Tobacco Mosaic Virus: Pioneering Research for a Century

One century ago, M.W. Beijerinck contended that the filterable agent of tobacco mosaic disease was neither a bacterium nor any corpuscular body, but rather that it was a contagium vivum fluidum (Beijerinck, 1898). Beijerinck's contribution followed A. Mayer's path-breaking work on tobacco mosaic disease and D. Ivanowski's demonstration in 1892 that the agent of tobacco mosaic disease could pass through a filter capable of retaining bacteria (Mayer, 1886; Ivanowski, 1892; Zaitlin, 1998). The claim that viruses, lacking cells, were nonetheless living, set off a scientific debate about the nature of life that animated biology for decades. Tobacco mosaic virus (TMV), as we now know the agent that Beijerinck and others were studying, was the first virus to be identified. Perhaps because of this, research on TMV and other plant viruses has continued to be of profound significance in addressing fundamental questions about the nature of viruses in general. Indeed, TMV as a model system has been at the forefront of virology research to the present time. For example, TMV was the first virus to be chemically purified (Stanley, 1935; Bawden et al., 1936), to be detected in an analytical ultracentrifuge and in an electrophoresis apparatus (Eriksson-Quensel and Svedberg, 1936), and to be visualized in an electron microscope (Kausche et al., 1939). TMV RNA was used in the first decisive experiments showing that nucleic acids carry hereditary information and that nucleic acid alone is sufficient for viral infectivity (Fraenkel-Conrat, 1956; Gierer and Schramm, 1956). The TMV coat protein (CP) was the first virus protein to be sequenced (Anderer et al., 1960; Tsugita et al., 1960), and TMV's particle structure was among the first to be elucidated in atomic detail (Bloomer et al., 1978; Namba et al., 1989). TMV's preeminence has extended into the recombinant era, when the first transgenic plants were constructed using TMV to demonstrate the concept of CP-mediated cross-protection (Abel et al., 1986). TMV was also the first virus shown to encode a cell-to-cell movement protein (MP; Deom et al., 1987). MP binds to RNA (Citovsky et al., 1990), associates with cytoskeletal elements (Heinlein et al., 1995; McLean et al., 1995), and increases the permeability of plasmodesmata to mediate cell-to-cell movement of the virus (Wolf et al., 1989; Waigmann et al., 1994). Several properties of TMV have made it particularly amenable to laboratory investigation. For example, infected tobacco plants produce TMV so abundantly that inclusion bodies of crystallized virions in the infected leaves are visible under the light microscope. Moreover, TMV is not transmitted by insects, nematodes, or other vectors; it infects cells via direct contact with wounded areas on plant surfaces. Virus infection causes disease by preventing chloroplast development, resulting in stunted plants with leaves showing a characteristic mosaic pattern of light and dark green. Furthermore, TMV is remarkably stable: its in vitro longevity in infected sap is 3000 days, and purified virions kept at 5°C remain viable for at least 50 years. Virus stability derives directly from the densely packed structure of the viral particles, which consist of a single genomic RNA molecule enclosed in a cylindrical protein coat. TMV virions have a regular length of 300 nm and a width of 18 nm; these rods comprise a tight array of 2130 identical CP subunits, each containing 158 amino acids. The TMV RNA genome is single stranded and linear, with a length of ~6400 bases. The complete TMV nucleotide sequence was first determined for the U1 strain (Goelet et al., 1982), and comparisons between this sequence and the RNA sequences of other TMV strains have revealed a tightly organized genome that encodes at least three nonstructural proteins (P126, P183, and the 30-kD MP), a putative 54-kD protein of unknown function, and the CP (Figure 1). Both P126 and P183 function as components of the TMV replicase (Palukaitis and Zaitlin, 1986) and are translated directly from the genomic TMV RNA; P183 is produced by read through of the amber termination codon of P126 (Pelham, 1978). In addition, there is a start codon within this read-through region and an open reading frame that could potentially encode the putative 54-kD protein. Translation of MP and CP occurs from the I2 RNA and CP subgenomic (sg) RNAs, respectively (Figure 1). Whereas P126, P183, and CP are continuously expressed (Watanabe et al., 1984), translation of MP is transient, occurring early in the infection process (Joshi et al., 1983; Watanabe et al., 1984). To celebrate the first century of TMV research, scientists from around the world gathered at the Royal College of Physicians of Edinburgh, Scotland on August 7 and 8, 1998, for a symposium sponsored by the Royal Society of Edinburgh in association with The Royal Society, London, UK. The meeting was organized by Professors Bryan D. Harrison and T. Michael A. Wilson (both of the Scottish Crop Research