the appropriate defocus (see above) and minimal astigmatism and image blurring owing to specimen movements (drift, vibration, etc.). The particle concentration should be high enough that a single scanned image will yield an adequate number of views but not so high that images of individual particles overlap. In this context, it is important to remember that the image of a particle includes the Fresnel rings at the periphery which arise from the CTF of the microscope. The effects of the CTF are most prominent where there are large differences in specimen density such as at the interface between the virus and the surrounding solvent. The resolutions at which the CTF has its first maximum and minimum should be noted, as these provide landmarks for use of the data in the reconstruction (Fig. 8).

Careful inspection of the cryo-EM images sometimes allows characteristic views to be recognized and also provides a sense of the heterogeneity of the preparation. If a large fraction of the particles appear to have adopted a preferred orientation, additional images should be recorded to expand the range of particle orientations. If necessary, a broader range of particle orientations can always be obtained by tilting the specimen holder by a small angle (~10 to 20°) (108, 296). Although the bulk of the effort in icosahedral reconstruction is spent on computation, most workers have had the humbling experience of realizing that an extra few minutes spent at this screening stage could have avoided hours of frustrating computational work.

The digitization of the micrographs should be optimized to capture the information in the images. The Shannon sampling theorem (270) states that one should scan the micrograph at a step size which is at least twice as fine as the resolution desired. In practice, most workers err a bit on the side of caution, and images which are intended to carry 25-Å information are usually scanned at about 5- to 8-Å spacing (i.e., one-fifth to one-third of the desired resolution). Cryo-electron micrographs usually have a very limited dynamic range (i.e., low contrast), so the scanner should be adjusted to optimally capture this range without truncation or compression of the density values. We have used an Optronics C4100 rotating drum scanner at a 25- or 12.5-µm step size; a Perkin-Elmer Micro-D flatbed scanner with a step size between 25 and 10 µm; or a Zeiss Phodis flatbed scanner at step sizes of 7, 14, or 21 µm to scan images taken at magnifications between 20,000 and 50,000.

Almost all cryo-electron micrographs exhibit a varying thickness of the water layer in the specimen by having a nonuniform background. Corrections for this varying background are made by fitting a least-squares plane to the average background value and subtracting it from the entire image. A mask is then generated to screen out other particles and debris within the box. The mask must be large enough to include all of the particle's features including the Fresnel defocus rings which surround the particle. The particle is then centered more accurately by use of cross correlation with a version of the same image rotated by 180° or with a low-pass filtered disk of the particle's diameter or with an azimuthally averaged radial profile of the particle (e.g., see references 228 and 296). The boxed image is then embedded in a larger square array of pixels and Fast Fourier transformed. A typical example is that of SFV with a scanned diameter of 700 Å (or 62 pixels in a 25-µm scan of a ×22,000 micrograph) (325). SFV was extracted in a 100- by 100-pixel box, masked within a circular window of radius 40 pixels to exclude other particles, centered by cross correlation with a low-pass, Fourier-filtered (0.1 pixel¹) disk of 62 pixels in diameter, and finally placed in a 512- by 512-pixel box for transformation. Representative transforms should always be examined to check that the process has not resulted in the production of artifacts such as streaks or rings in the transform, as these will overpower the information from the particle and lead to erroneous refinement of the , , , x, and y particle parameters.

The selection of large numbers of particles has been made easier by the availability of a number of automated and semiautomated programs for selecting and preprocessing images. These include PTOOLS (306) and EMMA (33) as well as the more general-purpose packages SPIDER (126), IMAGIC (322), and the MRC image processing package (92).

Once Fourier transforms of properly selected, centered, and boxed images have been generated, a series of programs are used to determine the center and orientation of each view by minimizing the common lines residuals. After the best , , and  parameters are found for the initial x,y center as described above, the center is recalculated to give the best common lines residual for that orientation. This center is then used to calculate a new orientation. This process is followed for 20 or more particles. Owing to the low signal-to-noise ratio in the images and systematic variation in common lines degeneracy, only a small fraction of the particles typically yield reliable orientations. A detailed description of the identification of these views and the explanation for the degeneracy in the common lines are presented in two recent reviews (9, 133). The identification of this apparently reliable subset is followed by comparison of the transforms by means of the cross-common lines residuals, allowing the refinement of particle orientations and origins against each other. Once a small, refined data set is available, a low-resolution 3D reconstruction is calculated from the available data set. If the reconstruction shows "reasonable" features such as continuity and yields projections which correspond with the original images, the set can be used as the basis for addition of further particle images. The additional data are refined against the original set until enough views have been collected to define the reconstruction to the desired resolution (9, 133). Modifications to the original common lines algorithms have recently been designed to improve and automate the orientation estimation search procedure and thereby enhance the potential resolution of computed reconstructions (307).

Once an initial reconstruction has been produced, it can be used to help with the orientation identification and refinement process. Two model-based methods are in widespread use. One uses a small set of projections (typically 6 to 10) to generate a fixed set of transforms for the calculation of cross-common lines residuals with the individual image transforms (93). The other calculates the complete set of projections for the asymmetric unit and then compares these with the images by using a polar Fourier transform method (9). This method has been further extended to help in the analysis of particles that prove particularly difficult to refine (54). Both methods allow rapid determination of image orientations for large numbers of images and give equivalent results (210). Their use has had a dramatic effect on the quality of reconstructions because they make it practical to combine hundreds (80) or even thousands (42) of images and to achieve an increased signal-to-noise ratio in the final reconstruction. A further advantage of model-based refinement is that it allows the determination of the relative scale of images in the data set. Magnification varies between and within images (3), and its correction is important for extracting the highest resolution from the data (210).

The CTF that enables the visualization of unstained specimens must be compensated in the reconstruction in order to achieve a reliable representation of the structure. The transfer function reverses, removes, and attenuates data in resolution ranges in the image transform (Fig. 8). The problem can be minimized by working at a resolution limit lower than the first zero of the transfer function since no phase reversal occurs in this range. For typical imaging conditions, this limits the reconstruction to worse than 20-Å resolution.

Higher resolution requires more careful treatment. Careful determination of the transfer function allows correction of the phases by multiplying the appropriate regions of the transform by 1.0. The transfer function is usually determined by locating its zeros as the points of minimum contrast in the average power spectrum of the image. This procedure is much more reliable when FEG or high-voltage data is used so that multiple zeros can be seen in the power spectrum. Often, it is helpful to include the edge of a hole in the image since the greater scattering from the support film makes visualization of the transfer function easier. One can correct the transfer function approximately by dividing by it (called Wiener filtering), but this ignores the fact that data are removed in the regions near the zeros. It is necessary to use multiple images with complementary defocuses to fill in these missing data (210). Two fundamentally different approaches have been used. The first generates a transfer function-weighted sum of the images of each particle taken at different defocuses so that a single corrected image is passed to the reconstruction programs (80, 314). The second generates a separate reconstruction from each image and then calculates a combined, corrected reconstruction as a sum of the reconstructions weighted by the CTF (40, 42). Both methods have been used successfully to achieve high-resolution reconstructions.

An alternative approach to filling in the missing data is to take advantage of information which is not affected by the imaging process of the microscope to correct the reconstruction. The transfer function for an image which has no astigmatism is radially symmetric. Hence, knowledge of the radial density distribution, for example, from a low-angle X-ray pattern (130), or accurate knowledge of the structure of a component can be used to calculate the appropriate correction to the image data. The availability of X-ray structures for the adenovirus hexon (298), the protein shell of CPMV (10, 62), and the HRV (62, 233, 235) has allowed very accurate difference imaging to be performed after CTF correction.

A number of the reconstructions which are presented in "Virus life cycles in three dimensions" are of objects which do not actually have complete icosahedral symmetry. The phages P2, P4 (101), and  (100) have a tail on one of their 12 fivefold vertices. The fiber of Ad2 (but not Ad3) has a bend which causes it to break icosahedral symmetry. The fibers of both viruses are trimeric and hence can never obey the fivefold symmetry required at an icosahedral fivefold vertex (239, 323). The effect of these departures from icosahedral symmetry is twofold. Firstly, they make it more difficult to orient the object. Usually, the nonicosahedral portion must be masked away to obtain reliable orientations (100, 101). Secondly, the nonicosahedral structure is smeared by the icosahedral averaging which takes place during reconstruction. Hence, we will never be able to use icosahedral reconstruction to visualize the trimeric structure of the adenovirus fiber (even in type 3 where it is shorter and straighter than in type 2) or to see the details of the site of tail interaction with bacteriophage capsids (100, 101).

The quality of the final reconstruction is, as described above, limited by the combined effects of the worst steps in the reconstruction process. The number of views needed to define the reconstruction to an isotropic resolution, d, depends on the distribution of views within the asymmetric unit and the diameter, D, of the particle. For evenly spaced views, the relation (D)/(60d) gives a lower estimate of the number of views required (91). A more precise answer is found by examining the eigenvalue spectrum for the determination of the coefficients of the expansion functions used to calculate the reconstruction (91). If all eigenvalues are one or greater, the reconstruction is well determined and the data from separate views will be averaged to improve the signal-to-noise ratio in the reconstruction. If eigenvalues are less than one, the corresponding expansion functions are ill determined and the reconstruction will contain artifacts due to undersampling of data. In practice, cryo-EM data are very noisy, and therefore enough data should be combined to yield an average eigenvalue significantly greater than 10 and no eigenvalues less than 1.0. For adenovirus (D = 1,100 Å), more than 20 views were used to achieve a resolution of 35 Å with an acceptable signal-to-noise ratio (296), although the lower-limit formula cited above would indicate that fewer than four views could be used to compute the 3D map to that resolution.

A first step toward the interpretation of a reconstruction is the definition of its absolute scale. This is not always straightforward, since magnetic lens hysteresis as well as changes in the height of the specimen in the objective lens causes small changes in magnification. One way to determine the absolute scale is comparison with a reconstruction of a standard virus whose precise dimensions have been determined from X-ray diffraction (26, 30). Direct comparison in three dimensions is much more precise than attempting a similar comparison with the images, particularly for a virion whose aspect changes with orientation.

Some authors refer to the symmetry of the reconstruction as part of the proof of its validity. Such analyses should be viewed with caution. The Fourier-Bessel expansion method constrains the reconstruction to have D5 point group symmetry (91). This imposes one fivefold axis and the five twofold axes in the plane perpendicular to the fivefold axis on the density map. Agreement between the separate views, and hence quality of the reconstruction, is reflected in the additional symmetry, i.e., the nonimposed threefold symmetry found in a truly icosahedral object. The agreement is also reflected by the phase residual calculated from the cross-common lines between images, the cross-common lines phase residual. The average phase error should be less than 90° for all data out to the resolution limit imposed on the reconstruction. This is a different criterion than that typically used for work with crystalline specimens. However, it has been demonstrated to be an appropriate one both theoretically and by comparison with X-ray data (296).

A more robust assessment of the reliability of the 3D density map is to compare independent reconstructions of the virus from the same or similar samples, from the same micrograph, or from different micrographs recorded under similar conditions of microscopy (19, 101, 251, 295).

The different resolution criteria in common use include weighted phase difference residuals (251), Fourier shell correlation (FSC) (321), and R-factor_AB (349). The formulas that define each of these criteria are as follows:

Phase residual
FSC
R-factor_AB
where F₁ and F₂ are the structure factors for the two maps, |F₁| and |F₂| are the structure amplitudes, F₂* is the complex conjugate of F₂, and the sums are taken separately over all structure factors within a given spherical resolution shell.

All three resolution criteria are computed for a series of concentric shells in the 3D transform and plotted as a function of spatial frequency (Fig. 13). The resolution limit normally defined by the phase residual is taken as the spatial frequency at which the residual exceeds 45°. For the FSC, the limit is considered to occur when the value falls below twice the value expected for noise with Gaussian correlation, which occurs at , where N is the number of Fourier terms included. In tests with icosahedral data, the cross-common lines phase residual often serves as a more-stringent criterion for resolution than the FSC, although all criteria show a sharp change indicating a lack of correlation between independent reconstructions at roughly the same resolution (210).

View larger version (21K): [in this window] [in a new window]   FIG. 13.   Measures of the resolution of a reconstruction of SFV with 80 particle images. The plot shows both the phase error (left scale) and the FSC (right scale) against the spatial frequency in inverse nanometers. The resolution of the reconstruction is approximately 27 Å as shown by the position at which the FSC crosses 0.5 and the phase error becomes greater than 45°. Notice that the two measures of resolution yield almost identical results.

