This project encompasses the determination of the three-dimensional molecular structures of a number of flexible filamentous plant viruses. It is part of a comprehensive, integrated structural analysis of the filamentous plant viruses, aimed at explaining the processes of viral infection, dissemination, and host interactions, and understanding the relationships among these important viruses. The viruses to be studied include members of the potexvirus, potyvirus, and closterovirus groups. The potexviruses are of great significance as models for fundamental virology and cell biology; they have enormous potential in biotechnology, and they are of considerable importance for the damage they cause in agriculture. The potyviruses are responsible for half the viral crop damage in the world, and are of interest because of their close relationship to the animal rhinoviruses (responsible for such diverse conditions as the common cold, polio, and foot-and-mouth disease). The closteroviruses are also of major agricultural significance. To utilize the full potential of the flexible filamentous viruses as models and in biotechnology, and to combat their effects in agriculture, knowledge of three-dimensional structures in atomic detail is required. Apart from this group's preliminary results, there are no three-dimensional structures known for any flexible filamentous virus. Virus structures to be determined will include the most important of the potexviruses, potato virus X, as well as the potyviruses soybean mosaic virus and wheat streak mosaic virus and the closterovirus beet yellows virus. Structures will be determined by cryo-electron microscopy and X-ray fiber diffraction; models derived from cryo-electron microscopy will be used as starting points for more detailed analysis using fiber diffraction data. Fiber diffraction specimen preparation methods developed in this laboratory have greatly improved the quality of data from potexviruses and potyviruses; before this project began, no useful diffraction data had been obtained from potyviruses.
Broader impacts will be through the integration of research and education, and through the application of this research to new developments in crop protection and plant biotechnology, particularly in the production of pharmacologically useful products. This laboratory has a strong program of involvement of undergraduates and K-12 teachers in research, and the project activities will include undergraduates, graduate students, and K-12 teachers and students.
This project concerns the determination of the three-dimensional structures of filamentous plant viruses, representing almost half of the known plant viruses. Filamentous plant viruses are important because of the enormous crop losses they cause, because they have the potential to be useful in the biotechnological production of useful molecules such as vaccines, hormones, and antibiotics, and because they can be considered as model systems for the filamentous components of much more difficult to study human viruses such as influenza and HIV. Knowledge of three-dimensional molecular structures is essential for all of these applications. Our principal tools for structural studies are X-ray fiber diffraction and cryo-electron microscopy. The pattern of X-rays scattered from a filamentous structure such as a fiber made from virus particles (Fig. 1) is mathematically related to the molecular structure. Images from low-temperature (cryo) electron microscopy (Fig. 2) are much more directly related to molecular structures, but each image contains random experimental errors, and thousands of images must be combined in a reconstruction to obtain an interpretable molecular image. We have combined the two approaches very effectively in our studies. In 2008, at the beginning of this project, we had established that the two most important families of filamentous plant viruses, the potyviruses (the most agriculturally devastating of all the plant viruses, responsible for half the viral crop damage in the world) and the alphaflexiviruses (including the agriculturally extremely important potexviruses) had very similar molecular structures, and can therefore be studied together with great effect for the purposes discussed in the previous paragraph. The potyviruses include potato virus Y (PVY) and soybean mosaic virus (SMV), while the alphaflexiviruses include the potexvirus potato virus X (PVX), which reduces world potato production by 10% every year. In coinfections with PVY, PVX can reduce crop yields by 90%. In a study of seven potyviruses, we showed that there is considerable variation in the helical symmetry and pitch (distance over which the molecular structure is regularly repeated), similar to the alphaflexiviruses, but in strong contrast to the well-studied rigid tobamoviruses. This variation is most likely to be a consequence of the flexibility of the potyviruses and the alphaflexiviruses. We reconstructed the structures of three potexviruses, PVX (previously determined by us, but this time with increased detail), papaya mosaic virus and narcissus mosaic virus (Fig. 3). These reconstructions resolved questions about the symmetry of PMV and NMV, and suggested a structural basis for the interactions between the protective coat proteins and the viral genomic RNA. We also reconstructed the structure of a potyvirus, wheat streak mosaic virus (WSMV) (Fig. 4), an important pathogen of wheat. WSMV belongs to the genus Tritimovirus, and is the first tritimovirus to yield to structural studies. We reconstructed the structure of a hordeivirus, barley stripe mosaic virus (BSMV) (Fig. 5). Our reconstruction defined the architecture (helical symmetry and distance parameters) of BSMV for the first time, and supported a structural relationship with the tobamoviruses. A key outcome of this project has been its importance in training undergraduates in research. Eight undergraduates worked on this project; four have already been co-authors of one or more published papers, and almost all of them are now in MD, PhD, or MD/PhD programs at major American universities. This project is the continuation of a series of projects, during which over 30 undergraduates received research training; over 20 of these graduated from Vanderbilt with Honors; every one of those Honors students went on to medical or graduate school, and either completed a postgraduate degree or is currently doing so. Students from the earlier years include current university faculty members and independent researchers.
