Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 182

Some naked viruses are considerably larger and more elaborate than the small RNA and DNA viruses described in the previous section. The characteristic feature of such virus particles is the presence of proteins devoted to specialized structural or functional roles. Despite such complexity, detailed pictures of the organization of this type of virus particle can be constructed by using combinations of biochemical and structural methods. Well-studied human adenoviruses and members of the Reoviridae exemplify these approaches. These two examples also illustrate distinct mechanisms by which large icosahedral capsids can be stabilized, via either specialized proteins that glue interactions among major capsid proteins or mutually reinforcing associations between protein layers.

Adenovirus. The most striking morphological features of the adenovirus particle (maximum diameter, 150 nm) are the well-defined icosahedral appearance of the capsid and the presence of long fibers at the 12 vertices (Fig. 4.15A). A fiber, which terminates in a distal knob that binds to the adenoviral receptor, is attached to each of the 12 penton bases located at positions of fivefold symmetry in the capsid. The remainder of the shell is built from 240 additional subunits, the trimeric hexons (Fig. 4.15B). Formation of this capsid depends on nonequivalent interactions among subunits: the hexons that surround pentons occupy a different bonding environment than those surrounded entirely by other hexons. The X-ray crystal structures of the trimeric hexon (the major capsid protein) established that each protein monomer contains two β-barrel domains, each with the topology of the β-barrels of the simpler RNA and DNA viruses described in the previous section (Fig. 4.15B). The very similar topologies of the two β-barrel domains of the three monomers facilitate their close packing to form the hollow base of the trimeric hexon. Interactions among the monomers are very extensive, particularly in the towers that rise above the hexon base and are formed by intertwining loops from each monomer. Consequently, the trimeric hexon is extremely stable.

Figure 4.15 Structural features of adenovirus particles. (A) The organization of human adenovirus type 5 is shown schematically to indicate the locations of the major (hexon, penton base, and fiber) and minor (IIIa, VI, VIII, and IX) capsid proteins and of the internal core proteins, V, VII, and μ. The locations of these proteins and some interactions were initially deduced from the composition of the products of controlled dissociation of viral particles and the results of cross-linking studies. This schematic is based on subsequent high-resolution structures of adenovirus particles. (B) Structure of the hexon homotrimer from PDB ID: 1P30. The monomer (left) is shown as a ribbon diagram, with gaps indicating regions that were not defined in the X-ray crystal structure at 2.9-Å resolution, and the trimer (right) is shown as a space-filling model with each monomer in a different color. The monomer contains two β-barrel jelly roll domains colored green and blue in the left panel. The trimers are stabilized by extensive interactions within both the base and the towers.

The adenovirus particle contains seven additional structural proteins (Fig. 4.15A). The presence of so many proteins and the large size of the particle made elucidation of adenovirus architecture a challenging problem. One approach that has proved generally useful in the study of larger viruses is the isolation and characterization of discrete subviral particles. For example, adenovirus particles can be dissociated into a core structure that contains the DNA genome, groups of nine hexons, and pentons. Analysis of the composition of such subassemblies identified two classes of proteins in addition to the major capsid proteins described above. One comprises the proteins present in the core, such as protein VII, the major DNA-binding protein. The remaining proteins are associated with either individual hexons or the groups of hexons that form an icosahedral face of the capsid, suggesting that they stabilize the structure.

The interactions of protein IX and other minor proteins with hexons and/or pentons were deduced initially by difference imaging (Fig. 4.5) and refined subsequently by X-ray crystallography and cryo-EM (Fig. 4.16A). The minor capsid proteins make numerous contacts with the major structural units. For example, on the outer surface of the capsid, a network formed by extensive interactions among the extended molecules of protein IX knit together the hexons that form the groups of nine (Fig. 4.16B). The function of protein IX as capsid “cement” has been confirmed by the much-reduced heat stability of altered particles that lack this protein. Other minor capsid proteins are restricted to the inner surface, where they reinforce the groups of nine hexons and their associations, or weld the penton base to its surrounding hexons. Not surprisingly, such protein “glues” also buttress other larger icosahedral structures, such as the herpes simplex virus nucleocapsid and the capsids of much larger viruses, such as Paramecium bursaria chlorella virus 1 (some 190 nm in diameter). During adenovirus assembly, interactions among hexons and other major structural proteins must be relatively weak, so that incorrect associations can be reversed and corrected. However, the assembled particle must be stable enough to survive passage from one host to another. It has been proposed that the incorporation of stabilizing proteins like protein IX allows these paradoxical requirements to be met.

Reoviruses. Reovirus particles exhibit an unusual architecture: they contain multiple protein shells. They are naked particles, 70 to 90 nm in diameter with an outer T = 13 icosahedral protein coat that contains the 10 to 12 segments of the double-stranded genome and the enzymatic machinery to synthesize viral mRNA. The particles of human reovirus (genus Orthoreovirinae contain eight proteins organized in two concentric shells, with spikes projecting from the inner layer through and beyond the outer layer at each of the 12 vertices (Fig. 4.17A). Members of the genus Rotavirus, which includes the leading causes of severe infantile gastroenteritis in humans, contain three nested protein layers, with 60 projecting spikes (Fig. 4.17B). Although differing in architectural detail, reovirus particles have common structural features, including an unusual design of the innermost protein shell.