Difference maps computed between independent and properly normalized reconstructions also give a measure of data reliability, because, ideally, the magnitude of the differences should be no larger than the noise level in the reconstructions. If the average fluctuation in the normalized difference map exceeds the noise level by more than 1 standard deviation, then the differences may be attributable to real differences between the maps. Such tests of reliability are therefore quite useful for establishing confidence in differences that are seen.

Information from biochemical data or complementary structural techniques such as X-ray diffraction can help support the reliability of the reconstruction. Obviously, the interpretation of the results is very much case dependent, as is their presentation. The least-squares fitting of the D5 expansion, which is used to represent the data averages, can lead to different ratios of signal to noise in different regions of the map for small numbers of particles (90) or when a preferential orientation of the particles in the water layer leads to an uneven distribution of orientation angles in the data used for reconstruction (108, 296). For this reason, the variation between data sets of the individual features in the map should be examined, particularly for those near symmetry axes.

It is possible in this age of computer graphics to make a result of any quality appear impressive. A publication of a reconstruction should be accompanied by all of the following to allow the reader to assess its validity: (i) magnification, beam dose, defocus, and relevant CTF extrema (these define the limits of resolution in the original data); (ii) step size for digitization and a description of any computational enhancement of the data such as contrast stretching, noise removal, median, or Fourier filtering (these define the reliability of transfer of data from micrographs to the reconstruction programs); (iii) description of the eigenvalue spectrum as a function of resolution (this defines the completeness of the data and acts as a control for the effects of preferential orientation); (iv) cross-common lines residual as a function of resolution or correlation coefficient as a function of resolution for the final data set (this shows the degree of agreement between data and its reproducibility); and (v) comparison of independent reconstructions by FSC, R factor, or variance map (this is the most widely accepted method of assessing the actual resolution of the final density map).

The success of the combination of icosahedral reconstruction and cryo-EM should not make us forget that the method is still relatively new. Its acceptance to date has been a consequence of the careful and conservative approach that has been used in interpreting and presenting results. As the method becomes more widespread, it is necessary to be even more careful that the limits of the reliability of the results are clearly defined not only for the users of the method but for our colleagues who rely upon our results for their own work.

Computer graphics make it easy to present a colored surface or volume representation of the reconstruction which will illustrate its features. A number of computer graphics packages are available for this purpose, including AVS, for which a number of nice user-written modules (273) are available for the presentation of biological macromolecular structure. The choice of the density (contour) level at which to represent a reconstruction is not always straightforward. Often people represent a structure at 1.5 or 2 standard deviations above the background to provide a somewhat objective presentation. Alternatively, they present the structure at a volume which they believe is appropriate to the molecular mass. This can be difficult because the volume is very sensitive to small changes in contour level, which in turn is sensitive to scaling and CTF correction. Experience in a number of systems indicates that the simple calculation of the molecular mass from the volume of the reconstruction rarely matches the expected value. This remains true for very highly refined reconstructions derived from large data sets. There are a number of potential explanations for this phenomenon. Limited resolution (85) will tend to cause the apparent volume to appear larger than the expected one. Typically, one derives the volume from the mass of the protein and ignores (with good reason, because we do not really know how to model them) the contributions of carbohydrate, lipid, or tightly bound nucleic acid. Further, there is the issue of disorder. Portions of a structure which are visible at low resolution may be too mobile to be visible at higher resolution. Taken together, these factors mitigate against placing enormous emphasis on the precise value of the volume derived from a reconstruction. As a guide, workers typically need to contour at about 120% of the expected volume in order to obtain a surface that makes biological sense. Roughly speaking, a structure with a volume near this level is reasonable; drawing conclusions based on volume fluctuations of 20% is not.

One of the difficult tasks facing the presenter of a reconstruction is conveying the same sense of the structure which has been gleaned from hours of examining it to other biologists who have less experience in interpreting such data. Stereo images (50, 205) are one way of conveying the 3D nature of the results. They are best for showing small portions of a structure. One needs to remember that roughly 15% of the readers (and seemingly 97% of referees) will not be able to visualize an image in stereo, so that an alternative presentation should be present. The use of different colors for chemically distinct components (64, 145, 243, 294, 352, 353), cut-open views (10, 132), radially cut surfaces (14, 15, 20), icosahedrally cut surfaces (40), and polar sections (132) helps to convey the structural details, but they should always be carefully described to allow the reader to distinguish representation from result. Another very useful technique is radial depth cueing (141, 264, 292), which conveys an immediate, and often quantitative, view of the radial placement of details in a map (145). Animation provides an alternative approach to enhance the viewer's perception of the 3D structure (106).

Difference imaging is one of the most powerful tools in the arsenal of the structural biologist. It has been used widely and successfully in many systems. One of the great advantages of cryo-EM over classical, negative-stain microscopy is that it allows reliable difference imaging (10, 17, 18, 38, 156, 162, 193, 240, 242, 243, 247, 267, 286, 297, 298, 308, 325, 352, 361, 362, 364). Many difference imaging results have been confirmed by biochemical and immunological means. Despite their potential, difference images must be calculated carefully to guarantee a reliable interpretation. The two maps whose difference is sought should be calculated to the same resolution and scaled in such a way that the differences are minimized. Propagation of error will guarantee that a difference image is always noisier than either of the two original images, and it is the noise in the difference map which determines the significance of features in it. Even if your understanding of the biology indicates that the only important difference is at a particular position, an understanding of statistics should inform your interpretation. One should always test to see whether a difference of a similar size can be found between independent reconstructions of the same structure before placing much weight on the result. Furthermore, one should always be very cautious about differences at symmetry axes, or equivalently at low radius, because noise tends to be higher at these locations. Finally, it is important to remember the caveats about the accuracy of molecular volume mentioned above, since a difference image is inherently more noisy and more sensitive to scaling errors.

The combination of X-ray and cryo-EM is particularly powerful (16). The ability to fit an X-ray model into the reconstructed density allows the definition of the position of the model with greater precision than the resolution of the cryo-EM map. There is no commonly agreed method that has been used for the fitting. Indeed, the same group will use different approaches to different systems. All approaches have the same general outline.

Firstly, the absolute magnification of the reconstruction must be established. The absolute magnification of an EM varies with the conditions of imaging and can vary from image to image. An error of a few percent completely destroys any possibility of a reliable fit. Workers check the scale by comparing the EM map with clear features in the X-ray structure of an individual component (298, 299), by using radial density profile information derived from scattering experiments (64), or by using the X-ray structure of an entire viral shell when this is available (156, 162, 166, 240, 290). When a single component is used to define the scale, it is necessary to recheck the scaling after the proper position and orientation of that component have been identified.

Secondly, the variation in density through the reconstruction must be matched to that in the X-ray structure. In the case of the fitting of the hexon to adenovirus, this was done by convolving the X-ray structure with the transfer function for the EM reconstruction (298). Although the original reconstruction (296) had been calculated by combining images at different defocus values, only the high-resolution data were used for the comparison with the X-ray data to avoid having to deconvolve each defocus separately. The effect of the imaging conditions was simulated by using a CTF of the form employed by reference 185 with both phase and amplitude components as well as attenuation:
where  is a decay factor which expresses the decrease in amplitude as a function of resolution due to the incoherence of the beam as well as the imprecision in aligning the images for the reconstruction. The defocus and decay values were fit empirically by convolving the X-ray map with the CTF and applying a Fermi filter to smoothly eliminate information beyond the resolution of the reconstruction (298, 299). Alternatively, when the extra density in the EM map can be cleanly masked away, the relative densities of the X-ray structure and the masked reconstruction can be adjusted by comparing the Fourier transforms of the projections of the two maps by using the smoothed ratio of the radially averaged amplitude to rescale the EM map (156, 162). Alternatively, one can simply normalize the EM map so that it is all positive and has the same range of density values as the corresponding X-ray map (166, 208, 344). An alternative, maximum-entropy approach (282) has been used for the treatment of CTF effects and has been shown to improve the correspondence with the X-ray structure for the adenovirus hexon by using the orientations and data from the original work (296, 298).

Thirdly, an interactive fit is made between the EM density and the X-ray structure by using a map-fitting program such as O (180) (Fig. 14). At this point, a subjective estimate of the quality of the fit is made. An important criterion for the quality of the fit is whether the hand of the structure can be identified. The EM density is generated from projected views of the structure, and although a relative hand is imposed during the refinement, the absolute hand must be determined from other data such as shadowing or comparison of the orientations of tilted images (28) of the same particle. The match with the X-ray data (for which the hand is known from the secondary structure and residue geometry) serves as an unambiguous determination of hand. Usually, both the correct hand and the wrong one are monitored through the rest of the analysis, and the difference between them is used as a criterion for the reliability of the data (205, 344). At this stage, one also needs to decide whether the X-ray structure should be fit as a single rigid body (298) or as two or more domains (145, 162, 166).

View larger version (66K): [in this window] [in a new window]   FIG. 14.   Atomic modeling of a 3D reconstruction of a virus-Fab complex shown in stereo. (Top) Reconstructed map of HRV14 (in white) complexed with Fab fragments from neutralizing MAb 17-IA (pink). (Middle and bottom) Orthogonal views of the atomic model of Fab 17-IA (light chain in grey; heavy chain in dark yellow) and the atomic model of HRV14 (VP1 to VP4 in blue, green, red, and cyan, respectively) fitted into the cryo-EM density envelope (wire mesh). The "hand-in-glove" fit of the atomic models into the cryo-EM envelope demonstrates that, at least in this example, the interacting surfaces can be defined to within a few angstroms (58, 285). See Fig. 55 for comparison.

Fourthly, the quality and uniqueness of the fit are assessed. This can be done simply by calculating an R factor between the two maps. A mask must be applied before calculation to remove the density which is not part of the structure being fit by the X-ray data (64, 205, 298). The uniqueness of the fit is then tested by rotating and shifting the X-ray structure in the EM density and monitoring the change in the R factor (58, 205).

Finally, an objective method of fitting is used to refine and check the uniqueness of the result of the interactive fitting. This refinement is performed either in reciprocal space or in real space. Reciprocal space refinement is usually performed with the program X-PLOR (47) after tailoring of the two maps to avoid ripple and edge effects due to masking and differences in contrast (145, 156, 162, 344). Some of the corrections compensate for the microscope imaging process, but others, such as the inclusion of a high-temperature factor (145, 298), correct for limitations in the reconstruction process such as alignment accuracy. Real space refinement has been performed with different procedures and programs by each group. The measure of the quality of fit is critical to the results. In the fit of SNV capsid protein to the RRV structure, the fitting was begun by superimposing the centers of mass of the capsid proteins on the centers of the capsid features in the map (64). The positions were refined by allowing a six-dimensional rotational and translational search to optimize the R factor between the two structures. The top 100 fits (which were reduced to 10 unique ones) were distinguished by two further criteria: the number of atoms placed in negative density (den) and the number of atoms in steric collision between monomers (nclash). The product of these (den*nclash) appeared to be the most sensitive test. Reassurringly, the best fit was obtained with the hand of the EM map which was shown to be correct by tilting experiments (64). A different real space method, implemented in the program GAP (145), which used a method of steepest-ascent optimization of the correlation coefficient between the two densities was employed for the fitting of VP7 into the BTV core. The scaling of the density maps was first optimized by tests with X-PLOR. The X-ray map was then filtered by using a mask which included all density within 10 Å of an atom. This mask, which includes a solvent contribution, was used to ensure that the shape of the structure rather than its internal features was used for fitting. The comparison between the results of real space fitting and those of reciprocal space fitting is informative. Reciprocal space fitting usually is not constrained to avoid interpenetration of the densities, an issue which is easy to address in real space fitting. When the two approaches yield different solutions, the possibility of a change in the X-ray structure upon binding should be considered.

