Image: University of Stuttgart/Sabine Eiben

Plant Viruses – beyond Good and Evil

Researchers show their work to Nobel Prize winners

Virus research at the University of Stuttgart paves the way into the next era: Viruses may lend wings to nanotechnology in future.

They're tiny, erect shells that make architects green with envy, and can still destroy their surroundings. A study in contrasts: plant virologist Dr. Katharina Hipp of the University of Stuttgart's Institute of Biomaterials and Biomolecular Systems (BIO) and its Department of Molecular Biology and Plant Virology studies the African cassava mosaic virus, a cassava plant parasite, while her colleague Dr. Sabine Eiben finds some good qualities in the tobacco mosaic virus. Viruses may lend wings to nanotechnology in future as a basis for sensors, a support framework for tissue substitutes, or even in cancer diagnostics.

Katharina Hipp has been fascinated by geminiviruses, since the day when she listened as a young student to a lecture at the Department of Molecular Biology and Plant Virology at the Institute for Biomaterials and Biomolecular Systems. Her eyes glow as she gently rotates and tilts a model of the cassava mosaic virus, a member of the geminiviruses, between her fingers. The model, a parting gift from a former doctoral student, was printed out on a 3D printer according to the high-definition structure the two had worked out from many blurred electron microscopic  projection images of a virus particle.

"What sets geminiviruses off from all other viruses is their protein envelope, which forms two incomplete icosahedra – whence the Latin name 'gemini', for 'twin'" explains biologist Hipp, who is now a post-doctoral researcher in the department headed by Holger Jeske. "Normally, we would expect these twin pairs to break apart easily, but that doesn't happen here." This still surprises Hipp. Viruses normally appear as rods, nearly spherical polyhedra, or – as here – as individual icosahedra.

Even though the geminiviruses are only about 20 to 35 nanometers in diameter (one nanometer = one millionth of a millimeter) and are thus pygmies among the viruses, and get by with only a minimum set of proteins, they cause considerable damage to many crop plants, primarily in the earth's tropical and subtropical zones. The African cassava mosaic virus initially betrays its presence by a "mosaic" pattern of light and dark-green areas on the leaves of the infected cassava plant, after which the entire plant wastes away. To make her point, Hipp says, "The cassava root is part of the basic diet of many in Africa and Southeast Asia, where it's as important as the potato once was for us." Many small farmers cultivate cassava behind their houses to feed their families. If the harvest fails due to a virus, the situation is dramatic for the people there."

Katharina Hipp with a model of the cassava mosaic virus. On the monitor in the background you can see the 3D reconstruction, which was made with an electron microscope.
Katharina Hipp with a model of the cassava mosaic virus. On the monitor in the background you can see the 3D reconstruction, which was made with an electron microscope.
Photo: Helmine Breitmaier

Understanding how it works

Whiteflies, which are endemic in warm zones, transmit the cassava virus from one plant to another by sucking their juices. As 39-year-old Hipp warns, "If climatic warming continues, these insect transmitters of geminiviruses could spread to the more temperate latitudes and bring many viruses with them." Even today, crop damage is found in Spain and Italy, for example, from the "tomato yellow leaf curl virus", a geminivirus which attacks tomatoes and paprika plants. Like the African cassava mosaic virus, it is one of the "top ten" most important plant viruses.

Hipp also points to a solution: "We can only prevent the virus from spreading if we understand how it works." As a biologist she wants to know how the 110 identical envelope proteins of the cassava mosaic virus interact with each other in order to form stable particle twins for transporting the viral genotype. Her tactic is to use a computer to analyze at which location on an electron microscopically reconstructed 3D-structure the individual amino acid building blocks of the envelope proteins are located. Hipp explains: "If I can find out which areas of the envelope proteins are important for this, I might be able to strategically infiltrate mutations that would prevent the virus from forming and transmitting particles."

Another point of attack against the virus might be to replicate the genetic information which it introduces into the plant cell. In doing so, the virus is dependent upon the host cell. Virologist Hipp has conducted experiments with yeast cells and found that a certain area of replication-associated proteins from the cassava mosaic virus trigger the cell to multiply the foreign genotype. But many questions about the structure and dissemination of geminiviruses remain to be answered. Hipp has a goal: "It would be wonderful if our basic research could help in developing a way to counter these viruses." In future, Hipp will be heading the electron microscope section of the Max-Planck Institute for Developmental Biology in Tübingen.

