Chapter 4: Structure

S Introduction Functions of the virion A primary function of an infectious virus particle (called the virion) is protection of the genome, which can be damaged irreversibly by a break in the nucleic acid or by mutation during passage through hostile environments In all virus particles, the nucleic acid is sequestered within a sturdy barrier formed by extensive interactions among the viral proteins that comprise the protein coat The highly folded nature of coat proteins and their dense packing to form shells render them largely inaccessible to proteolytic enzymes The envelope adds not only a protective lipid membrane but also an external layer of protein and sugar formed by the glycoproteins However, they must also attach to an appropriate host cell and deliver the genome to the interior of that cell, where the particle is at least partially disassembled The protective function of virus particles depends on stable intermolecular interactions among their components during assembly, egress from the virus-producing cell, and transmission Nevertheless, it is clear that contact of a virion with the appropriate cell surface receptor or exposure to a specific intracellular environment can trigger substantial conformational changes Virus particles are therefore metastable assemblies that have not yet attained the minimum free energy conformation Virions are not simply inert entities. Rather, they are molecular machines that play an active role in delivery of the nucleic acid genome to the appropriate host cell and initiation of the reproductive cycle Nomenclature Virus architecture is described in terms of structural units of increasing complexity, from the smallest biochemical unit (the polypeptide chain) to the infectious particle (or virion) Methods for studying virus structure Electron microscopy is the most widely used method for the examination of structure and morphology of virus particles The greatest contrast between virus particle and stain (negative contrast) occurs where portions of the folded protein chain protrude from the surface However, because these surface features are often formed by multiple proteins, their organization does not necessarily correspond to that of the individual proteins that make up the capsid shell The symmetry of most virus particles facilitates analysis of individual images obtained by cryoEM for reconstruction of two- or three-dimensional structure This approach can be complemented by cryo-electron tomography, in which two-dimensional images are recorded as the vitrified sample is tilted at different angles to the electron beam and subsequently combined into a three-dimensional density map The first descriptions of the molecular interactions that dictate the structure of virus particles were obtained by X-ray crystallography Not all viruses can be examined directly by X-ray crystallography: some do not form suitable crystals, and the larger viruses lie beyond the power of the current procedures by which X-ray diffraction spots are converted into a structural model Building a protective coat Regardless of their size and architectural sophistication, all virions contain at least one protein coat, the capsid or nucleocapsid, which encases and protects the nucleic acid genome The repetition of such interactions among a limited number of proteins results in a regular structure, with symmetry that is determined by the spatial patterns of the interactions Helical structures The nucleocapsids of some enveloped animal viruses, as well as certain plant viruses and bacteriophages, are rod-like or filamentous structures with helical symmetry A characteristic feature of a helical structure is that any volume can be enclosed simply by varying the length of the helix. Such a structure is said to be open In contrast, capsids with icosahedral symmetry are closed structures with fixed internal volume Repetitive interactions among coat protein subunits form disks, which in turn assemble as a long, rod-like s right-handed helix. In the interior of the helix, each coat protein molecule binds three nucleotides of the RNA genome The coat protein subunits therefore engage in identical interactions with one another and with the genome, allowing the construction of a large, stable structure from multiple copies of a single protein In all cases, these structures contain an RNA molecule, many copies of an RNA packaging protein (designated NP or N), AND the viral RNA polymerase and associated enzymes responsible for synthesis of mRNA and viral genomes Despite common helical symmetry and similar composition, the internal components of these (-) strand RNA viruses exhibit considerable diversity in morphology and organization X-ray crystallography of a ring-like N protein-RNA complex containing 10 molecules of the protein bound to RNA has revealed that each N protein molecule binds to 9 nucleotides of the RNA that is largely sequestered within cavities formed by the protein Furthermore, with the exception of terminal sequences, the RNA In these ribonucleoproteins is fully accessible to solvent, suggesting that the RNA is not sequestered in the interior of the ribonucleoprotein Because helical structures are "open", some property other than symmetry must limit the size of helical viruses, perhaps the nature of their genomes or susceptibility to shear forces Capsids with icosahedral symmetry General principles Icosahedral symmetry An icosahedron contains the largest number of faces (20), and 12 vertices related by two-, three-, and fivefold axes of rotational symmetry