Studies Of Tobacco Mosaic Virus Research Articles

Tobamoviruses as Models for the Study of Virus Evolution.

The study of tobacco mosaic virus and other tobamovirus species has greatly contributed to the development of all areas of virology, including virus evolution. Research with tobamoviruses has been pioneer, or particularly significant, in all major areas of research in this field, including: the characterization of the genetic diversity of virus populations, the mechanisms and rates of generation of genetic diversity, the analysis of the genetic structure of virus populations and of the factors that shape it, the adaptation of viruses to hosts and the evolution of host range, and the evolution of virus taxa and of virus-host interactions. Many of these continue to be hot topics in evolutionary biology, or have been identified recently as such, including (i) host-range evolution, (ii) predicting the overcoming of resistance in crops, (iii) trade-offs between virus life-history traits in virus evolution, and (iv) the codivergence of viruses and hosts at different taxonomical and spatial scales. Tobamoviruses may be particularly appropriate to address these topics with plant viruses, as they provide convenient experimental systems, and as the detailed knowledge on their molecular and structural biology allows the analysis of the mechanisms behind evolutionary processes. Also, the extensive information on parameters related to infection dynamics and population structure may facilitate the development of realistic models to predict virus evolution. Certainly, tobamoviruses will continue to be favorite system for the study of virus evolution.

Chapter 1 - A history of viral infections of the central nervous system: foundations, milestones, and patterns

For millennia, infectious diseases were viewed as the result of malignant supernatural forces or punishments by angry gods for human transgressions. In the 19th century several advances occurred. Medicine became based on clinical observations; Rudolph Virchow laid the foundation of cellular pathology; Louis Pasteur dismantled the notion of spontaneous generation, and Robert Koch established the fundamental techniques of bacteriology, together creating the revolutionary paradigm shift of germ theory. Virology was born in the last decade of the 19th century in the study of tobacco mosaic virus; the crucial characteristics were the capacity to pass through a bacteria-retaining filter and the need for living cells in which to replicate. In 1901 yellow fever was shown to be the first human disease to be caused by a “filterable virus.” In 1903 rabies would be the first disease of the nervous system to be determined to be viral in nature, and polio followed soon thereafter. Paradoxically, because no agent has yet been shown to be the cause, the modern study of epidemic encephalitis began with encephalitis lethargica in 1917. However, the flood gates of viral isolations opened in the 1930s for the arboviral encephalitides, using experimental primates and mice, starting with St. Louis encephalitis and Japanese encephalitis. Within a decade, the viral nature of aseptic meningitis and herpes simplex sporadic encephalitis was demonstrated. From the 1950s through the 1970s great interest in “slow viral diseases” arose as potential models for chronic diseases of the nervous system. The era of modern neurovirology has been characterized by two principal features. The first is the remarkable advances in technology – witness the use of polymerase chain reaction (PCR) for identifying infectious agents, magnetic resonance imaging (MRI) to image the nervous system, and the advance in therapeutics based on molecular medicine. The second has been the impact of emerging and re-emerging diseases – witness the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) epidemic, Nipah encephalitis, and the appearance of West Nile encephalitis in the western hemisphere.

Biobehavior in Normal and Tumor-Bearing Mice of Tobacco Mosaic Virus

Viral nanoparticles (VNPs) have shown great potential as platforms for biomedical applications. Before using VNPs for further biomedical applications, it is important to clarify their biological behavior in vivo, which is rare for rod-like VNPs. In this paper, a study of tobacco mosaic virus (TMV), a typical rod-like VNP, is performed on blood clearance kinetics, biodistributions in both normal and tumor-bearing mice, histopathology and cytotoxicity. TMV was radiolabeled with (125)I using Iodogen method for in vivo quantitative analysis and imaging purpose. In the normal mice, the accumulation of TMV in the immune system led to a rapid blood clearance. The uptake of TMVs in the liver was less than that in the spleen, which is opposite to the results observed in the case of spherical VNPs. No signs of overt toxicity were observed in examined tissues according to the results of histological analysis. In addition, similar biodistribution patterns were observed in U87MG tumor-bearing mice.