Institute, Dundee, UK) to consider how studies on TMV have contributed to the fundamental knowledge base of biology. A wide diversity of research fields—crystallography, plant pathology, immunology, biochemistry, genetics, and evolutionary biology—was represented at the meeting, with distinguished symposium speakers describing the contributions made by their respective disciplines to our current understanding of TMV biology. The overview they provided made clear that the special status of TMV as a research object has depended on its biological characteristics as well as upon its historical status as a virus of many “firsts.” In the remainder of this report, we describe symposium presentations focused on four central approaches to TMV research—structural biology, genetics and evolution, cell and molecular biology, and biotechnology—and we emphasize the impact that research on TMV has had among the life sciences over the course of the twentieth century. Current Molecular Biological Conception of TMV, as Represented by a Schematic Diagram of its Genome Organization. The genomic RNA is shown on top and the 3′ coterminal subgenomic (sg) RNAs are shown underneath. The predicted molecular weights of proteins encoded by the open reading frames (boxes) are given in kilodaltons, and the functions of individual genes are indicated (the function of the putative 54-kD protein is unknown). The asterisk denotes the amber read-through codon. Current Molecular Biological Conception of TMV, as Represented by a Schematic Diagram of its Genome Organization. The genomic RNA is shown on top and the 3′ coterminal subgenomic (sg) RNAs are shown underneath. The predicted molecular weights of proteins encoded by the open reading frames (boxes) are given in kilodaltons, and the functions of individual genes are indicated (the function of the putative 54-kD protein is unknown). The asterisk denotes the amber read-through codon. The intensive study of the structure of TMV has established it as one of the best-investigated models of macromolecular organization in biology. The classic reconstitution experiments, in which complete TMV was produced in vitro by mixing purified virus RNA and protein subunits (Fraenkel-Conrat and Williams, 1955), demonstrated that the information required for assembly is present in the structural components of the virus. Subsequent studies of the self-assembly of the virus have drawn from the wealth of biophysical and biochemical data on the various stable aggregates of TMV CP, as Donald L.D. Caspar (Florida State University, Tallahassee) noted in his contribution to the symposium. By the late 1950s, M. Lauffer's physicochemical studies of the TMV CP along with Caspar's own titration studies had provided evidence that disks comprising two cylindrical layers of 17 subunits each might serve as important intermediates for the assembly of virus helices (Lauffer et al., 1958; Caspar, 1960). During the ensuing four decades, a great deal of progress has been made in resolving the structure and function of these 34-subunit disks, although the degree to which disks are involved in virus assembly remains controversial. P.J.G. (Jo) Butler (MRC Laboratory of Molecular Biology, Cambridge, UK) recounted the evidence amassed by him, Klug, and their coworkers showing that virus disks are essential for self-assembly. According to their model, nucleation is initiated by the binding of an internal sequence of TMV RNA to a disk, which then dislocates into a helical structure. Other dislocated disks associate with the initiation complex to form nicked helices. Over time, the protein subunits realign and anneal into an uninterrupted helical rod (Butler, 1984). Butler presented kinetic evidence implicating the disks in rod elongation as well as nucleation. Butler and Klug's assertions did not go unchallenged, however. Marc H.V. Van Regenmortel (Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France) pointed out that only polar disks can form helices. The stacked disks produced by most in vitro experiments are bipolar, and thus cannot, in his view, represent intermediates in the assembly process. Recognizing that the problem of TMV self-assembly has not been entirely settled, Klug reminded the audience of A.N. Whitehead's famous dictum, “It is more important that an idea be fruitful than correct.” The desire to ascertain the structure of complete TMV, rather than only of smaller oligomeric subunits, has required the development of innovative crystallographic methods. Gerald Stubbs (Vanderbilt University, Nashville, TN) recalled a 1971 talk on TMV structure by K.C. Holmes that inspired his efforts to push the resolution of TMV structure below 10 Å. The conventional reliance on cylindrical averaging of the fiber diffraction data, although yielding the overall architecture of the virus, had effectively obscured more detailed structural information. Stubbs and his coworkers developed new isomorphous replacement and computational techniques to achieve atomic resolution of intact TMV, providing new structural details of both the