C Chapter 2: Particleshapter 2: Particles Slide 1/36 © Academic Press Particles Learning Objectives: On completing this session, you should be able to: Understand the reasons why viruses encode the proteins to make particles Identify the main structural types of virus particle Explain how the virus capsid interacts with both the host cell & virus genome during replication

C Chapter 2: Particleshapter 2: Particles Slide 2/36 © Academic Press Virus Particles One the function of the outer shells of a virus particle is to protect the fragile nucleic acid genome from physical, chemical, or enzymatic damage. On leaving the host cell, the virus enters a hostile environment which would quickly inactivate the unprotected genome, since nucleic acids are susceptible to physical damage such as shearing by mechanical forces & to chemical modification by ultraviolet light (from sunlight). The natural environment is heavily laden with nucleases either derived from dead or leaky cells or deliberately secreted by vertebrates as defence against infection. The protein subunits in a virus capsid are redundant, i.e. present in many copies per particle. Damage to one or more subunits may render that particular subunit non-functional, but does not destroy the infectivity of the whole particle.

C Chapter 2: Particleshapter 2: Particles Slide 3/36 © Academic Press Virus Particles The outer surface of the virus is also responsible for recognition of & the first interaction with the host cell. Initially, this takes the form of binding of a specific virus-attachment protein to a cellular receptor molecule. The capsid also has a role to play in initiating infection by delivering the genome in a form in which it can interact with the host cell.

C Chapter 2: Particleshapter 2: Particles Slide 4/36 © Academic Press Formation of virus particles To form infectious particles, viruses must overcome two fundamental problems: They must assemble the particle utilizing the information available from the components which make up the particle itself. Virus particles form regular geometric shapes, even though the proteins from which they are made are irregularly shaped.

C Chapter 2: Particleshapter 2: Particles Slide 5/36 © Academic Press Capsid Symmetry & Virus Architecture It is possible to imagine a virus particle, the outer shell of which (the capsid) consists of a single, hollow protein molecule, which as it folds to assume its mature conformation traps the virus genome inside. In practice, this arrangement cannot occur, since the triplet nature of the genetic code means that three nucleotides (or base pairs for viruses with double-stranded genomes) are necessary to encode one amino acid. Since the approximate molecular weight of a nucleotide triplet is 1000 & the average molecular weight of a single amino acid is 150, a nucleic acid can only encode a protein that is at most 15% of its own weight. Therefore, virus capsids must be made up of multiple protein molecules (subunit construction).

C Chapter 2: Particleshapter 2: Particles Slide 6/36 © Academic Press Formation of virus particles 1957: Fraenkel-Conrat & Williams showed that when mixtures of purified Tobacco mosaic virus (TMV) RNA & coat protein were incubated together, virus particles formed. The discovery that virus particles could form spontaneously from purified subunits without any extraneous information indicated that the particle was in the free energy minimum state & was therefore the favoured structure of the components. Stability is an important feature of virus particles. Although some viruses are very fragile & unable to survive outside the protected host cell environment, many are able to persist for long periods.

C Chapter 2: Particleshapter 2: Particles Slide 7/36 © Academic Press Formation of virus particles The forces which drive the assembly of virus particles include hydrophobic & electrostatic interactions - only rarely are covalent bonds involved in holding together the multiple subunits. Protein-protein, protein-nucleic acid, & protein- lipid interactions are used. There is now a good understanding of general principles & repeated structural motifs which appear to govern the construction of diverse, unrelated viruses: helical & icosahedral symmetry

C Chapter 2: Particleshapter 2: Particles Slide 8/36 © Academic Press Helical capsids Tobacco mosaic virus (TMV) represents one of the two major structural classes seen in viruses, those with helical symmetry. The simplest way to arrange multiple, identical protein subunits is to use rotational symmetry & to arrange the irregularly shaped proteins around the circumference of a circle to form a disk. Multiple disks can then be stacked on top of one another to form a cylinder, with the virus genome coated by the protein shell or contained in the hollow centre of the cylinder.