Structure of viruses: a short history

This review is a partially personal account of the discovery of virus structure and its implication for virus function. Although I have endeavored to cover all aspects of structural virology and to acknowledge relevant individuals, I know that I have favored taking examples from my own experience in telling this story. I am anxious to apologize to all those who I might have unintentionally offended by omitting their work. The first knowledge of virus structure was a result of Stanley's studies of tobacco mosaic virus (TMV) and the subsequent X-ray fiber diffraction analysis by Bernal and Fankuchen in the 1930s. At about the same time it became apparent that crystals of small RNA plant and animal viruses could diffract X-rays, demonstrating that viruses must have distinct and unique structures. More advances were made in the 1950s with the realization by Watson and Crick that viruses might have icosahedral symmetry. With the improvement of experimental and computational techniques in the 1970s, it became possible to determine the three-dimensional, near-atomic resolution structures of some small icosahedral plant and animal RNA viruses. It was a great surprise that the protecting capsids of the first virus structures to be determined had the same architecture. The capsid proteins of these viruses all had a 'jelly-roll' fold and, furthermore, the organization of the capsid protein in the virus were similar, suggesting a common ancestral virus from which many of today's viruses have evolved. By this time a more detailed structure of TMV had also been established, but both the architecture and capsid protein fold were quite different to that of the icosahedral viruses. The small icosahedral RNA virus structures were also informative of how and where cellular receptors, anti-viral compounds, and neutralizing antibodies bound to these viruses. However, larger lipid membrane enveloped viruses did not form sufficiently ordered crystals to obtain good X-ray diffraction. Starting in the 1990s, these enveloped viruses were studied by combining cryo-electron microscopy of the whole virus with X-ray crystallography of their protein components. These structures gave information on virus assembly, virus neutralization by antibodies, and virus fusion with and entry into the host cell. The same techniques were also employed in the study of complex bacteriophages that were too large to crystallize. Nevertheless, there still remained many pleomorphic, highly pathogenic viruses that lacked the icosahedral symmetry and homogeneity that had made the earlier structural investigations possible. Currently some of these viruses are starting to be studied by combining X-ray crystallography with cryo-electron tomography.

From Virus Structure to Chromatin: X-ray Diffraction to Three-Dimensional Electron Microscopy

Early influences led me first to medical school with a view to microbiology, but I felt the lack of a deeper foundation and changed to chemistry, which in turn led me to physics and mathematics. I moved to the University of Cape Town to work on the X-ray crystallography of some small organic compounds. I developed a new method of using molecular structure factors to solve the crystal structure, which won me a research studentship to Trinity College Cambridge and the Cavendish Laboratory. There I worked on the austenite-pearlite transition in steel. This is governed by the dissipation of latent heat, and I ended up numerically solving partial differential equations. I used the idea of nucleation and growth during the phase change, which had its echo when I later tackled the assembly of Tobacco mosaic virus (TMV) from its constituent RNA and protein subunits. I wanted to move on to X-ray structure analysis of large biological molecules and obtained a Nuffield Fellowship to work in J.D. Bernal's department at Birkbeck College, London. There, I met Rosalind Franklin, who had taken up the study of TMV. I was able to interpret some of Franklin's beautiful X-ray diffraction patterns of the virus particle. From then on, my fate was sealed. After Franklin's untimely death in 1958, I moved in 1962 to the newly built MRC Laboratory of Molecular Biology in Cambridge, which, under Max Perutz, housed the original MRC unit from the Cavendish Laboratory. I was thus privileged to join the Laboratory at an early stage in its expansion and consequently able to take advantage of, and to help build up, its then unique environment of intellectual and technological sophistication. There I have remained ever since.

Validation of microtubule‐associated Tobacco mosaic virus RNA movement and involvement of microtubule‐aligned particle trafficking

Functional studies of Tobacco mosaic virus (TMV) infection using virus derivatives expressing functional, dysfunctional, and temperature-sensitive movement protein (MP) mutants indicated that the cell-to-cell transport of TMV RNA is functionally correlated with the association of MP with microtubules. However, the role of microtubules in the movement process during early infection remains unclear, since MP accumulates on microtubules rather late in infection and treatment of plants with microtubule-disrupting agents fails to strongly interfere with cell-to-cell movement of TMV RNA. To further test the role of microtubules in TMV cell-to-cell movement, we investigated TMV strain Ni2519, which is temperature-sensitive for movement. We demonstrate that the temperature-sensitive defect in movement is correlated with temperature-sensitive changes in the localization of MP to microtubules. Furthermore, we show that during early phases of recovery from non-permissive conditions, the MP localizes to microtubule-associated particles. Similar particles are found in cells at the leading front of spreading TMV infection sites. Initially mobile, the particles become immobile when MP starts to accumulate along the length of the particle-associated microtubules. Our observations confirm a role for microtubules in the spread of TMV infection and associate this role with microtubule-associated trafficking of MP-containing particles in cells engaged in the cell-to-cell movement of the TMV genome.