RNA and the capsid subunits, and by 1989, they had obtained a 2.9-Å map of TMV (Namba et al., 1989). Stubbs pointed out that the techniques developed to extend the resolution of TMV have been useful for investigating other complex biological structures, including filamentous bacteriophages. As Yoshimi Okada (Teikyo University, Utsunomiya, Japan) reminded those attending the symposium, it was not until the 1950s that most biologists accepted that genes are constructed from nucleic acids. Experiments with TMV played an important role in this development by providing the first unequivocal demonstration that a viral RNA molecule—specifically the TMV RNA—was sufficient for infectivity and carried all of the information necessary for synthesis of the CP (Fraenkel-Conrat, 1956; Gierer and Schramm, 1956). Bea Singer (University of California, Berkeley) recounted how she and H. Fraenkel-Conrat (University of California, Berkeley) extended biochemical research of TMV genetics. They began with naturally occurring TMV strains to demonstrate that the progeny of mixed viruses (i.e., protein from one strain and RNA from another) were true-to-type for the TMV nucleic acid (Fraenkel-Conrat and Singer, 1957). Fraenkel-Conrat and Singer subsequently employed the mutagen nitrous acid (Gierer and Mundry, 1958) to generate novel variants of TMV that were then compared in terms of their nucleic acid content, CP composition, and disease symptoms. Due to the labile nature of the TMV RNA, these were difficult experiments. As Singer recalled, she and Fraenkel-Conrat protected the RNA from cellular RNases by adding the clay bentonite, leading their colleague C.A. Knight to remark that he “wouldn't put that mud in his stuff.” Singer asserted that her work with Fraenkel-Conrat represented the true beginning of chemistry applied to virology. However, one might well point out that this work drew on concurrent developments in bacteriology and bacterial genetics, beginning with research performed a decade earlier at the Rockefeller Institute, where Avery and his coworkers biochemically demonstrated the “transforming principle” of Streptococcus to be DNA. With the elucidation of the complete CP sequence in 1960 (Anderer et al., 1960; Tsugita et al., 1960) the collection of TMV mutants provided clues used to crack the genetic code. Only the startling development of cell-free translation systems the following year by H. Matthei and M. Nirenberg provided a less laborious means to decipher this code (reviewed in Kay, 1998), and even then TMV mutants were used to confirm the emerging codon dictionary. The TMV mutants shed light on other biological questions as well. As Singer also noted, almost all the mutants attributed to the nitrous acid treatment were less “fit” than was wild-type TMV, an observation suggestive of later developments in the arenas of virus diversity and evolution. Milton Zaitlin (Cornell University, Ithaca, NY) recalled how advances in molecular genetic techniques enabled researchers in the 1970s and 1980s to construct a detailed map of the TMV genome. Indeed, a significant clue to the genetic composition of TMV RNA came from studies in Zaitlin's laboratory showing that a low molecular–weight component termed sgRNA accumulated during viral infection (Jackson et al., 1972). This sgRNA was soon implicated as the mRNA that directs CP production (Hunter et al., 1976). By the mid-1970s, Zaitlin's group had correctly, albeit tentatively, placed the replicase-encoding gene at the 5′ end, the CP-encoding gene at the 3′ end, and a gene necessary for viral movement in the central portion of the genome (Beachy et al., 1976; see Figure 1). Nishiguchi, Okada, and coworkers (Nishiguchi et al., 1978; Ohno et al., 1983) then confirmed that the 30-kD MP was encoded by TMV using TMV strain L and a temperature sensitive variant, Ls-1. Several reverse genetics studies using infectious clones, first assembled in 1986 (Dawson et al., 1986; Meshi et al., 1986), have confirmed the gene functions assigned during these earlier studies. The initiation of TMV infection and disassembly of the TMV virion was reviewed by John G. Shaw (University of Kentucky, Lexington). Having entered its host cell, the TMV virion must remove its CP to enable viral replication. Shaw presented one model describing how this uncoating might take place bi-directionally, proceeding both from the 5′ and the 3′ ends of the TMV genomic RNA molecule. The 5′-to-3′ uncoating reaction may be cotranslational (Wilson, 1984), which would result in disassembly by a ribosome-mediated mechanism and concomitant protection of the uncoated viral RNA from cellular nucleases. Shaw suggested that the 3′-to-5′ uncoating reaction might occur in a coreplicational manner, because viral replicase mutants that are defective in 3′-to-5′ disassembly can be uncoated in plant by adding viral RNA with an intact replicase gene and studies of TMV infection at the molecular the of with virus replication. College of and London, UK) the process of TMV and he noted that although the viral proteins