However, most closed capsids look spherical, and they often possess prominent surface features or viral glycoproteins in the envelope that do not conform to the underlying icosahedral symmetry of the capsid shell As an icosahedron has 20 faces, 60 identical subunits (3 per face x 20 faces) is the minimal number needed to build a capsid with icosahedral symmetry Large capsids and quasiequivalent bonding In the simplest icosahedral packing arrangement, each of the 60 subunits (structural or asymmetric units) consists of a single molecule in a structurally identical environment As the viral proteins that form such closed shells are generally <~100 kDa in molecular mass, the size of the viral genome that can be accommodated In this simplest type of particle is restricted severely Indeed, the capsids of the majority of animal viruses are built from many more than 60 subunits and can house very large genomes The triangulation number, T A crucial idea introduced by Caspar and Klug was that of triangulation, the description of the triangular face of a large icosahedron in terms of its subdivision into smaller triangles, termed facets Because the minimum number of structural units required is 60, the total number of subunits in the structure is 60T A second cornerstone of the theory developed by Caspar and Klug was the proposition that when a capsid contains >60 subunits, each occupies a quasiequivalent position; that is, the noncovalent bonding properties of subunits In different structural environments are similar, but not identical Nevertheless, all subunits bond to their neighbors in similar (quasiequivalent) ways, for example, via head-to-head and tail-to-tail interactions The resulting large conformational differences between small regions of chemically identical subunits were not anticipated in early considerations of virus structure, because these principles were formulated when little was known about the conformational flexibility of proteins Furthermore, a capsid stabilized by covalent joining of subunits to form viral "chain mail" has been described Structurally simple capsids Adeno-associated virus 2: classic T = 1 icosahedral design The parvoviruses are very small animal viruses, with particles of ~25 nm in diameter that encase single-stranded DNA genomes of <5 kb The protein that forms the subunits of adenovirus-associated virus type 2, a member of the dependovirus subgroup of parvoviruses, contains a core domain commonly found in viral capsid proteins, in which beta-strands are connected by loops The prominent projections near the threefold axes of rotational symmetry, which have been implicated in receptor binding, and are formed by extensive interdigitation among the loops from adjacent subunits Adenovirus-associated virus vectors have proved valuable in human gene therapy, in part because variations In the sequences of these loops confer differences in tissue tropism Poliovirus: A T = 3 structure In contrast to the T = 1 parvoviruses, the ~ 30-nm diameter poliovirus particle is composed of 60 copies of a multimeric structural unit The poliovirus capsid is built from asymmetric units that contain one copy of each of three different proteins (VP1, VP2, and VP3), and is therefore described as a pseudo T = 3 structure That is, they [protein backbones in beta-barrel domains] possess the same topology, and the differences among these proteins are restricted largely to the loops that connect beta-strands and to the N- and C-terminal segments that extend from the central betabarrel domains It is well established that the three-dimensional structures of cellular proteins have been highly conserved during evolution, even though there may be very little amino acid sequence identity It is also possible that this domain topology represents one of a limited number commensurate with packing of proteins to form a sphere, and therefore an example of convergent evolution The overall similarity in shape of the beta-barrel domains of poliovirus VP1, VP2, and VP3 facilitates both their interaction with one another to form the 60 structural units of the capsid and the packing of these units The packing of the beta-barrel domains is reinforced by a network of protein-protein contacts on the inside of the capsid, which are particularly extensive about the fivefold axes Simian virus 40: an alternative icosahedral design The capsids of the small DNA polyomaviruses simian virus 40 and mouse polyomavirus, ~50 nm In diameter, are organized according to a different design that is not based on quasiequivalent interactions The capsid is built from 72 such pentamers engaged in one of two kinds of interaction. Twelve pentamers occupy positions of fivefold rotational symmetry, in which each is surrounded by five neighbors Furthermore, the VP1 molecules present in adjacent pentamers in the simian virus 40 capsid do not make extensive contacts via the surfaces of their beta-barrel domains In fact, there are just three kinds of interpentamer contact, which are the result of alternative conformations and noncovalent interactions of the long C-terminal arms of VP1 molecules Neither poliovirus nor simian virus 40 capsids conform to strict quasiequivalent construction: all contacts made by all protein subunits are not similar, and in the case of simian virus 40, the majority of VP1 pentamers are packed in hexameric arrays Structurally sophisticated capsids 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 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 very similar topologies of the two beta-barrel domains