The use of EM data in combination with X-ray data is not limited to cases in which the high-resolution structure from X-ray diffraction is used to enhance the interpretation of a reconstruction. In several cases the EM map was used to help solve the phase problem in the solution of the X-ray structure of the virus. The great advantage of this approach is that it avoids the classical use of heavy atom derivatives which produce only small changes in the scattering of a large object such as a virus and introduce problems of nonisomorphism. The first such example was the solution of CCMV, where the cryoreconstruction was used as a model for phasing the X-ray data (290). The atomic coordinates of southern bean mosaic virus were placed into the density of the cryoreconstruction. A rotation function calculated to 15 Å was used to orient the model with respect to the crystallogrpahic data. This oriented model was then used to calculate phases which were extended to 4 Å by the use of phase extension and noncrystallographic symmetry. This 4 Å envelope was then used to construct a polyalanine model which provided phases to 3.5 Å. In the solution of the core of BTV, the EM map (241) was used to position the X-ray-derived structure of the VP7 protein so that an atomic model could be generated (145), which was then used to calculate initial phases for the X-ray data for the whole core. The cryo-EM-derived map of HRV14 in complex with neutralizing Fab 17-IA (205) was used in the solution of the X-ray structure of the same complex. The structures of the isolated Fab and the uncomplexed virus had been solved previously. The cryo-EM map was used to position these components and provide a phasing model for the X-ray data (285). Interestingly, the solution of the X-ray structure showed an unprecented conformational change in the binding site region of the Fab which allowed a closer approach of the Fab into the receptor binding canyon than had been inferred from the EM data (205, 285).

VIRUS LIFE CYCLES IN THREE DIMENSIONS

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Shared Features of Virus Life Cycles

A virus is a parasite of its host. As a result, a virus's life cycle is inextricably tied to that of the host cell. Despite this dependence, all viruses share a number of essential tasks which they must accomplish for survival (Fig. 15). A successful virus must be able to find and recognize a cell in which it can replicate, release its genome into the cell, generate new viral components by transcription and translation, and assemble these components into precursors that mature into a stable progeny virion which is released from the host cell and transmitted to encounter a new host. Each of these tasks involves interactions between components in the context of the whole virion. Visualization of such interacting components is a job ideally suited to cryo-EM. Different viruses accomplish these tasks in different ways as a result of adaptation to different cellular environments. We will describe the results of cryo-EM and image reconstruction which illustrate the structural response of different viruses to these common tasks. Visualization of these structural solutions enhances our understanding of the deeper biology underlying them. One unexpected commonplace of structural virology is the relatedness of structures which accomplish these tasks in different hosts. The observation that structural similarity is found in the absence of detectable sequence homology indicates that it reflects fundamental features of the tasks. Several examples will make this general description of common tasks more concrete.

View larger version (21K): [in this window] [in a new window]   FIG. 15.   The common tasks faced by all viruses as exemplified by the replication of SFV, an enveloped virus, in a mammalian cell. The virus first binds to a cell surface receptor (Cell attachment). Entry ensues by receptor-mediated endocytosis, and the capsid-bearing genome is released into the cytoplasm via membrane fusion mediated by the low pH of the endosome (Release of genome). The capsid and envelope proteins are synthesized, and the spike is assembled in the secretory pathway as a heterodimer comprising the precursor of E2 (p62) and the fusion sequence-bearing protein E1. The capsid incorporates the genomic RNA in the cytoplasm (Pro-assembly). The envelope protein complex is processed in the late secretory pathway to yield the fusion active complex of E1, E2, and E3 (Maturation). The mature spike assembles into small patches on the plasma membrane which are complementary to the arrangement of capsid proteins, and the budding particle forms and is released from the cell (Release). Propagation of the infection involves evading the host immune system and, in some cases, replication in a second host, such as a mosquito for the arbovirus SFV (Transmission and host defense).

Many of the best-studied icosahedral viruses which replicate in animal cells enter by means of receptor-mediated endocytosis.

An alphavirus, such as SFV, is a relatively simple example of an enveloped virus. This positive-strand RNA virus comprises a nucleocapsid made of 240 copies of the C protein, which is surrounded by a lipid envelope in which 240 copies of each of two transmembrane glycoproteins, E1 and E2, and an extrinsic membrane protein, E3, are located. SFV, binds to a cell surface receptor by means of its envelope glycoproteins and is then recruited to a coated pit and endocytosed (Fig. 15). In the low pH of the endosome, the envelope proteins undergo a low-pH-mediated conformational change which results in the fusion of the viral envelop with the endosomal membrane. The binding to the cell surface receptor (cell attachment, Fig. 15) and the irreversible low-pH-mediated conformational change (activation, Fig. 15), which occurs after endocytosis, are the two aspects of cell recognition. Both are mediated by the envelope proteins. Fusion requires not only low pH but also the presence of cholesterol and sphingomyelin in the target membrane. The joining of the membranes allows release of the nucleocapsid into the cytoplasm, where the genome is transcribed to produce new copies of the genome and the mRNAs for the structural and nonstructural proteins. Copies of the genome are encapsidated by the C protein in the cytoplasm (proassembly, Fig. 15). The envelope proteins are synthesized by membrane-bound polysomes and translocated into the endoplasmic reticulum, where they oligomerize to form the spike complex. E2 and E3 are initially synthesized as a precursor, p62, which is cleaved by a host protease during the transport of the spike complex through the Golgi complex to the plasma membrane. This cleavage of the precursor p62 (proassembly, Fig. 15) renders the spike pH sensitive and is a necessary stage in the production of an infectious virion. Budding occurs when the assembled nucleocapsid is enveloped at the plasma membrane as a result of its interaction with the spike complexes (assembly and release, Fig. 15). The released virion then proceeds to infect other host cells. SFV is transmitted in the wild by means of a mosquito vector which is protected from the deleterious effects of infection by the lack of cholesterol in the insect.

Many of the details of the life cycle which were described above are unique to alphaviruses. Despite this specificity, analogous stages exist in the life cycles of other viruses. Rotavirus, for example, enters by the receptor-mediated endocytosis and utilizes the secretory pathway for assembly as does SFV. It buds, however, into the endoplasmic reticulum and loses its membrane after budding. Loss of the membrane and other modifications are necessary maturation steps which are required for the initial budded virus to become an infectious virion. Transport of the virion through the secretory pathway terminates in its secretion from the cell. A nonmembranous virus, such as rotavirus, does fuse with the endosome. However, binding of rotavirus to the cell surface receptor and exposure to the environment of the endosome cause dramatic conformational changes in the virion. Picornaviruses, for example, undergo a conformational change upon binding to the appropriate cell surface receptor, which results in their becoming much more hydrophobic. The altered capsid is more open, as seen by its inability to protect the RNA from nuclease digestion, which may facilitate the release of genome into the cytoplasm. Assembly of the structural proteins around the genomic RNA generates a protease active site which causes cleavage of the polyprotein product to generate the mature, infectious virion. Picornaviruses are typical of many other nonenveloped viruses in being released from the cell by lysis rather than by budding.

The first step in a productive viral infection is the binding of the virus to the correct host. Viruses recognize target cells by binding to specific receptor molecules at the cell surface. Binding involves specific interaction between a viral protein or glycoprotein and the receptor. Successful attachment usually ensues upon binding to one or more identical receptor molecules. Some viruses, like adenovirus (165), utilize at least two different receptors to achieve attachment. Abortive infection occurs if attachment does not lead to the subsequent activation step that triggers entry of the viral genome into the cell.

Viral receptor binding sites are typically comprised of highly conserved amino acid residues (257, 258), a requirement that appears to guarantee survival of the virus. Early crystallographic studies of picornaviruses indicated that these and other viruses may shield their receptor binding sites in cavities or surface depressions ("canyons") that are inaccessible to antibody molecules and would therefore provide viruses a selective advantage (258). Subsequent studies of other viruses have shown that receptor binding sites also occur in highly exposed regions of the viral surface, indicating that evolution of virus-receptor and virus-antibody interactions may be subject to independent constraints (76).

Detailed understanding of virus-receptor interactions ultimately requires structural analysis at high resolution. The first direct structural evidence for the nature of receptor interactions with complete virus capsids was accomplished by means of cryoreconstruction analyses and atomic modeling of two virus-receptor complexes: HRV16 complexed with the cellular receptor ICAM-1 (233) and the human parvovirus B19 complexed with the receptor globoside (67). The receptor molecule in each of these studies was shown to bind to a cavity or depression in the viral surface. A brief description of the HRV study illustrates the use of cryo-EM methods to examine virus-receptor interactions.

HRV16-ICAM complex. HRVs comprise more than 100 serotypes and are the major cause of the common cold. The icosahedral virus (~300-Å diameter) consists of 60 copies each of four proteins (VP1 to VP4) and a single copy of a positive-sense, ssRNA genome (Fig. 16). A 20-Å-deep and 12- to 15-Å-wide surface depression (canyon) encircles each of the 12 fivefold vertices and lies approximately halfway between the fivefold and three fold icosahedral axes.

View larger version (65K): [in this window] [in a new window]   FIG. 16.   Schematic diagram (left) and cryoreconstruction (right in radially depth-cued representation) of HRV14. The outer capsids of the more than 100 serotypes of HRV (family Picornaviridae) all contain 60 copies each of three viral proteins, VP1, VP2, and VP3. Four classes of neutralizing antibodies have been genetically mapped to viral epitopes on these surface proteins (274). NIm-IA and NIm-IB map to VP1, and NIm-II and NIm-III map to VP2 and VP3, respectively. The canyon, a prominent surface depression that completely surrounds each of the 12 fivefold vertices of the HRV capsid, was first recognized in the crystal structure analysis of HRV14 and hypothesized to be an important site of cellular recognition (258).

View larger version (174K): [in this window] [in a new window]   FIG. 17.   Cryoreconstruction study of native HRV16 (left) and an HRV16-D1D2 complex (right). (Top) Small fields of view showing vitrified samples of native HRV16 and HRV16 complexed with the D1D2 fragment of the cellular receptor ICAM-1. (Bottom) Isosurface representations of the HRV16 and HRV16-D1D2 reconstructions. The two-domain D1D2 structure binds radially to the HRV16 surface at the canyons that surround the star-shaped VP1 pentamers as predicted elsewhere (258).

Other cryo-EM studies concerning receptor-virus interaction. Indirect evidence about receptor binding to viruses has been obtained by means of cryoreconstruction studies of reoviruses, alphaviruses, adenoviruses, and the picornavirus FMDV. For example, the structure of VP4, the 88-kDa receptor recognition protein of rotavirus, has been the subject of several studies owing to its central importance to the rotavirus life cycle (242, 249, 271, 352, 353). VP4 manifests hemagglutinin activity and is a neutralization antigen as well as a determinant of virulence. It also mediates cell penetration and is susceptible to cleavage by trypsin, which enhances viral infectivity. VP4 has a role in the maturation of progeny virions through its interactions with VP6 and VP7 viral structural proteins (see above references for extensive citations concerning these functions). These studies showed that 60 dimers of VP4 ("spikes") extend ~100 Å above the viral capsid surface in a uniform arrangement that appears to facilitate binding interactions with cellular receptors (Fig. 18). The distal ends of the spikes are believed to contain the receptor binding sites, since neutralizing antibodies that bind near the distal ends inhibit viral penetration (242).

View larger version (109K): [in this window] [in a new window]   FIG. 18.   Cryoreconstruction of simian rotavirus (352). The prominent, dimeric VP4 spikes (yellow) project ~110 Å radially outward from the viral surface in a manner that may facilitate binding to cellular receptors. VP7 (blue), the major outer capsid protein and an antigenic determinant, forms a smooth outer shell with 132 channels, through which the second major capsid protein, VP6 (pink), and the core proteins, VP1 to VP3 (red; individual proteins could not be distinguished in this reconstruction), are accessible. Twelve (type I) channels occur at the fivefold axes, 60 (type II) are partially occluded by the VP4 subunits, and 60 (type III) have the rather prominent crescent-shaped openings. Figure courtesy of M. A. Yeager.

Enveloped virus entry. SFV, SNV, and RRV are three alphaviruses which have been subjects of extensive work with cryo-EM (64, 130-132, 186, 237, 238, 286, 325, 327) as well as cell biology and biochemistry (reviewed in reference 187), making this family the best characterized of the icosahedral, enveloped viruses. The life cycle for each follows the general scheme described above for SFV ("Shared features of virus life cycles"). Activation in these viruses refers to the process of pH-mediated fusion. This process is selective in the sense that the mature virus will encounter low pH only after endocytosis by the host cell. The combination of cryo-EM and biochemistry has been able to shed new light on the process by which the virion is activated for fusion by low-pH exposure. An alternative model for alphavirus infection (reviewed in reference 46) invokes disulfide shuffling without the requirement for endocytosis or low pH; however, we will adhere to the low-pH-endocytosis paradigm in this review.