involved in TMV are well the of host is the of their association with the viral replicase two host proteins that might be involved in TMV RNA which with the replicase and a of which with the In addition, genetic approaches to that have to the of the and mutants of and the of in which TMV is genetic studies with have also RNA viruses have the to more than viruses and TMV a of genetic University, compared sequences of of tobacco and TMV with those from infected in that these demonstrate that there has been in the genetic diversity of in over the years. Moreover, the in TMV to be because has the more tobacco virus in in that et al., from as as and to be of one world with This remarkably the viral genome remains as a result of In other there to be a of TMV sequence of which the host to and this is In this TMV to be from other viruses, as and which of nucleotide The TMV early were to from with have to into the of their and these for their own studies of are not only for understanding of the viral life and the of viral but they also shed light on cellular as the of gene and molecular TMV has as the system of for these of Indeed, as Harrison noted, Bawden and first suggested in that virus might be to the synthesis of cellular Several speakers at the symposium demonstrated that this has continued to research on plant cell biology. State University, viral functions involved in TMV from cell to cell through plasmodesmata until it the which The of TMV through the may be a occurring with the of In cell-to-cell movement between virus components and are in the of TMV by the MP (Figure which to permeability and to of viral genomic RNA through these TMV can also mediate the movement of viruses, as which are to the host and Research into the of TMV to virus cell-to-cell movement has focused on the TMV and this research has contributed to our understanding of plant of his that the TMV MP binds to nucleic and he that the MP directly to viral RNA in a to on to the of a tobacco cell protein which binds to TMV MP are involved in and these were shown to be required for viral movement and of plasmodesmata et al., Waigmann et al., 1994). also that the of MP on permeability is by of and its (Citovsky et al., with the of a single gene in termed which the of TMV et al., 1998), and he suggested that this might be useful in efforts to the of molecular movement in (University of an overview of by researchers during the century to the causes of with TMV recalled that Beijerinck first that TMV a disease of a that was by Bawden in the when he to of TMV infection and disease 1939). symposium with his to the of with an of about to may not be a direct of virus but rather can result from the that viral of the may to molecular genetic studies showing that the mosaic and is on the amino acid composition of the TMV replicase proteins and the by the and in TMV biology with the from cellular and molecular of more Donald the contributions that TMV research has made the mechanism of host a that the of on the one and on the reminded the audience that TMV in transgenic plants to be a process in which the of CP subunits the disassembly of TMV et al., a of reverse genetics and experiments in laboratory are beginning to shed light on the structural involved in CP-mediated the of his laboratory the 1980s have the for other for have in our understanding of and that have provided for many researchers with transgenic of an on studies of the which The significance of this upon the demonstration that the transgenic of the gene into plants the to a et al., is a single gene in that of the a role in and she the of molecular between the of the gene and the viral replicase protein. The of in the 1980s and has TMV research as Wilson pointed For example, the TMV sequence has been to the translation of et al., 1987). of progress in the development of virus it that TMV is a leading role in the of various to viruses as for genes in the current in TMV not only as a model but also as a The of TMV in infected plants and the stability of the virus to its for in production of that has used TMV to produce of and and that are in new in for the of proteins from tobacco One could not to upon the and of tobacco leaves for when it is in advances the of research on TMV, from its in an to its most University, The that one century the of over tobacco and the emerging of disease Beijerinck's of TMV its scientific impact the of virology as a TMV researchers a virus and the twentieth on this model virus has scientists to a understanding of both life and Current developments in TMV research are The of the TMV the molecular of of genetic and the of viruses as molecular represent and potentially of investigation. in this of for molecular TMV research its status as a to our understanding of biology. The research through from the to the of to the of to to and to the Research and to and the to and to might be to know that a collection of on contributions to the meeting be under the Research for a on Royal an of TMV One of to by and M. Zaitlin is for in the of by the Society