Figure 4.16 Interactions among major and minor proteins of the adenoviral capsid. (A) Cryo-EM reconstruction of the adenovirus type 5 capsid at 3.6-Å resolution radially colored by distance from the center, as indicated. This view is centered on a threefold axis of icosahedral symmetry. Only short stubs of the fibers are evident, as these structures are bent. For other views, see Movie 4.2 (http://bit.ly/Virology_AD5Cap). Courtesy of V. Reddy, The Scripps Research Institute. (B) Views of the outer (left) and inner (right) surfaces indicating the locations of the minor capsid proteins IX, IIIa, V, VI, and VIII (colored as in Fig. 4.15A) with respect to hexons (gray) and penton base (magenta). Data from Yu Y et al. 2017. Sci Adv 3:e1602670.

Removal of the outermost protein layer, a process thought to occur during entry into a host cell, yields an inner core structure, comprising one shell (orthoreoviruses) or two (rotaviruses and members of the genus Orbivirus, such as bluetongue virus). These subviral particles also contain the genome and virion enzymes and synthesize viral mRNAs in vitro under appropriate conditions. High-resolution structures have been obtained for bluetongue virus and human reovirus cores, some of the largest viral assemblies that have been examined by X-ray crystallography. Their thin inner layer contains 120 copies of a single protein (termed VP3 in bluetongue virus). These proteins are not related in their primary sequences, but they nevertheless have similar topological features and the same plate-like shape. Moreover, in both cases, the dimeric proteins occupy one of two different environments, and to do so, they adopt one of two distinct conformational states, indicated as green and red in Fig. 4.17C (right). Because of this arrangement, the green and red dimers are not quasiequivalent, and virtually all contacts in which the two monomer conformations engage are very different. However, these differences allow the formation of VP3 assemblies with either five- or threefold rotational symmetry and hence of an icosahedral shell. This VP3 shell of bluetongue virus abuts directly on the inner surface of the middle layer, which comprises trimers of a single protein organized into a classical T = 13 lattice (Fig. 4.17C, left). A large number of different (nonequivalent) contacts between these trimers and VP3 weld the two layers together and hence stabilize both. These properties of reoviruses illustrate that a classic quasiequivalent structure is not the only solution to the problem of building large viral particles: viral proteins that interact with each other and with other proteins in multiple ways can provide an effective alternative. The organization of the two protein shells described above appears to be conserved in most viruses with double-stranded RNA genomes. However, it is not yet known whether symmetry mismatch is also a feature of other large viruses that contain multiple protein layers.

Figure 4.17 Structures of members of the Reoviridae. The organization of mammalian reovirus (A) and rotavirus (B) particles is shown schematically to indicate the locations of proteins, deduced from the protein composition of intact particles and of subviral particles that can be readily isolated from them. dsRNA, double-stranded RNA. (C) X-ray crystal structure of the core of bluetongue virus, a member of the Orbivirus genus of the Reoviridae, showing the core particle and the inner scaffold. Trimers of VP7 (VP6 in rotaviruses; panel B) project radially from the outer layer of the core particle (left). Each icosahedral asymmetric unit, two of which are indicated by the white lines, contains 13 copies of VP7 arranged as five trimers colored red, orange, green, yellow, and blue, respectively. This layer is organized with classical T = 13 icosahedral symmetry. As shown on the right, the inner layer is built from VP3 dimers that occupy one of two completely different structural environments, colored green and red. Green monomers span the icosahedral twofold axes and interact in rings of five around the icosahedral fivefold axes in a T = 2 structure. In contrast, red monomers are organized as triangular “plugs” around the threefold axes. Differences in the interactions among monomers at different positions allow close packing to form the closed shell. As might be anticipated, VP7 trimers in pentameric or hexameric arrays in the outer layer make different contacts with the two classes of VP3 monomer in the inner layer. Nevertheless, each type of interaction is extensive, and in total, these contacts compensate for the symmetry mismatch between the two layers of the core. The details of these contacts suggest that the inner shell both defines the size of the virus particle and provides a template for assembly of the outer T = 13 structure. From Grimes JM et al. 1998. Nature 395:470–478, with permission. Courtesy of D.I. Stuart, University of Oxford.

Figure 4.18 Asymmetric capsids of retroviruses. (A) Variation in the morphology of retroviruses shown schematically. Although all retrovirus particles are assembled from the same components (see the text), the cores are primarily spherical, cylindrical, or conical in the case of gammaretroviruses (e.g., Moloney murine leukemia viruses), betaretroviruses (e.g., Mason-Pfizer monkey virus), and lentiviruses (e.g., human immunodeficiency virus type 1), respectively. (B) Cryoelectron tomographic slice of human immunodeficiency virus type 1 showing the conical core and the glycoprotein spikes projecting from the surface of the particle. © Jun Liu, Yale University School of Medicine, with permission. Courtesy of H. Winkler, Florida State University.