View larger version (110K): [in this window] [in a new window]   FIG. 19.   A cutaway representation of the 22-Å-resolution reconstruction of SFV (132) revealing the organization of this enveloped virus. The outer surface (blue) of this T=4 virus is studded with 80 trimeric spikes, each comprising three copies of the transmembrane glycoproteins E1 and E2 and the extrinsic membrane protein E3. The spikes are connected above the surface of the membrane by a protein skirt which is penetrated by holes at the two- and fivefold positions. The two leaflets of the lipid bilayer are seen below the skirt. The capsid protein (gold) forms a T=4 array of hexamers and pentamers which surrounds the RNA. The capsid is complementary to the spikes and connected to them by transmembrane regions which are shown in red.

View larger version (41K): [in this window] [in a new window]   FIG. 20.   An exploded view of the SFV structure (132) around a single spike showing the modular nature of the virion structure. The projecting regions of the spikes are shown in blue. The skirt of protein which connects the spikes is shown in yellow. The cavity at the center of the spike is indicated by an arrow. The outer and inner leaflets of the bilayer and the transmembrane regions which span them are shown in red. The portion of the capsid which interacts with these transmembrane regions is shown in green. Adapted from reference 132 with the permission of the publisher.

View larger version (23K): [in this window] [in a new window]   FIG. 21.   The conformational change which occurs within the first 50 ms of exposure to low pH is shown for a single SFV spike. Initially, the fusion sequence-bearing E1 protein which forms the edge of the spike alternates with the receptor binding E2 protein which forms the vertices around the cavity in the center. The triggering of the conformational change by low pH leads to the movement of the E2 domains away from the center and the formation of an E1 homotrimer in the center of the spike. The transmembrane regions and the skirt appear undisturbed at this stage of the conformational change. Adapted from reference 132 with the permission of the publisher.

View larger version (133K): [in this window] [in a new window]   FIG. 22.   Images of SFV contacting a target vesicle in the process of fusion. A defocus pair of images (nominally 1,000 Å and 4 µm) allows the visualization of the leaflets of the target vesicle as well as the nature of the contact on the side of the virion. The contact appears to be formed by several spikes acting in concert which would then cooperate in the forming of a fusion pore.

Nonenveloped virus entry. Adenovirus is a nonenveloped virus with apparent T=25 icosahedral (Fig. 23) symmetry. Cryo-EM and image reconstruction (296, 298) were used together with the high-resolution X-ray diffraction structure (255) of the major capsid protein (hexon) to identify the positions of all of the polypeptides by difference imaging. Many of these minor proteins appear to stabilize the complex structure of this large virus (see "T=25 structures: divide and conquer"). Hexon (polypeptide II) and the two proteins which comprise the fivefold vertex, penton base (polypeptide III) and the fiber (polypeptide IV), are known to have functions in the entry of the virion. Adenovirus utilizes at least two receptors to enter the cell (165, 340, 341). Entry is initiated by the attachment of the fiber to the primary cell surface receptor. For example, the fibers from Ad2 and Ad5 bind to CAR (coxsackievirus and adenovirus receptor), which is a 46-kDa protein expressed on the surface of many cells, and the Ad2 fiber also binds to major histocompatibility class I antigens (see reference 328 and references cited therein). The second receptor, a cellular integrin, binds to the penton base through its RGD sequence and triggers internalization by clathrin-coated vesicles (341) but not virus attachment. This second receptor must fulfill an activation function as shown by the fact that interaction with this receptor potentiates permeabilization of the endosome by the particle (340). Cryoreconstructions have provided a wealth of information which enhances our understanding of activation during the entry process.

View larger version (103K): [in this window] [in a new window]   FIG. 23.   A surface representation of a cryoreconstruction of Ad2 shows the T=25 arrangement of the trimeric hexon protein (inset). Two proteins form the fivefold vertex. Five copies of polypeptide III form the penton base, and three copies of polypeptide IV form the trimeric fiber. The fiber has an ~45° bend one-third of the way along its length, and hence only the proximal third is seen since the rest lacks full icosahedral symmetry. Reproduced from reference 298 with the permission of the publisher.

View larger version (64K): [in this window] [in a new window]   FIG. 24.   The change in the adenovirus penton base upon the removal of the fiber is shown by a comparison of cryoreconstructions of the dodecahedral structure formed by expressed Ad3 pentons. The structure of the dodecamer with the fiber attached is shown in blue for comparison with the fiberless structure shown in red. The top row shows a surface view (left) and sections through the surface for the two structures viewed from the side. The center of the virion would be toward the bottom of the page. The bottom row shows a surface view from the outside of the structure (left) and sections through the protrusions at the edge of the base and through the widest portion of the base. Reproduced from reference 267 with the permission of the author and the publisher.

View larger version (130K): [in this window] [in a new window]   FIG. 25.   The position and proposed structure of the RGD epitope on the surface of Ad2. The position of the epitope was established by Fab labeling. The structure was modeled from the sequence and shown to match the density seen at the appropriate position. Adapted from reference 297 with the permission of the author and the publisher.

Release of the genome from any virus is an asymmetric process. Thus, genome release from icosahedral viruses induces functionally important asymmetry in the form of large conformational changes. As valuable as icosahedral image reconstruction methods may be, they are ill suited for producing detailed structural pictures of asymmetric particles. The use of single-particle reconstruction methods (e.g., see reference 125) is likely to yield more-detailed analysis of such structures. Despite the disadvantages, image reconstruction studies of a few icosahedral viruses have provided new insights and hypotheses regarding the processes of genome release.

Model of genome release in FHV. Coordinated cryo-EM and X-ray crystallographic studies of FHV have provided information about genome organization (see "Genome organization") and also have led to a hypothesis concerning genome release in this insect virus (66). FHV (Nodaviridae family) has a T=3 capsid consisting of 180 copies of a single, 44-kDa protein (407 aa) which encapsidates an ssRNA genome (three genes on two RNA molecules) (Fig. 26). The capsid proteins undergo a postassembly cleavage required for infectivity. This cleavage produces 180  peptides (C-terminal 44 aa) on the interior of the virus. Crystallographic analysis of FHV virions (122) showed that 16 residues at the N terminus of each peptide form an amphipathic  helix and that these  helices lie in dramatically different environments owing to quasi-equivalent interactions among the  peptides (N-terminal 363 aa). A cryoreconstruction of virions at 22-Å resolution (66) revealed the organization of the bulk RNA that was invisible in the 3.0-Å crystal structure (122) and also showed density completely consistent with the highly ordered duplex RNA that was seen in contact with the inner capsid wall at the icosahedral twofold axes in the X-ray structure (Fig. 27).

View larger version (62K): [in this window] [in a new window]   FIG. 26.   Schematic diagram (left) and cryoreconstruction (right) of the ssRNA insect virus FHV (family Nodaviridae). The capsid of FHV consists of 180 copies of a single subunit arranged with T=3 icosahedral symmetry. Each icosahedral asymmetric unit consists of three quasi-equivalent subunits, labeled A, B, and C. A and B are related by a quasi-twofold axis (open ellipse). The positions of a peptide arm and a portion of the RNA genome that adopts a highly ordered, double-stranded loop are also indicated in the diagram. The isosurface rendering of the image reconstruction shows prominent peaks on the viral surface at local threefold positions (open triangle in diagram). Positions of four icosahedral symmetry axes (2, 5, and 3) are marked on the reconstruction. Adapted from reference 66 with the permission of the author and the publisher.

View larger version (46K): [in this window] [in a new window]   FIG. 27.   Positions of  helices in FHV. (Top) Cutaway view of FHV with density derived from cryo-EM in blue and density derived from a difference map (cryoreconstruction minus X-ray structure of capsid only) in red. The cylinders represent the three types of  helices, with A in blue, B in orange, and C in green. Asterisks mark the helices shown in the middle panel. (Middle) Close-up view of two pairs of  helices that are in close association with the bulk RNA (red mesh) and ordered RNA (space-filling model). (Bottom) Side view of region near the icosahedral fivefold axis (vertical white line) showing the bundle of A helices and A subunits (one in a green-, yellow-, and red-ribbon representation and the others in thin-yellow-line C backbone traces) fit into the EM envelope (blue mesh). The ellipsoidal difference density (red contours) is situated on the fivefold axis in a position consistent with unmodeled density from the X-ray map (white contours). Adapted from reference 66 with the permission of the author and the publisher.

View larger version (61K): [in this window] [in a new window]   FIG. 28.   Hypothetical entry pathway for FHV. (Top) Fivefold view of FHV cryoreconstruction. The pink circle marks the fivefold axis, and the two sets of blue, red, and green dots identify the exposed loops of the A, B, and C capsid subunits, respectively, at two of the five quasi-threefold positions on the viral surface adjacent to the marked fivefold axis. The arrow shows the view direction (perpendicular to fivefold axis) in the middle and bottom panels. (Middle) Initial interaction of six methionine residues (not depicted) at the tips of the loops in the A (blue), B (red), and C (green) capsid subunits with a bilayer membrane (grey balls). The bundle of A peptides (pink) and the RNA genome are poised just inside the viral surface at the fivefold axis. (Bottom) Capsid subunits rotate toward the membrane, allowing the A peptides to be inserted into the membrane and form a channel for release of the viral genome into the host cell. Middle and bottom panels courtesy of T. J. Smith.

Other cryo-EM studies concerning genome release. Environmentally induced particle expansion is believed to be a common mechanism for achieving disassembly of plant viruses in vivo. CCMV, a T=3, ssRNA plant virus, swells by 10% when metal ions are removed and the pH is raised to 7.0. Cryo-EM analysis of native and swollen CCMV particles along with X-ray crystallographic analysis of the native virion and atomic modeling of the swollen particle revealed that the coat subunits undergo a 29-Å radial expansion as a result of electrostatic repulsion of carboxylate groups (290) (Fig. 29). Preservation of RNA-protein contacts as well as intersubunit contacts (interwoven C termini) keeps particles from completely disassembling. Upon swelling, the hexamers and pentamers remain essentially intact but move to a higher radius (along quasi-sixfold and fivefold axes, respectively). They thereby separate from each other at the threefold axes, creating holes ~20 Å in diameter. These holes form large conduits for the radially expanded RNA genome which lies below the openings to them. The extent to which the observed swollen CCMV structure represents an in vivo particle state that is competent to release the genome for replication and translation is unknown. However, the viral RNA can act as a template for protein synthesis and as a substrate for RNase in the swollen particle (21, 45). Also, it is notable that the conditions that promote expansion (high pH, low Ca²⁺) are characteristic of intracellular conditions, whereas the conditions that promote the compact state of the virion (low pH, high Ca²⁺) are characteristic of extracellular sites in plants. Finally, an earlier crystallographic study of tomato bushy stunt virus, another T=3 plant virus, demonstrated that the capsid of this virus also undergoes an expansion that leads to opening of holes in similar sites (256).

View larger version (102K): [in this window] [in a new window]   FIG. 29.   Structures of native and swollen forms of CCMV determined by X-ray crystallography and cryoreconstruction (290). (Top left pair) Stereo view along twofold direction of the native CCMV protein shell rendered as a C tracing with the A, B, and C subunits colored blue, red, and green, respectively. The yellow cage depicts the edges of a truncated icosahedron. (Top right) Truncated icosahedral model of CCMV in the same view orientation and color coding as the stereo C model. Yellow symbols mark the locations of the strict two-, three-, and fivefold icosahedral symmetry axes, and white symbols mark the positions of quasi-two- and threefold axes. The central triangle of chemically identical A, B, and C subunits defines the asymmetric unit of the icosahedral structure. The subunits are colored differently to reflect the fact that they occupy slightly different geometric (and chemical) environments. Polygons with subscripts are related to A, B, and C by icosahedral symmetry (e.g., C and C2 are related by a strict twofold rotation and A and A5 are related by a strict fivefold rotation). The A, B, and C subunits of the asymmetric unit are related by a quasi- or local threefold axis (white triangle; vertices of yellow cage in stereo view to left). The three orange dots mark the putative calcium binding positions in one asymmetric unit of CCMV (290). (Middle row) CCMV capsid at pH 4.5. At the left is a magnified, stereo view of the native, 3.2-Å-resolution CCMV X-ray C model fit into the 23-Å-resolution cryoreconstruction envelope (blue mesh). The yellow, truncated icosahedral cage is the same as that depicted in the top row. Calcium ions (180/capsid) are bound between capsomers by residues associated with the helices (white cylinders) which appear as three pairs positioned around each quasi-threefold axis. An isosurface view of the cryoreconstruction is shown at the right (same size as models in the top row). (Bottom row) Same views as in middle row but for the swollen CCMV capsid at pH 7.5 in the absence of metal ions. The atomic models of the native capsid subunits were translated and rotated as rigid bodies to fit the 28-Å-resolution cryoreconstruction in the stereo view. Adapted from reference 290 with the permission of the author and the publisher.