C Chapter 2: Particleshapter 2: Particles Slide 9/36 © Academic Press Helical capsids Closer examination of the TMV particle by X-ray crystallography reveals that the structure of the capsid actually consists of a helix rather than a pile of stacked disks. A helix can be defined mathematically by two parameters: amplitude amplitude (diameter) pitch pitch (the distance covered by each complete turn of the helix) Helices are rather simple structures formed by stacking repeated components with a constant relationship (amplitude & pitch) to one another. Note that if this constraint is broken, a spiral forms rather than a helix, unsuitable for containing a virus genome.

C Chapter 2: Particleshapter 2: Particles Slide 10/36 © Academic Press Helical capsids click for animation

C Chapter 2: Particleshapter 2: Particles Slide 11/36 © Academic Press Helical capsids Helices are described by the number of subunits per turn of the helix, µ, & the axial rise per subunit, p. The pitch of the helix, P, is therefore equal to: P = µ x p For TMV, µ = 16.3, i.e coat protein molecules per helix turn, & p = 0.14 nm. Therefore, the pitch of the TMV helix is 16.3 x 0.14 = 2.28 nm.

C Chapter 2: Particleshapter 2: Particles Slide 12/36 © Academic Press Tobacco mosaic virus

C Chapter 2: Particleshapter 2: Particles Slide 13/36 © Academic Press Helical capsids Among the simplest helical capsids are those of the well-known bacteriophages of the family Inoviridae, such as M13 & fd. These phages are about 900 nm long & 9 nm diameter & the particles contain five proteins. The major coat protein is the product of phage gene 8 (g8p) & there are copies of this protein per particle, together with approximately five copies each of four minor capsid proteins, g3p, g6p, g7p & g9p, located at the ends of the filamentous particle.

C Chapter 2: Particleshapter 2: Particles Slide 14/36 © Academic Press Bacteriophage M13

C Chapter 2: Particleshapter 2: Particles Slide 15/36 © Academic Press Bacteriophage M13 Because the phage DNA is packaged inside the core of the helical particle, the length of the particle is dependent on the length of the genome. 'polyphage' 'miniphage' 'maxiphage'In all Inovirus preparations, 'polyphage' (containing more than one genome length of DNA), 'miniphage' (deleted forms containing phage genomes length of DNA), & 'maxiphage' (genetically defective forms but containing more than one phage genome length of DNA) occur. The plastic property of these filamentous particles has been exploited by molecular biologists to develop the M13 genome as a cloning vector - insertion of foreign DNA into the genome results in recombinant phage particles which are longer than the wild-type filaments. Unlike most viruses, there is no sharp cut-off at which the genome can no longer be packaged into the particle.

C Chapter 2: Particleshapter 2: Particles Slide 16/36 © Academic Press Bacteriophage M13 Inovirus phages are 'male-specific', i.e. they require the F pilus on the surface of Escherichia coli for infection. On infection, an interaction between g3p located at one end of the filament together with g6p, & the end of the F pilus. This interaction causes a conformational change in g8p. Initially, its structure changes from 100%  -helix to 85%  - helix, causing the filament to shorten. The end of the particle attached to the F pilus flares open, exposing the phage DNA. Subsequently, a second conformational change in the g8p subunits reduces its  -helical content from 85% to 50%, causing the phage particle to form a hollow spheroid ~40 nm in diameter & expelling the phage DNA, initiating infection of the host cell.

C Chapter 2: Particleshapter 2: Particles Slide 17/36 © Academic Press Helical plant viruses Many plant viruses show helical symmetry. These particles vary from approximately 100 nm (Tobravirus) to approximately 1000 nm (Closterovirus) in length. Quite why so many groups of plant virus have evolved this structure is not clear, but it may be related either to the biology of the host plant cell, or alternatively, to the way in which they are transmitted between hosts.

