VIRUS COURSAE

 The nucleic acid of a virus contains all the information needed to produce new virus particles. Some of this information is used directly to make virion components and some to make accessory proteins or to provide signals which allow the virus to subvert the biosynthetic machinery of a cell and redirect it towards the production of virus. Whereas the standard form of genetic material in living systems is double-stranded DNA, viruses contain a diverse array of nucleic acid forms and compositions. The nucleic acid content of a virus has been used as a basis for classifying viruses. The key aspect of this classification scheme is that it considers the nature of the virus genome in terms of the mechanisms used to replicate the nucleic acid and transcribe mRNA encoding proteins. A detailed consideration of the nature of virus nucleic acids and the mechanisms by which they are replicated and transcribed are to be found in Chapters 6–10. Here we will consider only how such features can b...

  The structural features of virus particles and the principles which underlie these structures have been described in Chapter 3. When viruses were first visualized in the electron microscope, defining classification groups on the basis of the observed particle shape or morphology was relatively simple. A key structural feature is whether or not the virus particle has a lipid envelope and this alone can be used as a designated feature, giving enveloped and nonenveloped viruses (see Section 3.4). If the virion is nonenveloped three morphological categories are defined, isometric, filamentous, and complex. Isometric viruses (see Section 3.3) appear approximately spherical but are actually icosahedrons or icosadeltahedrons. Filamentous viruses (see Section 3.2) have a simple, helical, morphology. The complex viruses are those which do not neatly fit within the other two categories. Complex shapes for virus particles may be made up of a combination of isometric and filamentous componen...

 An alternative approach has been to group viruses according to the host that they infect. This has the attraction that it emphasizes the parasitic nature of the virus–host interaction. However, there are several difficulties with this approach. This form of classification implies a fixed, unchanging, link between the virus and host in question. Some viruses are very restricted in their host range, infecting only one species, such as hepatitis B virus infecting humans, and so a designation based on the host is appropriate. However, others may infect a small range of hosts, such as poliovirus which can infect various primates, and the designation here must reflect this rather than name a single species. The most serious difficulty arises with viruses which infect and replicate within very different species. This can be seen with certain viruses which can infect and replicate within both plants and insects. Designation of a virus by the host it infects is therefore not always straigh...

  The first, and most common, experience of viruses is as agents of disease and it is possible to group viruses according to the nature of the disease with which they are associated. Thus, one Chapter 4 Outline 4.1 Classification on the basis of disease 4.2 Classification on the basis of host organism 4.3 Classification on the basis of virus particle morphology 4.4 Classification on the basis of viral nucleic acids 4.5 Classification on the basis of taxonomy 4.6 Satellites, viroids, and prions 50 PART I WHAT IS A VIRUS? can discuss hepatitis viruses or viruses causing the common cold. This is attractively simple. However, this method of grouping viruses, though reflecting an important characteristic, suffers from serious deficiencies. First, this approach is very anthropomorphic, focusing as it does on diseases that we recognize because they affect humans or our domestic livestock. This ignores the fact that most viruses either do not cause disease or cause a disease that we do not...

  Viruses represent one of the most successful types of parasite in the world and have been isolated from representatives of every known group of organisms from the smallest single-celled bacterium to the largest mammal. While in most cases the virus is specific for the host species in which it has been identified, some viruses are able to infect species from different phyla and even different kingdoms. The number of known viruses now reaches over 5000 with new viruses being discovered all the time. This very large number contains a diverse array of viruses which at first sight is very bewildering. To make easier the study of viruses and bring order to this apparent diversity, over the years a number of different systems has been proposed to generate classification schemes which will allow us to study representative viruses rather than each individually. All of the proposed classification schemes have different strengths and weaknesses but there is now general consensus. The Intern...