The family Reoviridae includes moderately large (700- to 900-Å), nonenveloped, T=13 icosahedral viruses with a complex, multilayered protein structure which encapsidates 10 to 12 segments of dsRNA (225). The strategies for infection and replication are similar for all Reoviridae viruses, and all viruses contain the enzymes required for transcription of the dsRNA.

Early cryoreconstruction work on the rotaviruses, orbiviruses, and orthoreoviruses led to a consensus hypothesis that large channels in these structures might serve as pathways for importing metabolites required for viral RNA transcription and also for exporting newly synthesized mRNA molecules used in subsequent viral replication (108, 157, 158, 241, 249, 353). A similar conclusion has been proposed for an aquareovirus (272) and also for dsRNA fungal viruses L-A and P4, which have much smaller (10- to 15-Å) holes (55, 63). Cryoreconstruction methods were recently used to visualize mRNA release from actively transcribing rotavirus particles (193). This study showed that nascent mRNA transcripts generated within the rotavirus core by endogenous transcriptase complexes are translocated through the intact capsid through a system of channels at the icosahedral fivefold axes (Fig. 30). Acridine orange, an ssRNA intercalating agent, was used to enhance the visibility of the exiting mRNA in the cryo-EM specimens. As expected, the particles remain structurally intact during transcription, which can continue indefinitely.

View larger version (33K): [in this window] [in a new window]   FIG. 30.   Transcribing rotavirus DLP. (Left) Cryoreconstruction at a 25-Å resolution of the actively transcribing rotavirus DLP, viewed along a threefold axis and rendered with radial depth cueing (color bar gives corresponding radii for various colors). The pink "bowling pins" represent discernible portions of newly transcribed mRNA molecules as they exit the channels along the virus fivefold axes, each of which is bounded by five trimers of VP6 (blue). The two middle images compare close-up views of transcribing (left) and nontranscribing (right; at ~28-Å resolution) DLPs. The rightmost image, in which one of the five VP6 trimers that surround the type I channel has been removed for clarity, depicts a hypothetical path (grey tube) that the mRNA transcript may take as it passes through the VP2 (green) and VP6 (blue) layers of the inner capsid. Adapted from reference 193 with the permission of the author and the publisher.

Additional information about transcription in dsRNA viruses has been obtained in cryoreconstruction studies of rotavirus (195, 247), orthoreovirus (107, 209), cytoplasmic polyhedrosis virus (356), bacteriophage 6 (48), and the fungal viruses L-A and P4 (55, 63). A common goal in these studies has been to probe the structure of the transcriptase complex in each virus. A noteworthy structural feature that appears to be unique to dsRNA viruses is that the fungal viruses and the cores of the other viruses all contain 120 copies of at least one protein subunit. This unique stoichiometry and the associated molecular architecture appear to be favored for the construction of specialized intracellular compartments for transcription and replication that protect the dsRNA genomes from host cell enzymes and may also serve to retain them in the cytoplasm.

In rotavirus, the viral dsRNA forms a dodecahedral structure in which the RNA interacts closely with 12 enzyme complexes located at the icosahedral fivefold axes. This arrangement is proposed to be conducive for an orchestrated movement of the RNA relative to the complex during transcription (247). Similar internal structures have been observed in orthoreovirus (107) and in the insect reovirus cytoplasmic polyhedrosis virus (356).

Heat and chymotrypsin treatment of reovirus core particles cleaves the 144-kDa 2 protein, which carries the guanylyltransferase activity (adds 5' caps to reovirus mRNAs), leaving a 120-kDa N-terminal fragment associated with the cores (209). Reconstruction of these modified cores revealed the loss of a distinctive outer region of 2. Because these particles were shown to retain all activities required to transcribe and cap reovirus mRNAs, the missing portion of 2 is dispensable for these functions.

Cryo-EM studies of the enveloped bacteriophage 6 revealed an outer, T=13 layer of protein P8 and a core consisting of four proteins (P1, P2, P4, and P7). P1 is present in 120 copies (48). The core, which has a structure strikingly similar to that seen in reovirus (108), acts as the RNA packaging and polymerase complex. Expressed core proteins assemble into replication-competent procapsids. Comparison of several procapsid-related particles (expressed and assembled in Escherichia coli) with virion-isolated nucleocapsids and cores suggested that the maturation process involves extensive structural rearrangements that produce expansion. The movements are coordinated with the packaging of the viral RNA and with the RNA polymerization reactions leading to virion assembly (48).

The L-A virus of yeast has a capsid consisting of 120 copies of Gag protein (76 kDa), of which approximately two contain the Pol moiety and in which the proteins are arranged as 60 asymmetric dimers (55, 63). A model of how RNA transcription occurs in L-A was formulated on the basis of a cryoreconstruction at 16-Å resolution (55). The capsid wall is perforated by 10- to 15-Å-diameter holes which function as a molecular sieve by allowing exit of nascent mRNA and influx of metabolites while retaining and protecting the dsRNA from degradative enzymes (Fig. 31).

View larger version (146K): [in this window] [in a new window]   FIG. 31.   Model of transcription in L-A virus (55). Approximately two copies of the RNA-dependent RNA polymerase (Pol; shown with an arbitrary shape) are present in each virion. Each Pol, which is covalently attached to one of the 120 Gag subunits that comprise the icosahedral capsid of L-A virus, presumably lies at the inner surface of the capsid close to the dsRNA substrate. In this diagram of the hypothetical transcription pathway, as the genome is processed by Pol, the ssRNA transcripts are extruded through small openings in the capsid. Adapted from reference 55 with the permission of the author and the publisher.

Harried parents on Christmas Eve (and others of us who never read manufacturers' instructions) cling to the belief that assembly proceeds through a series of stages which increasingly resemble the desired final product. Virus assembly rarely follows such an intuitively obvious path. Although the paradigm for understanding virus structures was based on the requirement for self-assembly (53), many virus capsids utilize chaperones and/or scaffolding proteins to ensure that the task of assembly is completed correctly and efficiently. All of the well-studied cases of assembly pathways include intermediate structures which need to undergo substantial conformational changes to reach the final state. Many of these states have been characterized by cryoreconstruction in combination with in vitro assembly and mutagenesis experiments. The results inform a more realistic view of general capsid assembly pathways (294).

Common features of capsid maturation in dsDNA viruses: from  to HSV-1. Bacteriophage  is one of the most widely used phages in molecular biology. The virion comprises a nearly icosahedral, isometric head that encapsidates 47 kbp of dsDNA and to which a helical tail is attached. The head contains 405 copies of the two major capsid proteins, gpD and gpE, clustered on a T=7laevo lattice as shown by conventional EM (51, 221, 265, 346). Protein gpE (38.2 kDa) is clustered as hexamers and pentamers, and gpD (11.6 kDa) is clustered as trimers (167, 183). Morphogenesis of  proceeds via the assembly of the prohead, an empty precursor capsid, which is subsequently filled with DNA. An initiator complex, which eventually gives rise to the portal vertex, begins prohead assembly and nucleates the polymerization of gpE to complete the procapsid shell. The packaging of DNA through the portal vertex is accompanied by a transition of the capsid to the expanded form which is stabilized by the addition of polypeptide gpD. Mutant virions which lack gpD can be propagated when stabilized by the presence of a high concentration of Mg²⁺. Mutations in the terminase, which recognizes and cleaves the  DNA and packages it into the procapsid (220), produce proheads that also lack gpD and can be isolated.

View larger version (51K): [in this window] [in a new window]   FIG. 32.   The structural transitions in the bacteriophage  capsid during maturation. The capsid has T=7l icosahedral symmetry and is formed of hexamers of protein gpE. The prohead reconstruction shows the capsid prior to the packaging of DNA and the accompanying expansion. Notice that the gpE hexamers are skewed in this form of the capsid. Genome packaging in the wt virion leads to a much smoother surface in which the individual hexamers have expanded. Expansion is also accompanied by the addition of protein gpD, which helps to stabilize the capsid structure. gpD is bound at the local threefold positions of the T=7 lattice as shown by the comparison of the wt reconstruction with that of a mutant (D) which lacks gpD. Adapted from reference 100 with the permission of the author and the publisher.

View larger version (81K): [in this window] [in a new window]   FIG. 33.   Procapsid maturation in bacteriophage HK97 (81). Shown are isosurface views along the twofold direction of cryoreconstructions of the T=7 HK97 precursor (left) and mature (right) capsids. HK97 capsid proteins (42 kDa) aggregate into pentamers and hexamers and form a prohead, but without scaffold proteins as often occurs in other viruses. A viral protease is incorporated into the prohead upon completion of assembly, and when activated, it cleaves the 11-kDa amino-terminal domain from the inner surface of the capsid protein. This reaction triggers transformation of the prohead into the expanded, mature capsid, in which adjacent subunits become covalently cross-linked. The hexamers undergo a dramatic change from their asymmetric, sheared state to a more symmetrical structure in the mature capsid. Adapted from reference 294 with the permission of the author and the publisher.

View larger version (163K): [in this window] [in a new window]   FIG. 34.   Procapsid maturation in HSV-1 (311, 312). Shown are isosurface views along a twofold direction of cryoreconstructions of the T=16 HSV-1 precursor procapsid at 28-Å resolution (left), mature capsid at 25-Å resolution (right), close-up views of each capsid along a fivefold axis (insets). The color images are overlaid on the unprocessed cryo-EM images of the corresponding vitrified specimens. Comparison of the two structures reveals the substantial changes that occur in the hexons (hexameric capsomers), which are distorted and nonsymmetric in the procapsid but become symmetric in the mature capsid. HSV-1 capsids contain three major proteins: VP5 (150 kDa; 960 copies), VP19c (50 kDa; ~320 copies), and VP23 (34 kDa; ~640 copies). The 320 "triplexes" (arrows in insets), which occupy local threefold axes of symmetry, consist of two copies each of VP23 and one copy of VP19c. The horns of density at the tips of the hexons, but not pentons, in the mature capsids are monomers of VP26 (12 kDa), an additional viral protein that is dispensable for capsid assembly but which binds to the capsid if present. Adapted from reference 294 with the permission of the author and the publisher.

Assembly proteins. Chaperones were first identified by their effects on the assembly of phage  in E. coli. Cryoreconstruction has provided direct views of these proteins in action. Traditionally, scaffolding proteins are described as sitting within the procapsid and being ejected upon the entry of the genome. This indeed appears to be true for P22 (308, 309) and HSV (311), where interior scaffolding protein density has been visualized. Unfortunately, much of the scaffolding protein density is not arranged with icosahedral symmetry (Fig. 35) and hence is not seen in its entirety. In contrast, bacteriophages P4 and X174 do have icosahedrally arranged scaffolding proteins on the outside of the virions (99, 169). The scaffolding protein density is seen in positions which constrain the geometry of the capsomers and define the procapsid diameter (Fig. 36).

View larger version (64K): [in this window] [in a new window]   FIG. 35.   The position of the scaffolding protein in the procapsid of the bacteriophage P22 scaffolding mutant is shown in outer surface and cut-open views of the reconstruction. (A) Outer surface of the structure; the density attributed to the internal scaffolding protein is colored white whereas that of the coat protein is colored yellow. One copy of each of the seven quasi-equivalent coat proteins is highlighted in a different color. (B) Cut-open view shows the distribution of the scaffolding protein (white) which extends to a radius of 206 Å within the outer layer (yellow). Adapted from reference 308 with the permission of the author and the publisher.

View larger version (123K): [in this window] [in a new window]   FIG. 36.   The role of the capsid-size-determining protein in the bacteriophage P2-P4 system is shown by a series of surface views of reconstructions. Bacteriophage P2 forms a T=7 particle of 600 Å in diameter (A) which packages its 33-kb genome efficiently. Bacteriophage P4 uses the proteins of P2 to form a 450-Å T=4 shell (B) which can package its smaller 11-kb genome but excludes the larger P2 one. The change in capsid size is controlled by the P4 gpSid protein. Reconstruction of a recombinant particle which resembles the 390-Å P4 procapsid (C) and the procapsid bearing a full complement of gpSid (D) shows that the size determination protein forms a 400-Å dodecahedral shell on the outside of the procapsid. Expansion after packaging brings the mature P4 to its final 450-Å diameter. Adapted from references 101 and 213 with the permission of the author and the publishers.