Tritium planigraphy comparative structural study of tobacco mosaic virus and its mutant with altered host specificity

Spatial organization of wild-type (strain U1) tobacco mosaic virus (TMV) and of the temperature-sensitive TMV ts21-66 mutant was compared by tritium planigraphy. The ts21-66 mutant contains two substitutions in the coat protein (Ile21-->Thr and Asp66-->Gly) and, in contrast with U1, induces a hypersensitive response (formation of necroses) on the leaves of plants bearing a host resistance gene N' (for example Nicotiana sylvestris); TMV U1 induces systemic infection (mosaic) on the leaves of such plants. Tritium distribution along the coat protein (CP) polypeptide chain was determined after labelling of both isolated CP preparations and intact virions. In the case of the isolated low-order (3-4S) CP aggregates no reliable differences in tritium distribution between U1 and ts21-66 were found. But in labelling of the intact virions a significant difference between the wild-type and mutant CPs was observed: the N-terminal region of ts21-66 CP incorporated half the amount of tritium than the corresponding region of U1 CP. This means that in U1 virions the CP N-terminal segment is more exposed on the virion surface than in ts21-66 virions. The possibility of direct participation of the N-terminal tail of U1 CP subunits in the process of the N' hypersensitive response suppression is discussed.

Вивчення вірусу тютюнової мозаїки за допомогою атомно-силового мікроскопу

Вивчення вірусу тютюнової мозаїки за допомогою атомно-силового мікроскопу

A comparative differential scanning calorimetric study of tobacco mosaic virus and of its coat protein ts mutant

The differential scanning calorimetry (DSC) ‘melting curves’ for virions and coat proteins (CP) of wild-type tobacco mosaic virus (strain U1) and for its CP ts mutant ts21–66 were measured. Strain U1 and ts21–66 mutant (two amino acid substitutions in CP: I21 → T and D66 → G) differ in the type of symptoms they induce on some host plants. It was observed that CP subunits of both U1 and ts21–66 at pH 8.0, in the form of small (3–4S) aggregates, possess much lower thermal stability than in the virions. Assembly into the virus particles resulted in a DSC melting temperature increase from 41 to 72°C for U1 and from 38 to 72°C for ts21–66 CP. In the RNA-free helical virus-like protein assemblies U1 and ts21–66 CP subunits had a thermal stability intermediate between those in 3–4S aggregates and in the virions. ts21–66 helical protein displayed a somewhat lower thermal stability than U1.

Infection of Plants by Tobacco Mosaic Virus

Plant virus diseases cause an estimated $15 billion crop loss worldwide (Matthews 1993). Plant viruses may infect organisms as diverse as fungi and maple trees. They exist in several different sizes and shapes, contain single or double-stranded RNA or DNA, and are transmitted mechanically and/or by an insect or arthropod (Figure 1). Disease symptoms arising from virus infection may be invisible (latent), small localized lesions (Figure 2A), or quite dramatic, such as the mosaic disease of tobacco (Figure 2B). While plant viruses are often economically destructive, they are otherwise harmless to humans and animals. In fact, information gleaned from the study of tobacco mosaic virus (TMV) has led to landmark discoveries in the field of science. Although many scientists and educators are aware of the events surrounding the discovery of the ubiquitous TMV, few are aware that the study of the biological and physicochemical properties of TMV provided a strong foundation in the basic sciences for later development of the field of molecular biology (Fraenkel-Conrat 1986). Tobacco mosaic virus, the type member (standard for comparison) of the tobamovirus group, is one of the world's most thoroughly studied viruses. The rod-shaped TMV particle (Figure 3) is 300 nanometers in length (0.000012 inch). Nearly 1700 particles could fit end-to-end to span the diameter of the period at the end of this sentence. It is composed of subunits of one coat protein that protectively en-

