Tobacco mosaic virus: the first century

Tobacco Mosaic Virus: Pioneering Research for a Century, organized by The Royal Society of Edinburgh, in conjunction with The Royal Society, was held at The Royal Society of Edinburgh, UK, 7–8 August 1998.     

Tobacco mosaic virus virulence and avirulence

In celebration of a century of research on tobacco mosaic virus that initiated the science of virology, I review recent progress relative to earlier contributions concerning how viruses cause diseases of plants and how plants defend themselves from viruses.     

Tobacco mosaic virus RNA as genetic determinant: genesis of a discovery.

It is generally held that the American geneticists Alfred Hershey and Martha Chase were the first to elucidate, in 1952, the genetic functions of phage DNA. The discovery of the genetic functions of RNA in a plant virus (Tobacco mosaic virus, TMV) is commonly attributed to the American plant virologist Heinz Fraenkel-Conrat, and to the Germans Alfred Gierer and Gerhard Schramm, who came to the same conclusion independently in 1956. In reality, the first understandings dated back to about 1940, when several scientists discovered that TMV infectivity was closely related to the presence of undamaged RNA in the virus particles. A very important but underestimated contribution came from the English group of Roy Markham, Kenneth Smith and Richard Matthews in 1948. This group purified and characterized an isometric plant virus, Turnip yellow mosaic virus, and first showed that virus infectivity depended on the presence of the RNA, concluding that nucleic acid was essential for virus multiplication. This finding was confirmed by the same group one year later but it laid neglected. After a five year period, in which several groups attempted to solve the question of the function of TMV RNA, the American electron microscopist Roger Hart offered, in 1955, further direct evidence which correlated RNA to TMV infectivity. One year later, three research groups (Fraenkel-Conrat; Gierer and Schramm; Max Lauffer, David Trkule and Anne Buzzell) obtained evidence that put an end to the question, which was (and is) fundamental to molecular Genetics because it demonstrated that RNA can function independently of DNA.     

Contact transmission of Tobacco mosaic virus: a quantitative analysis of parameters relevant for virus evolution

Transmission between hosts is required for the maintenance of parasites in the host population and determines their ultimate evolutionary success. The transmission ability of parasites conditions their evolution in two ways: on one side, it affects the genetic structure of founded populations in new hosts. On the other side, parasite traits that increase transmission efficiency will be selected for. Therefore, knowledge of the factors and parameters that determine transmission efficiency is critical to predict the evolution of parasites. For plant viruses, little is known about the parameters of contact transmission, a major way of transmission of important virus genera and species. Here, we analyze the factors determining the efficiency of contact transmission of Tobacco mosaic virus (TMV) that may affect virus evolution. As it has been reported for other modes of transmission, the rate of TMV transmission by contact depended on the contact opportunities between an infected and a noninfected host. However, TMV contact transmission differed from other modes of transmission, in that a positive correlation between the virus titer in the source leaf and the rate of transmission was not found within the range of our experimental conditions. Other factors associated with the nature of the source leaf, such as leaf age and the way in which it was infected, had an effect on the rate of transmission. Importantly, contact transmission resulted in severe bottlenecks, which did not depend on the host susceptibility to infection. Interestingly, the effective number of founders initiating the infection of a new host was highly similar to that reported for aphid-transmitted plant viruses, suggesting that this trait has evolved to an optimum value.     

Tobacco mosaic virus, not just a single component virus anymore

Taxonomy: Tobacco mosaic virus (TMV) is the type species of the Tobamovirus genus and a member of the alphavirus-like supergroup. Historically, many tobamoviruses are incorrectly called strains of TMV, although they can differ considerably in sequence similarities and host range from each other and from TMV. Physical properties: TMV virions are 300 × 18 nm rods with a central hollow cavity ( Fig. 1 ) and are composed of 95% capsid protein (CP), and 5% RNA. Each CP subunit interacts with 3-nts in a helical arrangement around the RNA. Virions are stable for decades; infectivity in sap survives heating to 90 °C.

Figure 1 Open in figure viewer PowerPoint Electron micrograph of TMV virions stained with uranyl acetate. Courtesy of Dr J.N. Culver, University of Maryland Biotechnology Institute.     

Tobacco mosaic virus as a carrier for small molecules: Artificial receptor antibodies and superhormones

Peptide agonists covalently attached to tobacco mosaic virus exhibit such unusual properties as superpotency, superaffinity, enhanced resistance towards enzymic degradation, and prolonged action at the target cell. These properties can be exploited for the isolation by density-gradient centrifugation of membrane vesicles bearing specific receptors for the peptides and for radioactive and fluorescent labeling of cell-surface receptors. Our observations can be explained by cooperative–affinity phenomena caused by the deployment in space of the agonist molecules.     

