Atlas Of Plant Viruses

Volume I

Robert G. Francki R.I.B; Milne

• Publisher: CRC Press
• Year: 2018
• ISBN:
  9781351086851
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Botany
• Index: Biological Sciences

This book assembles a comprehensive collection of plant virus electron micrographs of good quality, offers a consistent treatment, and backs the visual data with a consistent and comprehensive text. Although this book is primarily about the structure of virus particles and infected cells, the results of biochemical experiments are referred too when relevant, so that the virus particles described appear as part of a replicating complex. Similarly, infected cells are portrayed as active rather than static structures.

Chapter 1

Introduction

Who first “discovered” viruses or developed the concept “virus” as we understand it today is perhaps an idle question, as the idea took form over a number of decades.¹,² Flower-breaking in tulips was familiar in the middle of the 16th century, and by 1719 it was well established, at least in some circles, that flower-breaking in jasmine could be graft-transmitted.³ In 1883, Mayer¹ made a significant contribution when he found that tobacco mosaic virus (TMV) was sap-transmissible, and Ivanovski¹ in 1892 made another with his discovery that TMV was small enough to pass a Chamberland filter candle that prevented passage of ordinary bacteria. Although this discovery was important, Ivanovski did not appear to realize it, for he at first favored a bacterial toxin as the disease agent, and later suggested that it was a bacterium.² It was left to Beijerinck⁴ at the turn of the century to develop the concept that viruses differ basically from bacteria. Nevertheless, his choice of the term contagium vivum fluidum, or living infectious liquid, was unfortunate and probably hindered his ideas being taken seriously for some time.

In the mid 1930s research activity on viruses gained impetus when TMV was purified more or less independently in the U.S., Britain, and Australia^5–9 and was found to be a nucleoprotein.⁶ Various physical and chemical methods were used to investigate the size and shape of TMV particles. (Particles by now had superseded fluid, but for a long time arguments, that we now see as empty, continued about whether or not viruses were “living.”) Even before TMV was purified, it was concluded from rather ingenious stream double refraction experiments that the particles were rod-shaped.¹⁰ Subsequently, using crystalline purified virus preparations, X-ray diffraction studies confirmed that TMV particles were rod-shaped and that they were made up of regularly arranged, uniform subunits.⁶ Furthermore, it was concluded that the particles were about 18 nm in diameter and at least ten times as long.⁶ However, it was not until 1939 that the blindfold was finally removed from the eyes of researchers when a virus was examined in the electron microscope. TMV again had the distinction of being the first virus to be studied by this technique.¹¹

Although of obvious potential, electron microscopy was initially slow to contribute to virology. In the early days, the cost of the tricky and capricious instrument represented a disproportionate amount of the modest budgets of virus laboratories. Suitable specimen preparation methods had also to be developed before full advantage of the electron microscope could be taken. Today, most virologists, whether researchers or diagnosticians, would consider an electron microscope as one of the most basic instruments in their laboratories. Without it, they would feel inadequate to detect, identify, or characterize the viruses they work with as well as the impurities and contaminants they hope to avoid.

I. Virus Particle Structure

Early electron micrographs of isolated virus particles were not very informative because of the low contrast between particle and background. This is because neither proteins nor nucleic acids are opaque to the electron beam. However, even without any enhancement of image contrast, some valuable work was done. Kausche and his colleagues” confirmed the shape of the TMV particle and later Stanley and Anderson¹² obtained accurate size distributions of rods in purified virus preparations. Viruses with isometric particles were also examined, but the resolution was rather poor and little was achieved other than dispelling the then-prevailing view based on hydrodynamic data that particles of all viruses were anisometric.¹³

Electron microscopy of viruses was revolutionized when Williams and Wyckoff^14-17 introduced metal shadowing as a technique for increasing contrast. The method proved excellent for revealing the shapes and sizes of virus particles, and where the particle surface needs to be observed, metal shadowing yields unambiguous images uncomplicated by information coming from deeper layers of the particle. However, much of the fine detail is hidden due to the layer of metal deposited and the granularity of the resulting surface.

The introduction of negative staining in the 1950s^18-20 was an advance in technique as significant as metal shadowing. The images of virus particles could suddenly begin to be interpreted in molecular terms.²¹ The method largely replaced the more laborious and generally less informative shadow casting, but there are instances where shadowed preparations can be at least as informative as those negatively stained. Hatta and Francki²² used both methods to study Fiji disease virus particles, and found that some aspects of these complex structures were easier to understand from shadowed preparations. The presence of impurities is also best detected by shadowing.¹⁶

Negative staining has proved so useful because it was found to outline virus particles with nearly structureless electron-dense material, often also penetrating the surface to give contrast to the morphological subunits. Perhaps nearly as important, it was found to support the particle, to a greater or lesser extent, against the flattening and distortion experienced during drying.