View larger version (64K): [in this window] [in a new window]   FIG. 37.   The structure of the membrane-containing bacteriophage PRD1 is shown by three surface representations of PRD1 particles. The wt phage and a mutant phage which does not package the genomic DNA show that the T=25-like exterior is not changed by the packaging. The structures within the interior membrane of the virus increase in order after packaging. The structure of the P3 protein shell shows the organization of the major capsid protein and the removal of the fivefold vertex structures. The similarity to adenovirus (Fig. 23) is striking; both are T=25-like arrangements in which trimeric proteins fill hexavalent sites and have a separate protein complex that forms the vertex at the fivefold position and which also includes a trimeric protein. Adapted from reference 50 with the permission of the publisher.

View larger version (94K): [in this window] [in a new window]   FIG. 38.   Comparison of the structure of the wt mature SFV with that of the SFVmSQL mutant shows the effect of cleavage of the spike protein precursor (p62) on the spike structure. The spike coalesces from an open structure with separated E1E2 heterodimers to a closed structure containing a cavity at its center. The modular nature of the structure is reflected in the observation that the capsid, transmembrane regions, and spike skirts are unchanged by the cleavage. Adapted from reference 118 with the permission of the author and the publisher.

(a) Reoviridae. The family Reoviridae includes moderately large (700- to 900-Å), nonenveloped, icosahedral viruses that have a complex, multilayered protein structure that encapsidates 10 to 12 segments of dsRNA (225). Five genera infect vertebrates including humans (orthoreovirus, rotavirus, coltivirus, and orbivirus) and fish (aquareovirus), whereas other genera infect plants (phytoreovirus, fijivirus, and oryzavirus) and insects (cypovirus). The strategies for infection and replication are similar for all Reoviridae viruses, and all viruses contain the enzymes required for transcription of the dsRNA.

(b) Reoviridae: Orthoreovirus genus. Mammalian reoviruses, the prototype viruses of the genus Orthoreovirus, have two proteinaceous shells that encapsidate the genome (Fig. 39). The 10 genome segments encode eight structural and three nonstructural proteins. Four proteins, 1 (51 kDa), 3 (41 kDa), µ1 (76 kDa), and 2 (144 kDa), comprise the outer capsid, whereas 2 (47 kDa), µ2 (83 kDa), 1 (142 kDa), and 3 (142 kDa) comprise the bulk of the core (225). Although not a human pathogen, reovirus has been a model system for studying viral pathogenesis. Partial proteolysis of intact virions removes 3 and probably leads to the unfolding of 1, the cell attachment protein (136). The resulting ISVPs are infectious, and cleavage of µ1 appears to facilitate cell penetration (226). Continued proteolysis converts ISVPs to cores with the loss of µ1 and 1 (179), and 2 undergoes a dramatic change in conformation to form the core spikes.

View larger version (32K): [in this window] [in a new window]   FIG. 39.   Schematic diagrams of the compositions and structures of orthoreovirus virions, ISVPs, and cores. The structures of virions and ISVPs appear very similar for all serotypes studied, but the 2 spike of the serotype 1 Lang (T1L) core adopts a more open conformation than the cores of other serotypes, such as T3D (reovirus serotype 3, Dearing). The virion has a triple-layered structure, depicted as three concentric circles at the lower left. Outer and inner capsids, each comprised of multiple copies of four different proteins, encapsidate a central core that contains the segmented dsRNA genome. Adapted from reference 225 with the permission of the author and the publisher.

View larger version (96K): [in this window] [in a new window]   FIG. 40.   Cryo-EM of orthoreovirus T1L virions, ISVPs, and cores. Magnified views of three representative particles of each type are shown below. Bars represent 1,000 Å (low-magnification views) and 500 Å (higher-magnification views).

View larger version (60K): [in this window] [in a new window]   FIG. 41.   Isosurface views in the twofold orientation of the cryoreconstructions of the orthoreovirus T1L virion, ISVP, and core. These reconstructions were computed from images like those depicted in Fig. 40. The T1L virion is characterized by 600 finger-like projections (3 protein) and 12 depressions with flower-shaped features (2 protein) at the fivefold vertices and a knob of density (ascribed to the 1 protein) on the fivefold axis. The T1L ISVP has similar flower-like features centered on the icosahedral vertex positions and 200 trimeric structures (µ1 protein) distributed over the capsid surface. The 1 protein adopts a more extended conformation in the ISVP, though this is not apparent at the high contour level at which the isosurfaces are rendered (see Fig. 4 of reference 108). The 2 spikes (open, turret-like structures) are the most prominent feature of the T1L core. The composition of the knob-like features on the surface of the core is still undetermined (possibly a combination of 1 and 2 proteins), but the components of the viral RNA polymerase (3 and µ2 proteins) have been located inside the core shell and hence are not visible in this view (107).

(c) Reoviridae: Rotavirus genus. Rotavirus is the primary cause of infant gastroenteritis in numerous mammals and is the major cause of human infant mortality in developing countries (117). The rotavirus genome contains 11 dsRNA segments which encode six structural and five nonstructural proteins. The majority of the virion protein mass (VP4, 87 kDa; VP6, 45 kDa; VP7, 37 kDa) forms two T=13l capsid shells, whereas the remaining proteins (VP1, 125 kDa; VP2, 102 kDa; VP3, 98 kDa) are associated with the core (Fig. 42). VP6 and VP7 form two concentric capsid shells, with VP7 comprising the outermost layer. The viral cell attachment protein and hemagglutinin, VP4, interacts with both VP7 and VP6 (see below and "Cell attachment"). Proteolysis of VP4 (producing VP5* and VP8*) appears to play a central role both in viral entry into the cell (181) and in the enhancement of viral infectivity (206).

View larger version (39K): [in this window] [in a new window]   FIG. 42.   Organization and structure of rotavirus. A schematic diagram (left) and 3D reconstruction (right) illustrate the multilayered rotavirus structure and the disposition of its major components. The dsRNA genome and viral RNA polymerase (VP1 and VP3) are encapsidated by a core shell composed of VP2. This core is surrounded by two concentric shells of protein which include 260 trimeric-shaped columns of density (VP6) on the inside and 260 trimers of VP7 on the outside. VP7 forms a relatively smooth outer surface with 132 channels through which VP6 and VP2 are accessible (shown more clearly in Fig. 18). Sixty dimeric spikes (VP4), which contain the receptor recognition domain, project ~110 Å radially outward from the viral surface and ~90 Å beneath the surface and are in close contact with both VP7 and VP6 (352). Adapted from references 117 and 352 with the permission of the authors and the publishers.

View larger version (174K): [in this window] [in a new window]   FIG. 43.   Cryo-electron micrograph of a rotavirus sample containing both double-layered and triple-layered particles. The larger TLPs have a characteristic smooth circular profile whereas the smaller DLPs have a bristly outer profile. Magnified views of two TLPs (left) and two DLPs (right) are shown in the insets at the bottom.

View larger version (70K): [in this window] [in a new window]   FIG. 44.   Organization of rotavirus DLPs as visualized in radial-depth-cued surface representations, viewed along a threefold direction. (Top) DLP cryoreconstruction at a nominal resolution of 19 Å, depicted to show an exterior view of the entire particle (left), the internal organization in a cutaway view (middle), and the dodecahedral shell of ordered RNA (right), which represents ~25% of the total genome. The disposition of VP6 (blue) and VP2 (green) layers and the VP1-plus-VP3 RNA polymerase complex (red) and the ordered RNA (greyish green and yellow in the middle and yellow on the right) are denoted by arrows. (Bottom three rows) Radial cutaway views compare the internal features of the DLP (left column) and recombinant baculovirus-expressed VLPs composed of VP2 and VP6 (VLP-2/6 [middle column]) and of VP1, VP2, VP3, and VP6 (VLP-1/2/3/6 [right column]). The radial cutaways from cryoreconstructions computed to 23-Å resolution include only densities from radii of 160 out to 260 Å (top row), 235 Å (middle row), and 225 Å (bottom row). Features between radii 260 and 350 Å are invariant in these structures. RNA density (yellow) appears in the DLP at a radius of ~230 Å where corresponding regions of the VLPs are empty. The VP2 protrudes inward at the fivefold vertices from the main core shell to a radius of ~220 Å. A flower-shaped density, attributed to VP1 and VP3, attaches to the tips of VP2 at the fivefold axes in VLP-1/2/3/6, and similar density occurs in the DLP. Adapted from reference 247 with the permission of the author and the publisher.

(d) Reoviridae: Orbivirus genus. The Orbivirus genus is divided into 14 distinct serogroups (262) (Fig. 45). Orbiviruses, unlike reoviruses and rotaviruses, are transmitted between vertebrate hosts by arthropods and replicate in both vertebrates and arthropods. They cause diseases of economic importance in many livestock, though they are not considered to be important human pathogens. The orbiviruses are double-shelled structures with 10 segments of dsRNA that encode seven structural and four nonstructural proteins. BTV, the prototype and most-studied orbivirus, has an outer capsid composed of two proteins (VP2, 111 kDa; VP5, 59 kDa) and a core with five proteins, two of which are major components (VP3, 103 kDa; VP7, 38.5 kDa) and the remaining three of which are minor components (VP1, 150 kDa; VP4, 76 kDa; VP6, 36 kDa).

View larger version (52K): [in this window] [in a new window]   FIG. 45.   Comparison of the structures of BTV and BRDV reveals the common features of orbivirus structures. The views of the outer surface along the twofold axis show that these two viruses have a similar organization but quite different elaborations of the structure. The structure of the triskelion on the threefold axis (VP2 in BTV and VP4 in BRDV) shows this clearly. The core structure of BRDV is shown in a surface view masked at 250 Å to show the VP2 layer. The crystal structure of the BTV core (144) shows that this layer is composed of 60 pairs of nonequivalent copies of the major core structural protein which is described as a "T=2" structure. The T=13l arrangement of the VP7 trimers is shown in a surface representation of the structure masked at a radius of 290 Å. Adapted from reference 268 with the permission of the author and the publisher.

View larger version (29K): [in this window] [in a new window]   FIG. 46.   The arrangement of proteins in BTV is shown in a series of schematic diagrams. (A) Placement of the individual structural proteins in the multilayered virion. (B) Arrangement of the proteins in the core in which the T=13 VP7 layer sits atop the "T=2" VP2 shell which surrounds the 10 segments of dsRNA. (C) The outer surface of VP2 sits atop the VP5 layer which mediates its interaction with the VP7 layers. Adapted from reference 332 with the permission of the authors and the publisher.

(e) Reoviridae: Aquareovirus genus. Aquareoviruses, like other reoviruses, have a T=13l outer shell and a T=2 core. They are stable at pH 3 and are resistant to ether. They infect a variety of bony fish and crustaceans. The 11-segment genome codes for seven structural proteins (VP1 to VP7) and five nonstructural proteins. Though the stoichiometry and distribution of the structural proteins are unknown at this time, a recent cryo-EM structure determination of striped bass aquareovirus at 23-Å resolution displays a structure with an outer shell (~800-Å diameter) containing 600 trimeric morphological subunits that surround an inner shell of ~600 Å in diameter consisting of 120 subunits arranged as described previously (272) (Fig. 47). This 120-subunit stoichiometry and arrangement in the core shell appear to be a fundamentally important structural requirement of transcriptionally active dsRNA-containing particles. The same stoichiometry and organization have been found in or suggested for rotavirus (195, 247), reovirus (209), BTV (145), two fungal viruses (63), and bacteriophage 6 (48). Another set of proteins cluster at the fivefold axes and form continuous density that spans both capsid layers.

View larger version (146K): [in this window] [in a new window]   FIG. 47.   Comparison of aquareovirus (top) to orthoreovirus T1L ISVP (bottom). Shown are isosurface views of aquareovirus (in color and in perspective) and T1L ISVP (black and white) in threefold (left) and fivefold (right) view orientations. The threefold view of aquareovirus shows the incomplete T=13l surface lattice (white overlay) and the axes of icosahedral symmetry labeled. The distribution and orientations of the 200 trimeric morphological units are essentially identical in the two particles. The flower-like structures on the fivefold vertices are distinct (e.g., in aquareovirus there is an axial channel). Differences in the relative sizes of features in the two structures are mainly attributed to aquareovirus being viewed in perspective whereas T1L ISVP is not. Aquareovirus images are adapted from reference 272 with the permission of the author and the publisher.