 It is important to remember that all virus particles not only have to be constructed to protect the genome, but they also have to disassemble to permit the genome to enter a new target cell. This is supremely important to the virus particle as it has only the one chance to do this successfully and hence propagate its genome. The notion is developing that the particle is metastable, i.e. it can spontaneously descend to a lower energy level and, in doing so, releases its genome. Not surprisingly there are a number of fail-safe devices that tell the virus when it is safe to let go the genome. One of the simplest systems is used by enveloped animal viruses like HIV-1. This undergoes a succession of interactions between cell receptors and virus envelope protein binding sites, the passwords needed to gain entry to a high security establishment. If everything is in order, the metastable envelope protein then undergoes profound rearrangements that allow a hidden hydrophobic segment to ins...

The different virus morphologies discussed above do not occur with equal fr equency among animal, plant and bacterial viruses (Table 3.3). There are relatively few purely icosahedral viruses in bacteria (see Appendix 4); nonenveloped helical viruses are common and occur almost exclusively in plants; enveloped icosahedral viruses and enveloped helical viruses are common in animals and rare in plants and bacteria. Finally head–tail virus morphology, in which an isometric head and a helical tail are joined together, is found only in bacteria. Unfortunately there is no real explanation as to why there should be these restrictions. There also exist some very large, very complex viruses (e.g. poxviruses of animals and mimivirus of amoebae: see Appendix) whose morphogenesis is beyond our current comprehension

While the head–tail architectural principle is unique to bacterial viruses (Fig. 3.16), many bacterial viruses have other morphologies (Table 3.3 Fig. 3.15 The influenza virus hemagglutinin (HA). This is a homotrimer but only a monomer is shown here. The HA is synthesized as a single polypeptide which is proteolytically cleaved into the membrane-bound HA2 and the distal HA1. (a) An outline structure showing that HA1 and HA2 are both hairpin structures. (b) The crystal structure. The globular head of HA1 bears all the neutralization sites (A–E; shaded) and is made of a distorted jelly-roll β barrel like most of the icosahedral viruses. (From Wiley et al. (1981) Nature (London), 289, 373.) 46 PART I WHAT IS A VIRUS? and Appendix 4). There is a large variation on the head–tail structural theme and bacteriophages can be subdivided into those with short tails, long noncontractile tails, and complex contractile tails (see Appendix 4). A number of other structures, such as base plates, collar...

Although they appear complex, these viruses have a conventional isometric or helical structure that is surrounded by a membrane – a 4-nm-thick lipid bilayer containing proteins. Examples include many of the larger animal viruses, but only a few plant and bacterial viruses. Traditionally these viruses were distinguished from nucleocapsid viruses by treatment with detergents or organic solvents, which disrupts the membrane and destroys infectivity. Thus they were sometimes referred to as “ether-sensitive viruses.” The envelope, which is derived from host cell membranes, is obtained by the virus budding from cell membranes, but most contain no cell proteins. How cell proteins are excluded and why retroviruses, the exception, do not exclude cell proteins from their virions are not understood (see Section 11.6). An isometric core surrounded by an isometric envelope: Sindbis virus particles Sindbis virus (a togavirus) has an icosahedral nucleocapsid that comprises a single protein, surrounde...

A second way of constructing a symmetrical particle would be to arrange the smallest number of subunits possible around the vertices or faces of an object with cubic symmetry, e.g. tetrahedron, cube, octahedron, dodecahedron (constructed from 12 regular pentagons), or icosahedron (constructed from 20 equilateral triangles). Figure 3.3 shows possible arrangements for objects with triangular and square faces. Multiplying the minimum number of subunits per face by the number of faces gives the smallest number of subunits which can be arranged around such an object. The minimum number of subunits is determined by the symmetry element of the face, i.e. a square face will have four subunits, a triangular face will have three subunits, etc. For a tetrahedron the smallest number of subunits is 12, for a cube or octahedron it is 24 subunits, and for a dodecahedron or icosahedron it is 60 subunits. Although it may not be immediately apparent, these represent the few ways in which an asymmetric...