C Chapter 2: Particleshapter 2: Particles Slide 18/36 © Academic Press Helical animal viruses Helical, naked (i.e. non-enveloped) animal viruses do not exist. This probably reflects aspects of host cell biology & virus transmission, but the reasons are not clear. There are a large number of animal viruses based on helical symmetry, but all have the addition of an outer lipid envelope. There are too many viruses with this structure to list individually, but this category includes many of the best known human pathogens, e.g. influenza virus, mumps & measles viruses, & Rabies virus.

C Chapter 2: Particleshapter 2: Particles Slide 19/36 © Academic Press Rhabdovirus particle

C Chapter 2: Particleshapter 2: Particles Slide 20/36 © Academic Press Helical animal viruses All helical animal viruses possess single-stranded, negative-sense RNA genomes. The molecular design of all of these viruses is similar. The virus nucleic acid & a basic, nucleic acid-binding protein condense together in the infected cell to form a helical nucleocapsid. This protein-RNA complex serves to protect the fragile virus genome from physical & chemical damage & in some instances also provides other functions associated with virus replication. The envelope & its associated proteins are derived from the membranes of the host cell & are added to the nucleocapsid core of the virus during replication.

C Chapter 2: Particleshapter 2: Particles Slide 21/36 © Academic Press Helical Symmetry (Summary) Many different groups of viruses have evolved around helical symmetry. Simple viruses with small genomes use this architecture to provide protection for the genome without the need to encode multiple capsid proteins. More complex virus particles utilize this structure as the basis of the virus particle, but elaborate on it with additional layers of protein & lipid.

C Chapter 2: Particleshapter 2: Particles Slide 22/36 © Academic Press Icosahedral (isometric) capsids An alternative way of building a virus capsid is to arrange protein subunits in the form of a hollow quasispherical structure, enclosing the genome within. The criteria for arranging subunits on the surface of a solid are more complex than those for building a helix. tetrahedron cube octahedrondodecahedron icosahedron 20 triangular faces arranged around the surface of a sphereIn theory, a number of solid shapes can be constructed from repeated subunits, e.g. a tetrahedron (four triangular faces), cube (six square faces), octahedron (eight triangular faces), dodecahedron (12 pentagonal faces) & an icosahedron - a solid shape consisting of 20 triangular faces arranged around the surface of a sphere.

C Chapter 2: Particleshapter 2: Particles Slide 23/36 © Academic Press Icosahedral (isometric) capsids Direct examination of a number of small 'spherical' viruses by electron microscopy revealed that they have icosahedral symmetry. Although in theory it is possible to construct virus capsids based on simpler symmetrical arrangements, such as tetrahedra or cubes, there are practical reasons why this does not occur - it is more economic in genetic terms to design a capsid based on a large number of identical, repeated protein subunits rather than fewer, larger subunits. It is unlikely that a simple tetrahedron consisting of four identical protein molecules would be large enough to contain even the smallest virus genome. If it were, the gaps between the subunits would be so large that the particle would be leaky, & fail to carry out its primary function of protecting the virus genome.

C Chapter 2: Particleshapter 2: Particles Slide 24/36 © Academic Press Icosahedral Symmetry For an icosahedron the rules of symmetry are based on the rotational symmetry of the solid, known as symmetry: An axis of twofold rotational symmetry through the centre of each edge An axis of threefold rotational symmetry through the centre of each face An axis of fivefold rotational symmetry through the centre of each corner (vertex)

C Chapter 2: Particleshapter 2: Particles Slide 25/36 © Academic Press Icosahedral Symmetry click for animation

C Chapter 2: Particleshapter 2: Particles Slide 26/36 © Academic Press Icosahedral Symmetry Since protein molecules are irregularly shaped & not regular equilateral triangles, the simplest icosahedral capsids are built up by using three identical subunits to form each triangular face. This means that 60 identical subunits are required to form a complete capsid. A few simple virus particles are constructed in this way; for example, bacteriophages of the family Microviridae, such as  X174. An empty precursor particle called the procapsid is formed during assembly of this bacteriophage. Assembly of the procapsid requires the presence of the two scaffolding proteins which are structural components of the procapsid, but are not found in the mature virion.