(f) Reoviridae: Cypovirus genus. A significant pathogen of arthropods is a reovirus commonly referred to as cytoplasmic polyhedrosis virus. Infectious particles occur inside characteristic crystalline inclusion bodies (called polyhedra) composed of viral proteins (222). Insects become infected when they ingest polyhedra contained on contaminated food. The high pH of the insect gut dissolves the polyhedra and releases virus particles. These then infect the epithelial cells that line the gut wall, where they replicate in the cytoplasm.

(g) Reoviridae: Pytoreovirus genus. RDV, which replicates in both insects and graminaceous plants, is transmitted only by insects (e.g., leafhoppers). The widespread infection of rice plants in Southeast Asia causes severe reduction in crop production and hence poses a major economic threat to people in this region. The structure of the 700-Å-diameter RDV capsid was recently solved to 25-Å resolution by use of cryoreconstruction methods (207). The results revealed an outer T=13l capsid, composed of 260 trimers of the 48-kDa P8 viral protein and an inner T=1 shell composed of 60 dimers of the 114-kDa P3 protein. Atomic models of the VP7 trimer of BTV in the two crystal conformations REF and HEX (23, 143) were docked into the outer capsid density of the RDV cryoreconstruction and revealed a better fit of the HEX structure. This and a sequence comparison between the RDV and BTV P8 and VP7 proteins, respectively, led to a proposal that P8 may have an upper domain with a -sandwich motif and a lower domain of  helices (207).

(h) Birnaviridae. The family Birnaviridae includes nonenveloped, icosahedral viruses that have a worldwide geographic distribution and are characterized by having a two-segment dsRNA genome. IBDV, an important pathogen of young chickens, has a genome that codes for four structural proteins. These include two major components, VP2 (40 kDa; carries major neutralizing epitopes) and VP3 (32 kDa; contains a very basic C-terminal region believed to interact with the viral RNA), and two minor components, VP4 (28 kDa) and VP1 (90 kDa; the viral RNA polymerase) (222).

(i) Fungal viruses. The family Totiviridae includes RNA viruses of simple eukaryotes (342). These viruses have a genome that is a single segment of dsRNA encapsidated in an icosahedral shell and that encodes a major coat protein and an RNA-dependent RNA polymerase. L-A virus, the most-studied member of the group, has a 76-kDa Gag coat protein and an estimated one or two copies per particle of a minor 180-kDa Gag-Pol fusion protein. Cryoreconstruction studies of two totiviruses, L-A of Saccharomyces cerevisiae and P4 of Ustilago maydis, showed that both viruses have similar 430-Å-diameter structures (Fig. 48), consisting of 120 subunits arranged as 60 asymmetric dimers in a T=1 lattice and with 60 small holes (10- to 15-Å diameter) that extend through the capsid wall (55, 63). The two subunits in each dimer occupy nonequivalent bonding environments. The holes act as molecular sieves, being large enough to allow influx of metabolites needed for RNA synthesis and replication and for exit of new transcripts but small enough to shield the genome from degradative enzymes (Fig. 31).

View larger version (83K): [in this window] [in a new window]   FIG. 48.   Reconstructions of two fungal viruses, L-A (left) and P4 (right), as viewed along an axis of twofold symmetry. The two viruses exhibit very similar features with 120 morphological units packed on a T=1 lattice (63). The capsids consist of 12 pentons, each of which has 10 elongated and curved subunits arranged in two sets of five. An inner ring of five subunits is encircled by an outer ring of five subunits that are offset like fish scales from the inner ring. Despite occupying nonequivalent packing environments, the inner and outer ring subunits are similar in appearance. The known capsid stoichiometry indicates that each subunit is a monomer of the Gag protein (55, 63). The images are rendered at an isosurface threshold too low to reveal the small channels that penetrate the capsid wall and are presumed to be exit portals through which nascent mRNA transcripts can pass (see Fig. 31).

(j) Bacteriophage 6. 6 is an enveloped dsRNA phage of the plant pathovar Pseudomonas syringae. Virions consist of four concentric structures: an outer lipid bilayer, a lysozyme layer, a nucleocapsid (NC), and a polymerase complex (core) which encapsidates a three-segment genome of dsRNA (Fig. 49). The outer lipid bilayer contains five proteins, two of which form a spike complex which mediates binding to the pilus of the host. Virus fuses with the outer bacterial membrane, releasing the NC and phage lysozyme into the periplasmic space which interacts with the inner bacterial membrane, resulting in penetration of the NC into the cytoplasm. NC contains the RNA polymerase complex (P1, 85 kDa; P2, 75 kDa; P4, 35 kDa; P7, 17 kDa) which is covered by a layer of a single protein (P8, 16 kDa) (219, 278, 319, 320). Loss of P8 activates the polymerase complex which initiates transcription and replication of the three-segment genome.

View larger version (34K): [in this window] [in a new window]   FIG. 49.   Stages of the bacteriophage 6 life cycle. The virus enters the bacterium by attaching to a pilus (A) and crossing the outer membrane (B), digesting the proteoglycan layer with a viral lysozyme, and then crossing the inner membrane (C) to deposit the nucleocapsid in the cytoplasm (D). Plus RNA strands are synthesized and lead to the production of proteins P1, P2, P4, and P7 (E), which assemble to form the prohead (F). The prohead packages a single copy of each of the three plus strands, which leads to expansion and minus-strand synthesis (G). Later events in the transcription cycle lead to the production of protein P8, which attaches to the expanded prohead to form the nucleocapsid (H). This is followed by envelopment (I) and lysis (J).

View larger version (146K): [in this window] [in a new window]   FIG. 50.   The internal structures of bacteriophage 6 are shown in surface views of the exterior and cut-open reconstructions of the polymerase complex (proteins P1, P2, P4, and P7) in its unexpanded (A) and expanded (B) forms as well as the nucleocapsid (C). The expansion of the polymerase complex allows the attachment of protein P8 (white in panel C) to form the T=13 shell of the nucleocapsid. The major structural protein of the polymerase complex (P1) forms a T=2-like shell similar to that of BTV. The remaining proteins contribute to the turret structure which extends from the vertex of the polymerase complex and projects through the holes of the T=13 shell of the nucleocapsid. Recent work (97) shows that this structure does not have the fivefold symmetry imposed by the icosahedral reconstruction and hence is blurred in the average shown. Adapted from reference 48 with the permission of the publisher.

(ii) Modulation of T number. (a) Bacteriophage P2 and the assembly parasite P4. The P2-P4 system represents an opportunity to use cryo-EM to study the mechanism by which a capsid protein can be directed to form two different-sized shells (for a review, see reference 32). This addresses a common problem in phage assembly. In phages such as , the information which controls the size of the capsid appears to reside in the capsid proteins themselves. Mutations in the major  capsid protein alone can result in variations of the assembled capsid size (115). In others, such as P22 (182), the presence of a scaffolding protein is necessary to ensure the final correct capsid size.

(b) Size control in hepatitis B virus. Size control in the P2-P4 system allows the parasitic P4 capsids to exclude the full-length P2 genome. In contrast, the importance of dimorphism in the HepBc is obscure. Hepatitis B virus consists of an inner nucleopcapsid or core surrounded by a viral envelope containing virus envelope glycoproteins. Cores can be formed either by expression in E. coli or by reassembly after purification, urea treatment, and refolding. When a full-length capsid protein is used, the resulting cores are a mixture of small and large shells which correspond to T=3 and T=4, respectively (93). Capsids isolated from infected human liver tissues show that same dimorphism, although the ratio is skewed heavily toward the larger T=4 form (13:1) (184). Careful analysis of the effect of mutations showed that truncation of the carboxy-terminal tail caused loss of nucleic acid packaging and an increase in the fraction of T=3 reassembled cores (365). Only 5% of cores, which extended to residue 149, formed T=3 shells, whereas 85% of shells truncated to 140 residues were reassembled as T=3 structures. Truncated sequences shorter than 138 residues did not assemble at all. The results with cores isolated directly after expression show a similar trend, although there is greater variability (184). Recently, cryoreconstruction has achieved unprecedented resolution for the cores formed from the 149-residue protein (42, 80). Reconstructions at 7.4-Å (42) and 9-Å (80) resolution (Fig. 51) have demonstrated that the bulk of the assembly domain of HepBc protein is a four-helix bundle. This result opened the way toward understanding the mechanism of size determination. Undecagold maleamide labeling of Cys-150 (364) demonstrated that this residue lies at the crowded dimer interface inside the core shell. Hence, the C termini of adjacent subunits crowd each other around the fivefold vertices and, to a lesser degree, quasi-sixfold vertices. The larger fraction of dimers in quasi-sixfold positions would explain the preference of longer-tailed variants for the T=4 structure.

View larger version (90K): [in this window] [in a new window]   FIG. 51.   Cryoreconstructions of the T=4 HepBc computed to 7.4-Å (42) and 9-Å (80) resolution (left and right, respectively). Both reconstructions revealed details of the tertiary structure of the capsid protein. In particular, dimer clustering of two core protein subunits produces the prominent surface spikes, which consist of four long  helices arranged in a radial bundle. A series of subsequent labeling studies have further defined details of the HepBc structure at similar resolutions (41, 78, 79, 364). Adapted from references 42 and 80 with the permission of the authors and the publisher.

(iii) Big viruses. The hypothesis of quasi-equivalence (53) suggested that regular but small changes in the binding between subunits would solve the problem of self-assembly of virions containing more than 60 subunits. Such a scheme of regular alteration of binding patterns of chemically identical subunits is compelling for low triangulation numbers but becomes less and less tenable for larger ones. How can a virus with T=25 symmetry and hence 25 separate potential sets of binding interactions complete its assembly in an efficient and rapid manner? The issue is even more extreme for the 1,900-Å-diameter algal virus Paramecium bursaria Chlorella virus type 1 with triangulation number T=169 (351). The classical answer to this paradox is that assembly of large and complex viruses utilizes scaffolding and accessory proteins to guide the decisions which the capsid protein must make during its complicated assembly. The details of such a solution have been revealed for several systems by cryo-EM and image reconstruction.

(a) T=25 structures: divide and conquer. Adenovirus can be viewed as an assembly of a set of relatively inflexible bricks (hexons) into a complex structure by the application of the appropriate cement in the form of a series of accessory proteins. The hexon itself assembles as an extremely stable trimer which forms all of the hexa-coordinated capsomers on the virion. The fivefold positions are formed by a separate protein, penton base. Hence, the intimidating task of assembling the 1,500 subunits of a true T=25 structure is reduced to the much simpler one of modulating the 240 hexon trimers which sit in four distinct environments on the surface of the virion. The analysis of the structure by cryoreconstruction is made much simpler because the structure of the hexon trimer is known to high resolution from X-ray crystallography (255). Further, the stoichiometry of all of the components of the virion has been established by careful quantitation (323). Together, these two results allow the use of difference imaging to assign locations to the minor proteins of the capsid and to localize the hexon sequences which form contacts in the structure.

View larger version (90K): [in this window] [in a new window]   FIG. 52.   The positions of the proteins of adenovirus are shown by difference imaging with an atomic model based on the high-resolution structure of the adenovirus hexon protein (blue in panels A and B). An external view of the virion along the twofold axis (A) shows the position of the trimers of polypeptide IX which link the group of nine hexons in a single facet and polypeptide IIIa which links adjacent facets. The penton base I is represented in yellow with the fiber being shown in green. The view from the interior of the virion shows polypeptide IIIa, which passes through the layer of hexons, and of polypeptide VI hexamers near the penton base. These positions are shown schematically in panel C, where the triangular top and hexagonal base of hexon and the pentagonal penton base are shown in white. The polypeptide IX trimers are shown as black triangles. The polypeptide VI hexamers are shown as black circles, and the hexon layer spanning polypeptide IIIa is shown as filled black bars. The fit between the hexon density seen in the high-resolution structure and that in the reconstruction is shown for the region surrounding the penton base in panel D. Adapted from reference 298 with the permission of the publisher.