  One of the simplest ways of symmetrically arranging nonsymmetrical components is to place them round the circumference of a circle to form discs (Fig. 3.2). This gives a two-dimensional structure. If a large number of discs is stacked on top of one another, the result is a “stacked-disc” structure. Thus a symmetrical three-dimensional structure can be generated from a nonsymmetrical component such as protein and still leave room for nucleic acid. Examination of published electron micrographs of viruses reveals that some of them have a tubular structure. One such virus is the tobamovirus, tobacco mosaic virus (TMV) (Fig. 3.1). However, closer examination reveals that the TMV subunits are not arranged cylindrically, i.e. in rings, but helically. There is an obvious explanation for this. A helical nucleic acid could not be equivalently bonded in a stacked-disc structure. However, by arranging the subunits helically, the maximum number of bonds can still be formed and each subunit eq...

While proteins may have regular secondary structure elements in the form of α helix and β structure, the tertiary structure of the protein is not symmetrical. This is a consequence of hydrogen bonding, disulfide bridges, and the intrusion of proline in the secondary structure. Although it may be naïvely thought that the nucleic acid could be covered by a single, large protein molecule, this cannot be the case since proteins are irregular in shape, whereas most virus particles have a regular morphology (Fig. 3.1). However, that viruses must contain more than a single protein can also be deduced solely from considerations of the coding potential of nucleic acid molecules. A coding triplet has an Mr of approximately 1000 but specifies a single amino acid with an average Mr of about 100. Thus a nucleic acid can at best only specify one-tenth of its mass of protein. Since viruses frequently contain more than 50% protein by mass, it is apparent that more than one protein must be present. Obv...

All virus genomes are surrounded by proteins which: • Protect nucleic acids from nuclease degradation and shearing.   • Contain identification elements that ensure a virus recognizes an appropriate target cell (but plant viruses do not, and enter the cell directly by injection or injury).   • Contain a genome-release system that ensures that the virus genome is released from a particle only at the appropriate time and location.   • Include enzymes that are essential for the infectivity of many, but not all, viruses.   • Are called structural proteins, as they are part of the virus particle. All viruses contain protein and nucleic acid with at least 50%, and in some cases up to 90%, of their mass being protein. At first sight it would appear that there are many ways in which proteins could be arranged round the nucleic acid. However, viruses use only a limited number of designs. The limitation on the range of structures is due to restrictions imposed b

  As discussed above, all techniques have their advantages and limitations. For example, serological methods of virus detection are effective and quick but tell us nothing about the virus genome. Neutralization tests are simple, but are confined to viruses that can be cultivated, and are slow to give a result. This depends on the time a virus takes to kill a detectable number of cells, and this can range from several days to several weeks. Such a situation is far from ideal, and the problem was solved by the discovery of a technique which makes many, many copies of a chosen part of the virus genome. This is the polymerase chain reaction (PCR) which synthesizes DNA from a DNA template, and was devised in 1985 by Kari Mullis. If the virus of interest has an RNA genome, the region of interest has first to be converted into DNA using a primer (see below) and the retrovirus enzyme, reverse transcriptase (Section 8.3). If a unique sequence is chosen and there is a positive result, the vi...

 Antibodies are proteins produced by the immune system of higher vertebrates in response to foreign materials (antigens) which those cells encounter. Such antibodies have a region that recognizes and binds specifically to that same antigen. Antibodies are secreted into the body fluids and are most easily obtained from blood. Blood is allowed to clot and antibodies remain in the fluid part (serum) which remains after clotting has removed cells and clotting proteins. This is then known as an antiserum. The principle of identifying infectious virus by using an antibody of known specificity is shown in Fig. 2.6. If the antibody recognizes and binds to the virus, virus infectivity will be inhibited (Fig. 2.6, top line). Infectivity is only one of several virus properties that can be affected by antibody binding, and hence can be monitored in this type of assay. Another is inhibition of the agglutination of red blood cells by virus. This is a property of some viruses, like the influenza ...

  The culture system for growing a virus always consists of living cells, and the choice is outlined in Box 2.1. Which culture system is used depends on the aims of the experiment, for example isolation of viruses, biochemistry of multiplication, structural studies, and study of natural infections. Often a virus is first noticed because it is suspected of causing disease. By definition, disease can only be studied in the whole organism, preferably the natural host. However, this may be ruled out for humans on ethical or safety grounds. Alternatively, organ cultures and cells can be used. Logically, these should be from the natural host and obtained from those sites where the virus multiplies in the whole animal. However, it may be that cells from unrelated animals are susceptible, e.g. human influenza viruses were first cultivated by inoculating a ferret intranasally and found to grow best in embryonated chicken eggs. Usually, viruses grow poorly on initial isolation but a...