Negative staining was introduced at a time when much interest in virus particle structure had been generated following the purely theoretical considerations on the subject by Crick and Watson.²³ These workers suggested that since viruses with small particles had limited amounts of nucleic acid, their protein shells must be built of numerous identical subunits. There was already evidence that this was so with some viruses from X-ray crystallographic and chemical data.²⁴ In a paper accompanying that by Crick and Watson,²³ it was shown by X-ray crystallography that the protein shell of the tomato bushy stunt virus particle consists of subunits arranged with 5:3:2 symmetry.²⁵

Examination of negatively stained particles of many plant, animal, and bacterial viruses by Home and Wildy²⁶ caused great excitement. This was especially so for viruses with isometric particles because what had previously been seen as roughly spherical blobs became intricate structures belonging to a number of geometrical families. (Though a large particle, that of tipula iridescent virus, had already been elegantly shown by shadowing to have icosahedral form.²⁷) With hindsight, it is ironic that the first manuscript fully describing the negative staining method was rejected by a leading virological journal. The circumstances of the rejection have been described in a review by Horne and Wildy.²⁸ Fortunately, the editors soon realized the error of their ways and in 1961 the same journal, in expiation, published an unusually long and interesting paper on virus architecture based on the results of negative staining.²⁶ The extent to which the negative stain technique contributed to the understanding of virus structure in a very short period of time can be gauged from a review written less than 4 years after introduction of the technique.²⁹

The method of Brenner and Horne,²⁰ using neutralized solutions of phosphotungstic acid (PTA) was in fact so successful that it acquired elements of dogma. Many workers, particularly those in the medical field, used and still use neutral PTA exclusively and somewhat indiscriminately: this, despite the fact that Hall¹⁸ had originally used PTA at pH 4.6, Huxley and Zubay³⁰ had introduced uranyl acetate, and Home at various times has emphasized that other negative stains have merit. As early as 1963 it was reported by Markham in a personal communication to Home and Wildy²⁹ that some plant viruses disrupt in neutral PTA. It appears that one type of virus unstable in the stain is that whose stability depends on electrovalent bonding between protein and RNA. The problem can usually be overcome by fixing the virus, lowering the pH of the PTA, or using a stain such as unbuffered uranyl acetate.³¹,³²

With enveloped viruses, PTA can produce artifacts of another kind in that the particles become distorted. The cause of this is unknown but it has been suggested that at least with the Rhabdovirus, lettuce necrotic yellows virus, osmotic and imbibition effects are responsible.³³ The exact shapes assumed by the stained particles depend largely on the pH of the PTA. At neutrality the bacilliform particles become bullet-shaped due to the invagination of the envelope at one end. The effect of the pH of PTA on the particle morphology of lettuce necrotic yellows virus has been discussed in detail.³⁴,³⁵

It has been our experience that unbuffered uranyl acetate (pH ~4.2) is a better general negative stain for plant viruses and has been used for the majority of the micrographs in this book. Uranyl formate,³⁶ though otherwise behaving like uranyl acetate, offers a definite advantage in revealing the fine structure of rod-shaped viruses. However, staining artifacts have been encountered when uranyl acetate was used to stain Rhabdoviruses.³⁷,³⁸ It should also be mentioned that there is a potential danger of obtaining positively stained particles because of the affinity of uranyl ions for nucleic acids. This is not a problem unless staining times are inordinately long or the support film is very hydrophobic.

In addition to PTA and uranyl acetate, solutions of sodium tungstate and tungstoborate, lithium tungstate, ammonium molybdate, lanthanum acetate, and uranyl formate have been used³⁹ as well as methylamine tungstate, sodium silicotungstate, uranyl oxalate, and sodium zirconium glycollate. The subject of negative staining often provokes fierce brand loyalties among microscopists, sometimes obscuring the fact that an ideal negative stain has not yet been discovered. There is good reason to try out new heavy metal salt formulations, but these should give exceptional results, repeatable in other laboratories, and should not just be novelties, in order to replace the generally satisfactory portfolio of PTA at neutral and low pH, uranyl acetate, uranyl formate, and ammonium molybdate.

One of the most serious problems encountered when examining virus particles in the electron microscope is that biological objects (and their negative-stain images) exposed to the electron beam are very readily burnt up, with a consequent drastic loss of structural detail. This was recognized by Williams and Fisher,⁴⁰ who devised the minimal exposure technique and showed that using it can lead to significantly improved capture of fine detail. The procedure is very simple in that part of the specimen near the region of interest, rather than the actual field to be photographed, is used for focusing and other adjustments. This leaves the important region unirradiated until everything is ready for the photograph to be taken. The method presents problems where the subject is unique or occurs rarely on the support film. In this case the necessary preliminary search can be made at very low illumination. Wrigley and his colleagues⁴¹ have recently suggested refinements to the minimal exposure technique.

Although electron images of negatively stained particles possess high contrast, their resolution is not very good when compared to that attainable by modern electron microscopes. To extract more information, a number of techniques have been d...

Table of contents

• Cover
• Title Page
• Copyright Page
• Contents
• Chapter 1 Introduction
• Chapter 2 Caulimovirus Group
• Chapter 3 Geminivirus Group
• Chapter 4 Plant Reoviridae
• Chapter 5 Plant Rhabdoviridae
• Chapter 6 Tomato Spotted Wilt Virus Group
• Chapter 7 Maize Chlorotic Dwarf Virus Group
• Chapter 8 Tymovirus Group
• Chapter 9 Luteovirus Group
• Chapter 10 Sobemovirus Group
• Chapter 11 Tobacco Necrosis Virus Group
• Chapter 12 Tombusvirus Group
• Virus Abbreviations and Taxonomic Groups
• Index