Increased CSF and brain quinolinic acid in schizophrenia and bipolar disorder

For millennia, infectious diseases were viewed as the result of malignant supernatural forces or punishments by angry gods for human transgressions. In the 19th century several advances occurred. Medicine became based on clinical observations; Rudolph Virchow laid the foundation of cellular pathology; Louis Pasteur dismantled the notion of spontaneous generation, and Robert Koch established the fundamental techniques of bacteriology, together creating the revolutionary paradigm shift of germ theory.Virology was born in the last decade of the 19th century in the study of tobacco mosaic virus; the crucial characteristics were the capacity to pass through a bacteria-retaining filter and the need for living cells in which to replicate. In 1901 yellow fever was shown to be the first human disease to be caused by a “filterable virus.” In 1903 rabies would be the first disease of the nervous system to be determined to be viral in nature, and polio followed soon thereafter.Paradoxically, because no agent has yet been shown to be the cause, the modern study of epidemic encephalitis began with encephalitis lethargica in 1917. However, the flood gates of viral isolations opened in the 1930s for the arboviral encephalitides, using experimental primates and mice, starting with St. Louis encephalitis and Japanese encephalitis. Within a decade, the viral nature of aseptic meningitis and herpes simplex sporadic encephalitis was demonstrated. From the 1950s through the 1970s great interest in “slow viral diseases” arose as potential models for chronic diseases of the nervous system.The era of modern neurovirology has been characterized by two principal features. The first is the remarkable advances in technology – witness the use of polymerase chain reaction (PCR) for identifying infectious agents, magnetic resonance imaging (MRI) to image the nervous system, and the advance in therapeutics based on molecular medicine. The second has been the impact of emerging and re-emerging diseases – witness the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) epidemic, Nipah encephalitis, and the appearance of West Nile encephalitis in the western hemisphere.

Immunochemical studies of tobacco mosaic virus—V. Localization of four epitopes in the protein subunit by inhibition tests with synthetic peptides and cleavage peptides from three strains

Two antigenic determinants have been located in the vicinity of residues 1–10 and 34–39 of the coat protein of tobacco mosaic virus by means of ELISA and complement fixation inhibition experiments with synthetic peptides. The determinant present in residues 1–10 is also expressed at the outer surface of the subunits when they are assembled into virions. Two other determinants expressed in the dissociated protein subunit have been located in the vicinity of residues 55–61 and 80–90 by means of inhibition tests with tryptic peptides. These results bring to seven the number of antigenic determinants of the protein subunit that have been located.

Studies of virus structure by laser Raman spectroscopy. IX. Structural studies of tobacco mosaic virus and its components by laser Raman spectroscopy

Raman spectra have been recorded for oriented gels of tobacco mosaic virus, aqueous solutions of TMV protein in the form of capsid and disk, and protein-free TMV RNA. The protein-free RNA molecule contains about 80% ordered type-A structure, which is similar to other RNA molecules in solution. RNA in the virus particle does not exhibit the base stacking and pairing normally found in protein-free RNA but instead shows at least two distinct structures characterized by different phosphodiester group frequencies in the Raman spectrum. Approximately one out of three such linkages retains the type-A geometry characteristic of aqueous RNA, while two out of three appear to have a different geometry not encountered previously for RNA. The secondary structure of the TMV coat protein molecule is similar for the three aggregation states studied (virus, capsid, and disk) and consists of 40-50% alpha helix, 40-50% irregular structure, and 0-20% beta sheet. Two of the three tryptophan residues per protein molecule reside in hydrophobic regions, and the third is in contact with water. The distribution of the four tyrosine residues per protein molecule among hydrogen-bonding states is the same for capsid and virus but different for the disk. The latter could be a consequence of the presence of Cl- ions which may form hydrogen bonds with one or more p-hydroxyl groups of tyrosine residues.

Immunochemical studies of tobacco mosaic virus—III.: Demonstration of five antigenic regions in the protein sub-unit

The antigenic determinants of the protein sub-unit of tobacco mosaic virus have been studied by inhibition of complement fixation experiments. The viral sub-unit has been found to possess five epitopes. Three of them situated in tryptic peptides 1.4 and 8 are cryptotopes, i.e. epitopes absent from the outer surface of the assembled viral capsid. Two other epitopes situated in tryptic peptides 1 and 12 are located in a region that is recognized by both anti-sub-unit and anti-viral antibodies. When the results are analyzed in terms of the three-dimensional structure of the viral sub-unit, it appears that all the antigenic reactive regions occupy highly accessible locations on the surface of the protein.