Viruses are too small to be seen except by electron microscopy (EM) and this requires concentrations in excess of 1011 particles per ml, or even higher if a virus has no distinctive morphology, some fancy equipment, and a highly skilled operator. Thus viruses are usually detected by indirect methods. These fall into three categories: (i) multiplication in a suitable culture system and detection of the virus by the effects it causes; (ii) serology, which makes use of the interaction between a virus and antibody directed specifically against it; and (iii) detection of viral nucleic acid. However these days the polymerase chain reaction (PCR) is more likely to be employed as it is much quicker provided that the appropriate oligonucleotide primers are available (Section 2.3). Many viruses are uncultivatable, particularly those occurring in the gut, but some of these occur in such high concentration that they were actually discovered by EM. This chapter is not intended to be a technical man...

  The question of the origin of viruses is a fascinating topic but as so often happens when hard evidence is scarce, discussion can generate more heat than light. There are two popular theories: viruses are either degenerate cells or vagrant genes. Just as fleas are descended from flies by loss of wings, viruses may be derived from pro- or eukaryotic cells that have CHAPTER I TOWARDS A DEFINITION OF A VIRUS 15 dispensed with many of their cellular functions (degeneracy). Alternatively, some nucleic acid might have been transferred accidentally into a cell of a different species (e.g. through a wound or by sexual contact) and, instead of being degraded, as would normally be the case, might have survived and replicated (escape). Although half a century has elapsed since these two theories were first proposed, we still do not have any firm indications if either, or both, are correct. Rapid sequencing of viral and cellular genomes is now providing data for computer analysis that is giv...

 With the assumption that the features of virus growth just described for particular viruses are true of all viruses, it is possible to compare and contrast the properties of viruses with those of their host cells. Whereas host cells contain both types of nucleic acid, viruses only contain one type. However, just like their host cells, viruses have their genetic information encoded in nucleic acid. Another difference is that the virus is reproduced solely from its genetic material, whereas the host cell is reproduced from the integrated sum of its components. Thus, the virus never arises directly from a pre-existing virus, whereas the cell always arises by division from a pre-existing cell. The experiments of Hershey and his collaborators showed quite clearly that the components of a virus are synthesized independently and then assembled into many virus particles. By contrast, the host cell increases its constituent parts, during which the individuality of the cell is continuously ...

  One of the easiest ways to understand the steps involved in a particular reaction within an organism is to isolate mutants which are unable to carry out that reaction. Like all other organisms, viruses sport mutants in the course of their growth, and these mutations can affect all properties including the type of plaque formed, the range of hosts which the virus can infect, and the physicochemical properties of the virus. One obvious caveat, however, is that many mutations will be lethal to the virus and remain undetected. This problem was overcome in 1963 by Epstein and Edgar and their collaborators with the discovery of conditional lethal mutants. One class of these mutants, the temperature-sensitive mutants, was able to grow at a lower temperature than normal, the permissive temperature, but not at a higher, restrictive temperature at which normal virus could grow. Another class of conditional lethal mutants was the amber mutant. In these mutants a DNA lesion converts a codon ...

  SIMILAR The growth curves and other experiments described above have been repeated with many animal viruses with essentially similar results. CHAPTER I TOWARDS A DEFINITION OF A VIRUS 13 Treatment with 7mol/L urea Treatment with 7mol/L urea RNA RNA Protein subunits Protein subunits Make hybrid virus Infect plants Harvest virus Strain B virus Strain B virus Strain A virus Strain A virus Fig. 1.5 The experiment of Fraenkel-Conrat and Singer which proved that RNA is the genetic material of tobacco mosaic virus. Bacterial and animal viruses both attach to their target cell through specific interactions with cell surface molecules. Like the T4 bacteriophage, the genomes of some animal viruses (e.g. HIV-1) enter the cell and leave their coat proteins on the outside. However, with most animal viruses, some viral protein, usually from inside the particle, enters the cell in association with the viral genome. In fact it is now known that some phage protein enters the bacterial cells with ...