of the three monomers facilitate their close packing to form the hollow base of the trimeric hexon The interactions of protein IX and other minor proteins with hexons and/or pentons were deduced initially by difference imaging and refined subsequently by X-ray crystallography and cryo-EM 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 Reoviruses 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 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 or two Other capsid architectures The capsid of all retroviruses surrounds a nucleoprotein that contains the diploid (+) strand RNA genome. The capsid is surrounded by the viral matrix (M) protein, which is encased in the viral envelope However, pentamers are not spaced at regular intervals throughout the structure. Rather, they are restricted to the terminal caps and separated by spirals (a variant of helical symmetry) of CA hexamers that form the body of the cone Packaging the nucleic acid genome A definitive property of a virion is the presence of a nucleic acid genome. Incorporation of the genome requires its discrimination from a large population of cellular nucleic acid Such confinement of the genome can result in high internal pressure, equivalent to that generated in locomotive steam engines, some 18 and 25 atm within herpes simplex virus type 1 and phage capsids, respectively Packaging of nucleic acids is an intrinsically unfavorable process because of the highly constrained conformation imposed on the genome In some cases, the force required to achieve packaging is provided, at least in part, by specialized viral proteins that harness the energy released by hydrolysis of ATP to drive the insertion of DNA Direct contact of the genome with a protein shell In the simplest arrangement, the nucleic acid makes direct contact with the protein(s) that forms the protective shell of the virus particle Much of the RNA genome forms an outer layer in which it makes extensive contact with the inner surface of the capsid Use of the same protein or proteins both to condense the genome and to build a capsid allows efficient utilization of limited genetic capacity. It is therefore an advantageous arrangement for viruses with small genomes Remarkably, as much as 80% of the RNA genome appears highly ordered within the core, with strong elements of icosahedral symmetry Neutralization might be accomplished by proteins that form the inner surface of the capsid, or by the incorporation of small, basic peptides made by the host cell, such as spermine and spermidine Packaging by specialized viral proteins In many virus particles, the genome is associated with specialized nucleic acid-binding proteins, such as the nucleocapsid proteins of (-) strand RNA viruses and (+) strand retroviruses, or the core proteins of adenoviruses This mode of binding is exemplified by the structure of the vesicular stomatitis virus N protein, in which 9 nucleotides of RNA are tightly but nonspecifically bound in a cavity formed between the two domains of each N protein molecule However, how the viral DNA genome is organized and condensed by the core proteins is not known: the nucleoprotein was not observed in the high-resolution structures of adenovirus particles, and the structures of core proteins have not been determined The fundamental DNA packaging unit is a multimer of protein VII, which appears as beads on a string of adenoviral DNA when other core proteins are removed Protein VII and the other core proteins are basic, as would be expected for proteins that bind to A negatively charged DNA molecule without sequence specificity Packaging by cellular proteins The circular, double-stranded DNA genomes released from these virus particles are organized into nucleosomes that contain the four cellular core histones, H2A, H2B, H3 and H4, to form a minichromosome This packaging mechanism is elegant, with two major advantages: none of the limited viral genetic information needs to be devoted to DNA binding proteins, and the viral genome, which is transcribed by cellular RNA polymerase II, enters the infected cell nucleus as a nucleoprotein closely resembling the cellular templates for this enzyme Viruses with envelopes However, viral envelopes vary considerably in size, morphology, and complexity, and they differ in lipid composition, the number of proteins they contain, and their location The membranes form the outermost layer of enveloped animal viruses, but in some bacteriophages and archaeal viruses the membrane lies beneath an icosahedral capsid Viral envelope components The lipid composition is variable, because viral envelopes can be derived from different kinds of cellular membranes Embedded in the membrane are viral proteins, the great majority of which are glycoproteins that carry covalently linked sugar chains, or oligosaccharides Intra- or interchain disulfide bonds, another common chemical feature of these proteins, are also acquired during transport to assembly sites Envelope glycoproteins Viral glycoproteins are integral membrane proteins firmly embedded in the lipid player by a short membrane-spanning domain Internal domains, which make contact with other components of the virus particle, are often essential for virus assembly With few if any exceptions, viral membrane glycoproteins form oligomers, which can comprise multiple copies of a single protein or may contain two or more protein chains The hemagglutinin (HA) protein of human influenza A virus is a trimer that contains a globular head with a top surface that is projected ~135 A from the viral membrane