Protein‐RNA interactions during TMV assembly

A review of the structural studies of tobacco mosaic virus (TMV) is given. TMV is essentially a flat helical microcrystal with 16 1/3 subunits per turn. A single strand of RNA runs along the helix and is deeply embedded in the protein. The virus particles form oriented gels from which high-resolution X-ray fiber diffraction data can be obtained. This may be interpreted by the use of six heavy-chain derivatives to give an electron density map at 0.4 nm resolution from which the RNA configuration and the form of the inner part of the protein subunit may be determined. In addition, the protein subunits form a stable 17-fold two-layered disk which is involved in virus assembly and which crystallizes. By the use of noncrystallographic symmetry and a single heavy-atom derivative, it has been possible to solve the structure of the double disk to 0.28 nm resolution. In this structure one sees that an important structural role is played by four alpha-helices, one of which (the LR helix) appears to form the main binding site for the RNA. The main components of the binding site appear to be hydrophobic interactions with the bases, hydrogen bonds between aspartate groups and the sugars, and arginine salt bridges to the phosphate groups. The binding site is between two turns of the virus helix or between the turns of the double disk. In the disk, the region proximal to the RNA binding site is in a random coil until the RNA binds, whereupon the 24 residues involved build a well-defined structure, thereby encapsulating the RNA.

Immunochemical studies of tobacco mosaic virus—I: Refutation of the alleged homogeneous binding of purified antibody fragments

Immunochemical studies of tobacco mosaic virus—I: Refutation of the alleged homogeneous binding of purified antibody fragments

Immunochemical studies of tobacco mosaic virus—I: Refutation of the alleged homogeneous binding of purified antibody fragments

Binding experiments with tobacco mosaic virus and specific Fab fragments were analysed by means of two types of Scatchard plot. The first type, which makes no assumption regarding antigen valence, yielded a series of curves which, when extrapolated, revealed that the effective antigenic valence of the virus was 800. The various plots obtained with different Fab concentrations showed that the affinity constants of TMV antibodies depended on antibody concentration, and they did not substantiate earlier claims that TMV antibodies are homogeneous. The second type of Scatchard plot, which is commonly used for monovalent haptens, yielded a series of lines, the slope of which varied with Fab concentration. The linearity of these plots appears to be an artefact produced by the large antigen valence of tobacco mosaic virus, and does not indicate homogeneity of association constants. The forward binding reaction between Fab and TMV is practically irreversible and is largely dependent on an increase in entropy. It appears that previous workers who investigated this system and reported that anti-TMV antibodies are homogeneous were misled because they assumed an incorrect antigenic valence and used equations that are unsuitable for antigens with a very large valence.

Direct visualization of the RNA location in cucumber green mottle mosaic virus and tobacco mosaic virus protein disk

The location of RNA in cucumber green mottle mosaic virus and tobacco mosaic virus protein disks was visualized by a negative staining method as a narrow ring localized at a radius of 4 nm, which corresponds to the location of RNA obtained by X-ray diffraction studies of tobacco mosaic virus. The same ring-shaped stains were observed in the end views of helical rods prepared in acidic solutions from viral protein without RNA. Since such a ring-shaped image could not be observed in end views of natural particles and reconstituted particles composed of protein and RNA, the narrow ring was concluded to indicate the RNA location on the basis of X-ray analysis.

An Apparatus for the Measurement of Dichroism in a Pulsed Electric Field

An instrument is described for the measurement of the dichroism produced in solutions of macromolecules by pulsed electric fields. Measurements can be made with monochromatic radiation in the wavelength range from 240 to 600 nm. The pulse generation equipment employed is similar to that used by previous workers but the optics and signal detection system are novel and allow a direct measurement of the dichroism. The relation between the output signal of the apparatus and the dichroism of the sample is derived. Representative experimental data from a study of tobacco mosaic virus are presented to demonstrate the performance of the apparatus. Limitations of the instrument are noted and possible improvements are discussed.

Continuous filter paper electrophoresis of tobacco mosaic virus

1. The technique of continuous electrophoresis as applied to a study of tobacco mosaic virus (TMV) has been described.2. Virus isolated by differential centrifugariton from plants mixedly infected with two strains of TMV has been separated into two components with this technique. The components corresponded in biological activity to the strains used to infect the plants.3. The biological activity per unit protein of one strain (U2) was unchanged by continuous electrophoresis. The other strain was not analysed in this way.4. TMV purified directly from leaf homogenates by continuous electrophoresis was an inferior preparation when compared with TMV purified by differential centrifugation.5. The low molecular weight protein associated with TMV infection (soluble antigen) has been purified and was found to be similar in ultra-violet absorption spectrum to the nucleic acid-free protein obtained by the degradation of TMV.6. The usefulness of continuous electrophoresis as a technique for the purification of plant viruses and related proteins has been discussed.