C Chapter 2: Particleshapter 2: Particles Slide 27/36 © Academic Press Icosahedral Symmetry Most icosahedral virus capsids contain more than 60 subunits, for reasons of genetic economy. A regular icosahedron composed of 60 identical subunits is a very stable structure because all the subunits are equivalently bonded, i.e. they show the same spacing relative to one another & each occupies the minimum free energy state. With more than 60 subunits it is impossible for them all to be arranged completely symmetrically with exactly equivalent bonds to all their neighbours, since a true regular icosahedron consists of only 20 subunits. 1962: Caspar & Klug proposed the idea of quasi-equivalence - subunits in nearly the same local environment form nearly equivalent bonds with their neighbours, permitting self- assembly of icosahedral capsids from multiple subunits.

C Chapter 2: Particleshapter 2: Particles Slide 28/36 © Academic Press Icosahedral symmetry

C Chapter 2: Particleshapter 2: Particles Slide 29/36 © Academic Press Icosahedral Symmetry In higher order icosahedra, the symmetry of the particle is defined by the triangulation number of the icosahedron. triangulation numberThe triangulation number, T, is defined by: T = f 2 x P where f is the number of subdivisions of each side of the triangular face, f 2 is the number of subtriangles on each face & P = h 2 + hk + k 2, where h & k are any distinct, non-negative integers.

C Chapter 2: Particleshapter 2: Particles Slide 30/36 © Academic Press Triangulation Numbers

C Chapter 2: Particleshapter 2: Particles Slide 31/36 © Academic Press Picornavirus capsids Atomic structures of the capsids of a number of different picornaviruses have been determined, including polioviruses, Foot-and-mouth disease virus (FMDV), Human rhinovirus & a number of others. The structure of these virus particles is remarkably similar to those of many other unrelated viruses, such the family Nodaviridae & the Comovirus group. All these virus groups have icosahedral capsids ~30 nm diameter with triangulation number T = 3. The capsid is composed of 60 repeated subassemblies of proteins, each containing three major subunits, VP1, VP2, & VP3, i.e. 60 x 3 = 180 surface monomers in the particle. All three proteins are based on a similar structure, consisting of amino acid residues in what has been described as an '8-strand anti-parallel  -barrel' which has been found in all T = 3 icosahedral RNA virus capsids.

C Chapter 2: Particleshapter 2: Particles Slide 32/36 © Academic Press strand anti-parallel  -barrel

C Chapter 2: Particleshapter 2: Particles Slide 33/36 © Academic Press Picornavirus capsids

C Chapter 2: Particleshapter 2: Particles Slide 34/36 © Academic Press Picornavirus capsids Picornavirus capsids contain four structural proteins. In addition to the three major proteins VP1-3, there is a small fourth protein, VP4. VP4 is located predominantly on the inside of the capsid & is not exposed at the surface of the particle. VP4 is formed from cleavage of the VP0 precursor into VP2+VP4 late in assembly & is myristylated at its amino terminus, i.e. it is modified after translation by the covalent attachment of myristic acid, a 14-carbon unsaturated fatty acid. Five VP4 monomers form a hydrophobic micelle, driving the assembly of a pentameric subassembly. The chemistry, structure, & symmetry of the proteins which make up the picornavirus capsid reveal how assembly is driven.

C Chapter 2: Particleshapter 2: Particles Slide 35/36 © Academic Press Picornavirus Assembly

C Chapter 2: Particleshapter 2: Particles Slide 36/36 © Academic Press Part 2 (click on this link) Particles