(b) A T=16 structure: organizing 960 identical subunits. The capsid of HSV-1 has T=16 symmetry and differs from adenovirus in that both its five-coordinated and six-coordinated capsomers are made from the same protein, VP5. In contrast to adenovirus, VP5 does not form stable hexamers and pentamers which would simplify the assembly problem. Image reconstruction (17, 18, 34, 223, 224, 269, 363) confirms that the pentavalent and hexavalent capsomers are indeed pentamers and hexamers (Fig. 34). Reconstructions also show that nodules of density, called triplexes, are at all of the local threefold axes of symmetry. The T=16 symmetry and the presence of triplex nodules have also been demonstrated for other members of the herpesvirus family, such as channel catfish herpesvirus (37) and human cytomegalovirus (49). In HSV-1, a further protein, VP26, has been shown to be localized to the tops of the hexavalent capsomers. VP26 is not present on the pentavalent ones (38, 312, 313, 348, 362). HSV appears to make use of a scaffolding protein which organizes a procapsid. Assembly of the procapsid requires three components: the major capsid protein (VP5), the triplex proteins (VP19c and VP23), and a scaffolding protein (VP22a and/or pre-VP21). In vitro assembly experiments show that VP5, the triplex proteins, and the scaffolding protein form a complex which can pack to define the procapsid size. Examination of procapsid structures (311, 359) suggests that the majority of the scaffold protein is not icosahedrally arranged but may act as a loose micelle on whose surface VP5 can move and interact with the triplex proteins to define the interactions between capsomers (Fig. 34) which survive into the mature virion. A recent examination of whole virions (average diameter, ~2,000 Å) has revealed the presence of extensive interactions between the tegument and the icosahedrally ordered capsid (359).

View larger version (243K): [in this window] [in a new window]   FIG. 53.   Cryo-electron micrograph of a densely packed monolayer of bacteriophage T7 heads recorded at 2.5-µm defocus (56). The particles exhibit characteristic fingerprint patterns with 25-Å spacings associated with closely packed strands of dsDNA. The pattern motifs vary according to the viewing direction, with concentric rings revealed in axial views and punctate arrays revealed in side views. In the particular double mutant sample shown here, the T7 heads adopt a preferred axial orientation. Modeling analysis of these and other images established that the T7 chromosome is spooled around an axis in approximately six coaxial shells in quasi-crystalline packing (56). Adapted from reference 56 with the permission of the author and the publisher.

View larger version (67K): [in this window] [in a new window]   FIG. 54.   Ordered duplex RNA in FHV as viewed from the side toward the C-C intersubunit contact (see Fig. 26). (Top) Thin (~20-Å) cross-sectional view of FHV cryo reconstruction (blue mesh) superimposed on the FHV X-ray C backbone model (yellow), the contoured difference density map (red mesh; cryo-EM density map minus X-ray capsid structure only), and the locally ordered RNA (space-filling representation). The vertical white line is a twofold axis. The other lines mark the locations of two adjacent fivefold axes. (Bottom) Same view as above with ribbon representations of two complete B (red) and C (green) capsid subunits in close association with the dsRNA (space-filling model) and the peptide arm. Adapted from reference 66 with the permission of the author and the publisher.

Virus budding in animal cells must overcome two further problems beyond the ones of assembly that occur in nonenveloped viruses. Firstly, budding must be constrained to the appropriate membrane of the many that exist in an animal cell. Budding of an alphavirus into the lysosomes would not be an effective strategy for propagation. Secondly, the budding must incorporate the viral envelope proteins and exclude the host proteins. Even at very late stages of infection, viral envelope proteins represent around 1% of the cellular membrane proteins, and so the incorporation must have great selectivity. The solutions to these two problems must lie in the interaction of the viral envelope proteins and the internal proteins of the virion. The presence (or their lack) of sorting signals on a viral glycoprotein can cause it to be directed to a particular cellular membrane (276). The first problem then reduces to one of constraining virus budding to compartments in which the envelope proteins exceed a critical threshold concentration. The availability of structures from cryoreconstruction of several alphaviruses provides an opportunity to examine the interactions of the envelope proteins and of the spikes for clues to the control of budding.

Examination of the alphavirus structures shows that four types of independently modulable interactions need to be considered for the spike proteins. First is the interaction between the E1 and E2 proteins within the heterodimer. This interaction is first formed in the endoplasmic reticulum (Fig. 38) and remains the most important one until the precursor p62 is cleaved in the formation of the compact spike complex (118, 186). This interaction is lost completely after exposure to low pH (Fig. 21) (132). The second is the interaction of the heterodimers in forming the spike trimer which is initiated by p62 cleavage and replaced by the formation of the E1 trimer after low-pH treatment. The third is the lateral interaction of spikes via their skirts (64, 132). Finally, the spikes interact with the nucleocapsid via their transmembrane regions (64). These last two interactions are not affected by cleavage of p62 nor changed during the early times after low pH (118, 132).

The independence of these interactions suggests a way in which virion assembly can be dependent on a critical concentration of envelope proteins and exclude the host proteins which fill the membrane from which it buds. The capsid binding sites are complementary to the arrangement of spikes on the surface. Although the spike protein/capsid protein ratio is 1:1, the three heterodimers of a spike always interact with three separate nucleocapsid capsomers. Binding needs to be cooperative as a result. The envelope proteins can pack on the surface of the membrane to form hexagonal arrays which exclude host membrane proteins by means of their lateral interactions. The skirts extending between spikes would allow relatively little space for other membrane proteins. The hexagonal packing produces a relatively flat region of envelope proteins which would be stabilized by the binding of the complementary capsid. Reorganization of the spikes must occur upon introduction of the fivefold positions. This allows the interaction of spikes which are partially bound by capsid hexamers to completely bind their third cytoplasmic domain to the capsid pentamer. The use of cryoreconstruction not only has defined the broad outlines of this scheme but, by fitting the high-resolution structure of the capsid protein into the nucleocapsid density (64), also has begun to define potential atomic interactions which accomplish the individual steps.

VIRUS TRANSMISSION AND HOST DEFENSE

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Virus transmission. Little is known from cryoreconstruction studies concerning the structural basis of transmission. The limited clues that we have thus far suggest that virion structure is adapted to the requirements of transmission. For example, the alphaviruses use an arthropod host as a vector to transmit viruses between mammalian hosts. The evidence suggests a feature of the virion structure which may be important for adaptation to this task, i.e., for replication in two hosts. In mammalian cells, alphavirus budding occurs at the plasma membrane. In insect cells, virus buds into the early compartments of the secretory pathway (reviewed in reference 187). Hence, budding in insect cells occurs before the cleavage of the spike precursor, which activates the spike complex for fusion after endocytosis in the mammalian cell. Because this is such a major functional change, it is likely to be accompanied by a major structural change that might be incompatible with budding. The structure of the SFVmSQL mutant indicates that an answer to this question lies in the unchanged spike-spike interactions mediated by the spike skirt in the uncleaved virus (118). Hence, the alteration in the projecting regions of the spike by cleavage, interactions between the spikes, and interactions between the spikes and the capsid, which are important for budding, remain unaffected. This property would allow the virion to bud efficiently in the early secretory pathway in the insect cell, prior to cleavage, as well as at the plasma membrane in the mammalian cell after cleavage of the precursor in the late Golgi phase.

Host defense. The immune system in vertebrates provides a first line of defense by which infectious agents like viruses are detected and neutralized. Though several theories have been proposed to explain how neutralization occurs, the mechanisms by which it occurs are not well understood. Neutralization may involve antibody-induced structural changes in the virus, or antibodies may interfere with virus-receptor interactions by competing for common binding sites or by steric hindrance. Alternatively, antibodies may prevent viral uncoating, by aggregation (intercapsid cross-linking) or by stabilizing the virus capsid with bivalent (intracapsid) cross-linking. A given host probably employs different mechanisms for different viruses. The antibody-mediated response to an infectious challenge must act in synergy with other defense mechanisms such as opsonization (antibody-mediated phagocytosis).

Cryoreconstruction methods have proved to be extremely effective tools for examining fundamental structural aspects of viral neutralization in several different viral systems. This is true partly because these methods provide a rapid means to visualize directly virus-antibody complexes, which are typically too large (400-Å diameter) to be routinely studied by high-resolution methods such as X-ray crystallography or nuclear magnetic resonance spectroscopy. In addition, if high-resolution structures of the virus and antibody Fab molecules (or closely related ones) are known, a pseudoatomic model of the virus-antibody complex can be produced by docking the atomic structures together with the cryo-EM density map as a guide (

Antibody neutralization of HRVs. The structure of HRV has already been introduced ("Cell attachment" and Fig. 16). Based on sequence analysis of escape mutants, four areas on the surface of the virion were identified as NIm sites (for a review, see reference 260). The three major classes identify epitopes in the VP1 (NIm-IA and NIm-IB), VP2 (NIm-II), and VP3 (NIm-III) viral coat subunits. These epitopes lie either on the "north" (NIm-I) or "south" canyon rims (NIm-II and NIm-III) where the fivefold vertex points north.

View larger version (63K): [in this window] [in a new window]   FIG. 55.   Complexes between HRV14 and NIm-IA neutralizing MAbs (58). (Top) Isosurface representations, all viewed along a twofold axis of symmetry, of cryoreconstructions of HRV14 complexed with a neutralizing MAb (MAb 17-IA in red) and with Fab fragments from strongly neutralizing MAbs 17-IA (blue) and 12-IA (purple) and weakly neutralizing MAb 1-IA (green). (Bottom) Pseudoatomic fitting of different Fab models into the cryo-EM density envelopes of the HRV-Fab complexes (wire mesh) illustrates in side views the close interactions that each Fab makes with the HRV structure. Fab 17 and Fab 12 bind in essentially identical, tangential orientations to the viral surface, which favors bidentate binding over icosahedral twofold axes. Fab 1 binds in a more radial orientation that makes bidentate binding unlikely. All three Fabs contact a significant portion of the viral canyon and directly overlap much of the ICAM-1 receptor binding site.

Antibody-mediated neutralization of human Ad2. A recent cryo-EM and X-ray modeling study of the binding of Fab molecules to the highly mobile integrin binding sequence (RGD) on the penton base of Ad2 revealed a novel mechanism for evasion of antibody-mediated neutralization (297). The Fab fragment from a MAb that recognizes a nine-residue linear epitope, including the RGD motif, when bound to virus adopts a wide range of conformations. Hence, the epitope is quite flexible even with bound Fab. Careful inspection and interpretation of the image reconstruction provided a structural basis for explaining why Fabs but not IgGs inhibit virus infection. The position of Fab binding and the high mobility of the RGD loop as well as the smaller size of a Fab compared to MAb permit saturation of viral epitopes with Fabs but not IgGs. When only a few of the five potential RGD sites on each penton base have IgGs bound, the remaining sites remain accessible to cell integrins, but the bound IgGs and the adenovirus fiber cause steric hindrance and hence block further binding of IgGs.

Published results obtained for structural studies of spherical viruses that have been examined with cryoreconstruction procedures are summarized in Table 1. The success and popularity of cryoreconstruction have guaranteed a wealth of new information about the structures and functions of many spherical viruses, and unfortunately, we are unable to highlight each of these. Consequently, Table 1 provides a more comprehensive, referenced catalog of the physical characteristics of viruses studied by these procedures by our laboratories and others.

The power of cryoreconstruction procedures for studying icosahedral virus structure is evident. As illustrated in "Virus life cycles in three dimensions," the coordinated application of complementary structural, biochemical, and molecular biology methods will continue to yield further insight into structure-function relationships in viruses. The progress in this field has been extremely rapid and shows no sign of abating. More than a hundred reconstructions have been published since R. A. Crowther's insight (86-89, 91) into common lines produced the first in 1970. The quality and resolution of the reconstructions have continued to improve. For many of the better-established systems, the limits are computational and instrumental. The two reconstructions of HepBc which broke the 10-Å resolution barrier and allowed the first identification of a novel protein fold by cryo-EM and single-particle reconstruction are a landmark. They will not stand alone for very long. We are aware of other groups who have reached similar levels in their work on other viral systems. Improvements in microscopes and the increasing availability of computation resources will bring improved resolution in the near future. Work on the less well characterized systems is directed less at higher resolution than at specific biological questions. As the methods have become better understood and refined, workers have become more willing to attempt less homogeneous and stable samples. The past 10 years have seen this method pass from a difficult methodological challenge to a standard and reliable tool in the structural biologists' repertoire.