The first virus was purified in 1933 by Schlessinger using differential centrifugation. Chemical analysis of the purified bacteriophage showed that virus in paracrystalline form, and this crystallization of a biological material thought to be alive raised many philosophical questions about the nature of life. In 1937, Bawden and Pirie extensively purified tobacco mosaic virus and showed it to be nucleoprotein containing ribonucleic acid (RNA). Thus virus particles may contain either DNA or RNA. However, at this time it was not known that nucleic acid constituted genetic material. The importance of viral nucleic acid In 1949, Markham and Smith found that preparations of turnip yellow mosaic virus comprised two types of identically sized spherical particles, only one of which contained nucleic acid. Significantly, only the particles containing nucleic acid were infectious. A few years later, in 1952, Hershey and Chase demonstrated the independent functions of viral ...

  We now know a great deal about the processes which occur during the multiplication of viruses within single cells. The precise details vary for individual viruses but have in common a series of events marking specific phases in the multiplication cycle. These phases are summarized in Fig. 1.3 and are considered in detail in section II of this book. The first stage is that of attachment when the virus attaches to the potential host cell. The interaction is specific, with the virus attachment protein(s) binding to target receptor molecules on the surface of the cell. The initial contact between a virus and host cell is dynamic and reversible, and often involves weak electrostatic interactions. However, the contacts quickly become much stronger with more stable interactions which in some cases are essentially irreversible. The attachment phase determines the specificity of the virus for a particular type of cell or host species. Having attached to the surface of the cell, the virus...

Although methods of assaying viruses had been developed, there were still considerable doubts as to the nature of viruses. d’Hérelle believed that the infecting phage particle multiplied within the bacterium and that its progeny were liberated upon lysis of the host cell, whereas others believed that phage-induced dissolution of bacterial cultures was merely the consequence of a stimulation of lytic enzymes endogenous to the bacteria. Yet another school of thought was that phages could pass freely in and out of bacterial cells and that lysis of bacteria was a secondary phenomenon not necessarily concerned with the growth of a phage. It was Delbruck who ended the controversy by pointing out that two phenomena were involved, lysis from within and lysis from without. The type of lysis observed was dependent on the ratio of infecting phages to bacteria (multiplicity of infection). At a low multiplicity of infection (with the ratio of phages to bacteria no greater than 2 : 1), then the phag...

   Much of the early analytical virus work was carried out with bacterial viruses. Virologists of the time would much rather have worked with agents that caused disease in humans, animals, or crop plants, but the technology was not sufficiently advanced. It is simply not possible to analyze the details of virus growth in whole animals or plants, although viruses could be assayed in whole organisms (see below). Animal cell culture was not a practicable proposition until the 1950s when antibiotics became available for inhibiting bacterial contamination; plant cell culture is still technically difficult. This left bacterial viruses which infect cells that grow easily, in suspension culture, and quickly – experiments with bacterial viruses are measured in minutes, rather than the hours or days needed for animal viruses. The observations of d’Hérelle in the early part of the twentieth century led to the introduction of two important techniques. The first of these was the preparatio...

it is instructive and interesting to consider how this knowledge came about. It was only just over 100 years ago at the end of the nineteenth century that the germ theory of disease was formulated, and pathologists were then confident that a causative microorganism would be found for each infectious disease. Further they believed that these agents of disease could be seen with the aid of a microscope, could be cultivated on a nutrient medium, and could be retained by filters. There were, admittedly, a few organisms which were so fastidious that they could not be cultivated in vitro (literally, in glass, meaning in the test tube), but the other two criteria were satisfied. However, a few years later, in 1892, Dmitri Iwanowski was able to show that the causal agent of a mosaic disease of tobacco plants, manifesting as a discoloration of the leaf, passed through a bacteria-proof filter, and could not be seen or cultivated. Iwanowski was unimpressed by his discovery, but Beijerinck repeate...