From parasite to nano-tool

In contrast to geminiviruses, tobacco mosaic viruses are among the best researched of all viruses. In fact, the first proof that viruses exist came at the end of the 19th century with the finding that tobacco mosaic disease is triggered by germs which pass through bacteria-proof filters. Today, Sabine Eiben even finds a lot that's good, precisely in the tobacco mosaic viruses: they can be useful nanobiotechnological tools. Eiben is a post-doctoral student and team leader in the "NanoBioMater" Project House of the Carl-Zeiss Foundation and the University of Stuttgart and works in the research group of Prof. Christina Wege, in the same department as Katharina Hipp. Eiben's group tests tobacco mosaic virus-based nanostructures for different applications - a difficult task, since such structures are technically difficult to produce from biological material.

It sounds like a simple kitchen recipe: mix the genotype, a ribonucleinic acid strand, with the envelope proteins of the tobacco mosaic virus, and presto! Protein rings appear almost magically and package the genotype into a 300 nanometer-long tube. Then the virus substance can be manipulated to create new kinds of structures: tobacco mosaic viruses of different lengths which can be bent like boomerangs or made to branch out like stars. "We can even put together virus-like particles in a test tube that no longer have an infectious viral genotype," reports Eiben. As always, however, the most effective producers are the tobacco plants that flourish in the greenhouse on the roof of Natural Sciences Center II on the University of Stuttgart's Stuttgart-Vaihingen campus, where Sabine Eiben studies how viruses infect the plants via defects in their leaves. Eiben is working here with basic elements of nature's nano-toolbox by triggering bacteria to produce tobacco mosaic virus envelope proteins or genetically modifying these proteins so that enzymes, peptides or nanoparticles bind to them, which allows biologist Eiben to assign new functions to these virus-like particles. For example, her work team recently showed that when coupled with enzymes, virus particles can greatly improve the action of biosensors in detecting glucose. As Eiben explains, "The viruses stabilize the enzymes and increase the surface area, resulting in a high level of sensitivity."

Sabine Eiben (on the left) and Christina Wege (in the center) presenting their research during the Lindau Meeting of Nobel Laureates.
Sabine Eiben (on the left) and Christina Wege (in the center) presenting their research during the Lindau Meeting of Nobel Laureates.
Photo: Staatsministerium Baden-Württemberg / Uli Regenscheit

Different functions

Theoretically, a functional group, such as an enzyme, can bind to each of the more than 2,000 envelope proteins of the tobacco mosaic virus. "What's more, we can combine different envelope protein variants in a virus particle in such a way as to spatially separate different functional areas from each other," says Eiben triumphantly. Whether this will someday take the place of the conventional blood sugar measurement swabs used today remains to be seen, says Eiben; but new, more sensitive biosensors will be eagerly welcomed in other areas too.

This might include, for example, virus-based sensors which can detect drug residues or toxic agents in foodstuffs or environmental samples. And for field effect transistors like those found as switches in every computer, Eiben has also produced virus particles upon which electrically conductive metal oxides are deposited even at room temperature. Previously this required temperatures above 400 degrees centigrade, so that only temperature-resistant materials were feasible as transistor boards. The gas-sensitive metal oxide layer might make it possible in future to use such virus-bearing field effect transistors to detect, for example, methane in the surrounding air.

Eiben is working in the interdisciplinary NanoBioMater Project House with materials scientists, technology specialists, and chemists to learn more about the uses of tobacco mosaic viruses in hydrogels with regard to biocompatible materials - for example, to produce tissue substitutes. It might be possible to use the viruses as a support framework for cells and growth factors or to precipitate calcium phosphate, the basic substance in our skeleton.

A virus like a rock star

The tobacco mosaic virus continues to inspire Sabine Eiben with new ideas for its application, but also in the form of artistic images, like those hanging above her office desk. This year, Eiben was privileged to present, together with her boss Christina Wege, her task force's research at the year-end Nobel Prize Winners' Meeting at the invitation of the Competence Network for Functional Nanostructures of the Land of Baden-Württemberg, which gave the two their own booth at the round trip by ship on the Lake of Constance on July 1. Eiben fondly remembers the friendly, pleasant atmosphere on that occasion: "Although the conference was devoted this year to physics, we were visited by a great many people who were interested in our biological work in the border area between physical, chemical and technical applications." For her part, she stands out as nearly unique in the world of research as one of the world's few plant virologists in the area of nanotechnology. Helmine Braitmaier

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