Tobacco mosaic virus as a new carrier for tumor associated carbohydrate antigens

Tumor-associated carbohydrate antigens (TACAs) are being actively studied as targets for antitumor vaccine development. One serious challenge was the low immunogenecity of these antigens. Herein, we report the results of using the tobacco mosaic virus (TMV) capsid as a promising carrier of a weakly immunogenic TACA, the monomeric Tn antigen. The copper(I) catalyzed azide-alkyne cycloaddition reaction was highly efficient in covalently linking Tn onto the TMV capsid without resorting to a large excess of the Tn antigen. The location of Tn attachment turned out to be important. Tn introduced at the N terminus of TMV was immunosilent, while that attached to tyrosine 139 elicited strong immune responses. Both Tn specific IgG and IgM antibodies were generated as determined by enzyme-linked immunosorbent assay and a glycan microarray screening study. The production of high titers of IgG antibodies suggested that the TMV platform contained the requisite epitopes for helper T cells and was able to induce antibody isotype switching. The antibodies exhibited strong reactivities toward Tn antigen displayed in its native environment, i.e., cancer cell surface, thus highlighting the potential of TMV as a promising TACA carrier.     

Detection of Tobacco mosaic virus and Tomato mosaic virus in pepper and tomato by multiplex RT-PCR

Aims: To develop a highly sensitive and rapid protocol for simultaneous detection and differentiation of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) in pepper and tomato. In this study, we use the multiplex PCR technique to detect dual infection of these two viruses. Methods and Results: A multiplex RT–PCR method consisting of one‐tube reaction with two primer pairs targeted to replicase genes was developed to simultaneously detect TMV and ToMV in seed samples of pepper and tomato. Specific primers were designed from conserved regions of each of the virus genomes, and their specificity was confirmed by sequencing PCR products. RT–PCR detected up to 10^?6 dilution of total RNA extracted from infected leaves. Multiplex RT–PCR revealed the presence of both TMV and ToMV in three of 18 seed samples of tomato and one of 18 seed samples of pepper. Conclusions: The multiplex PCR assay was a cost effective, quick diagnostic technique, which was helpful in differentiating TMV and ToMV accurately. Significance and Impact of the Study: The multiplex PCR assay described in this study is a valuable tool for plant pathology and basic research studies. This method may facilitate better recognition and distinction of TMV and ToMV in both pepper and tomato.     

Tobacco mosaic virus movement protein-mediated protein transport between trichome cells.

Tobacco mosaic virus movement protein (TMV MP) is required to mediate viral spread between plant cells via plasmodesmata. Plasmodesmata are cytoplasmic bridges that connect individual plant cells and ordinarily limit molecular diffusion to small molecules and metabolites with a molecular mass up to 1 kD. Here, we characterize functional properties of Nicotiana clevelandii trichome plasmodesmata and analyze their interaction with TMV MP. Trichomes constitute a linear cellular system and provide a predictable pathway of movement. Their plasmodesmata are functionally distinct from plasmodesmata in other plant cel types; they allow cell-to-cell diffusion of dextrans with a molecular mass up to 7 kD, and TMV MP does not increase this size exclusion limit for dextrans. In contrast, the 30-kD TMV MP itself moves between trichome cells and specifically mediates the translocation of a 90-kD beta-glucuronidase (GUS) reporter protein as a GUS::TMV MP fusion. Neither GUS by itself nor GUS in the presence of TMV MP moves between cells. These data imply that a plasmodesmal transport signal resides within TMV MP and is essential for movement. This signal confers selectivity to the translocated protein and cannot function in trans to support movement of other molecules.     

Tobacco mosaic virus movement protein functions as a structural microtubule-associated protein

The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.     

Tobacco mosaic virus and the study of early events in virus infections

In order to establish infections, viruses must be delivered to the cells of potential hosts and must then engage in activities that enable their genomes to be expressed and replicated. With most viruses, the events that precede the onset of production of progeny virus particles are referred to as the early events and, in the case of positive-strand RNA viruses, they include the initial interaction with and entry of host cells and the release (uncoating) of the genome from the virus particles. Though the early events remain one of the more poorly understood areas of plant virology, the virus with which most of the relevant research has been performed is tobacco mosaic virus (TMV). In spite of this effort, there remains much uncertainty about the form or constituent of the virus that actually enters the initially invaded cell in a plant and about the mechanism(s) that trigger the subsequent uncoating (virion disassembly) reactions. A variety of approaches have been used in attempts to determine the fate of TMV particles that are involved in the establishment of an infection and these are briefly described in this review. In some recent work, it has been proposed that the uncoating process involves the bidirectional release of coat protein subunits from the viral RNA and that these activities may be mediated by cotranslational and coreplicational disassembly mechanisms.