by a long stem Other viral glycoproteins that mediate cell attachment and entry adopt a quite different orientation (and structure); the external domain of E protein is a flat, elongated dimer that lies on the surface of the viral membrane rather than projecting from it Simple enveloped viruses: direct contact of external proteins with the capsid or nucleocapsid On the outer surface of the membrane, the external portions of these glycoproteins, together with the E3 protein, form an unexpectedly elaborate structure: a thin T = 4 icosahedral protein layer covers most of the membrane and supports the spikes, which are hollow, three-lobed projections Enveloped viruses with an additional protein layer Enveloped viruses of several families contain an additional protein layer that mediates interactions of the genome-containing structure with the viral envelope In the simplest case, a single viral protein, termed the matrix protein, welds an internal ribonucleoprotein to the envelope. This arrangement is found in members of several groups of (-) strand RNA viruses Because the internal capsids or nucleocapsids of these multilayered enveloped viruses are not in direct contact with the envelope, the organization and symmetry of internal structures are not evident from the external appearance of the surface glycoprotein layer Large viruses with multiple structural elements Such particles comprise obviously distinct components with different symmetries and/or multiple layers and in some cases exhibit architectures that do not appear to be based on helical or icosahedral symmetry Particles with helical or icosahedral parts Bacteriophage T4 The T4 particle, which is built from ~50 of the proteins encoded in the ~170-kbp doublestranded DNA genome, is a structurally elegant machine tailored for active delivery of the genome to host cells The most striking feature is the presence of morphologically distinct and functionally specialized structures, notably the head containing the genome and a long tail that terminates in a base plate from which six long tail fibers protrude One of the 12 vertices is occupied by a unique structure termed the connector, which joins the head to the tail The tail is connected to the head via a hexameric ring and at its other end to a complex, dome-shaped structure termed the baseplate, where it carries the cell-puncturing spike Herpesvirus The latter are the nucleocapsid surrounding the DNA genome and the proteinaceous layer encasing this structure, called the tegument The large herpesviral capsid, like that of adenoviruses, is further stabilized by additional proteins, including two that form triplexes that link the major structural units Such a network of covalent bonds must greatly increase the stability of the large nucleocapsid and may also be necessary to counter the high pressure exerted on this protein shell The portal comprises 12 copies of the UL6 protein and is a squat, hollow cylinder that is wider at one end and surrounded by a two-tiered ring at the wider end Tegument proteins are not uniformly distributed around the capsid, but are concentrated on one side, where they form a well-defined cap-like structure Mimiviruses Characteristic features of members of the mimiviridae, which infect single-cell eukaryotes, are their very large, double-stranded DNA genomes and correspondingly huge particles Despite their large size, mimivirus particles exhibit some familiar structural features, notably icosahedral symmetry and a capsid built from a major capsid protein with the double beta-barrel jelly roll topology Alternative architectures Poxviruses Particles of poxviruses such as vaccinia virus also comprise multiple, distinct structural elements, but none of these exhibit obvious icosahedral or helical symmetry A second distinctive feature is that two forms of infectious particles are produced in vaccinia virus-infected cells, termed mature virions and enveloped extracellular virions, which differ in the number and origin of membranes Pithoviruses The internal nucleoid, which contains the double-stranded DNA genome of some 0.6 Mbp, is enclosed within what is thought to be a lipid membrane, in turn encased in a thick protein layer (the tegument) Following uptake of virus particles into host cells by phagocytosis, this unusual cork structure is expelled to allow fusion of the viral nucleoid membrane with that of the cellular vacuole Other components of virions Enzymes These enzymes generally catalyze reactions unique to virus infected cells, such as synthesis of viral mRNAs from an RNA template or of viral DIA from an RNA template This complement of enzymes is necessary because transcription of the viral double-stranded DHA genome takes place in the cytoplasm of infected cells, whereas cellular DNA dependent RNA polymerases and the RNA processing machinery are restricted to the nucleus Other viral proteins Among the best characterized are the protein primers for viral genome replication that are covalently linked to the genomes of picornaviruses such as poliovirus and adenovirus Other herpesvirus tegument proteins induce the degradation of cellular mRNA or block cellular mechanisms by which viral proteins are presented to the host's immune system Cellular macromolecules Because they are formed by budding, enveloped viruses can readily incorporate cellular proteins and other macromolecules Consequently, it can be difficult to distinguish cellular components specifically incorporated into enveloped virus particles from those trapped randomly or copurifying with the virus