Exploring the World of Viruses
Any story about virus research has to begin Nile virus and avian flu. So in society's collective with one simple truism: not all viruses are bad.
It's a tough sell, though. Most people react negatively to the thought of viruses because they've never heard of or experienced a virus in a positive way. The only time a virus truly captures society's attention is when it is wreaking havoc on the human population. In fact, the movement of viruses has become a focal point of fascination and fear, driven by instant accessibility to global news. Even before the H1N1 "swine flu" virus began rooting around in the world's daily headlines, the last decade had brought the world unwelcome encounters with the West consciousness, viruses are bad.
Drs. Marilyn Roossinck and Rick Nelson know that viruses defy such a simple label. As virologists and principal investigators at the Noble Research Institute, they have indeed seen firsthand how viruses can systematically break down an organism. They've also witnessed how viruses can promote life instead of taking it. Most of all, they know the key to combating viruses is in understanding them. Through the past two decades, Roossinck and Nelson have pioneered specific fields of virus research, unraveling the complex processes of how viruses evolve and move. They are distinguishing fact from myth as they learn how viruses impact vital agricultural crops.
In essence, they are exploring the world of viruses. Turns out, it is not such a scary place after all.
Rick Nelson: Virus movement and accumulation
Rick Nelson has spent a career chasing viruses through plants. By understanding how plant RNA viruses move and accumulate, he hopes to mitigate their influence on agriculture. "You can stop disease caused by viruses in three ways," Nelson said. "You can prevent viruses from replicating in the first cell they encounter. You can stop them from moving cell to cell and into other parts of the plant. Or you can mitigate disease symptoms after virus movement and accumulation."
In more than 25 years of research, Nelson has investigated all three phases of virus control. After earning a doctorate in biology from the University of Illinois, Nelson took a postdoctoral fellowship with acclaimed scientist Roger Beachy at Washington University in St. Louis (Beachy was recently appointed as the first director of the National Institute of Food and Agriculture). It was 1985 and research directed toward the production of transgenic plants - the procedure by which genetic information from an organism is inserted and expressed in plants - was just beginning.
Beachy's group cloned a small piece of a virus gene and inserted it into the plant genome. They theorized that the plant would become resistant to the virus, somewhat similar to injecting a human with a viral vaccine. The experiment led to one of the earliest plant transformations in the United States and the first demonstration of transgenically derived virus resistance in plants. "It was like science fiction," Nelson said. "No one had ever done this before. No one had taken a viral gene, put it into a plant and shown its potential for practical usefulness in such a striking way."
The transformation led to another historic event - the first field test of virus-resistant plants. In 1987, Beachy's group collaborated with researchers from Monsanto to grow virus-resistant tomatoes along with tomatoes Monsanto had modified to be Roundup Ready- and caterpillar-resistant (two other technologies that are commercially available today). "We had extraordinary resistance to viruses in the field, but later aspects of this work have been frustrating," Nelson said. "We can produce virus-resistant plants in almost all crops using this and related transgenic technologies developed 20 years ago, but we have yet to take full advantage of this technology because of public resistance to transgenic plants."
Less than a year later, Nelson joined the Noble Research Institute, which had initiated its fundamental plant research a few months prior with the founding of the Plant Biology Division. He continued to build on his postdoctoral work, using a strain of tobacco mosaic virus (TMV) that exhibited mild symptoms in infected plants. TMV is a particularly devastating pathogenic virus so Nelson's initial question was simple: Why is this strain of TMV milder? Ten years later, he found his answer.
Nelson discovered this particular TMV strain possessed 50 substitutions in its 6,400-nucleotide build- ing blocks (molecules that when joined together form DNA or RNA), any one of which could cause the mild effects. Nelson's group took a piece of the mild strain and put it into a severe strain of TMV and vice versa, and observed the disease produced by these chimerical viruses.
The goal was to identify the gene responsible for the mild symptoms. After almost a decade of moving genetic material back and forth, and characterizing the activities of the different viral proteins, they pinpointed a gene that encoded a key protein responsible for moderating the disease symptoms. All plants have a defense system - called an RNA silencing system - that protects them against viruses. It turns out Nelson's protein was responsible for defeating this defense system. In the mild strain of TMV, this protein was naturally defective, resulting in milder disease symptoms since it could not defeat the RNA silencing system. The knowledge gained revealed how other TMV strains operated to damage their plant hosts. "To understand how to inhibit the virus, we had to understand the function of this particular protein," Nelson said. "It was clearly a marker for disease."
The protein became the focal point of Nelson's research while also paving the way for a unique way of studying gene function.
Walk the line
While Nelson's laboratory was identifying this specific TMV protein as a marker for disease, another research group in Japan determined the protein played a role in virus movement between cells. Nelson furthered this concept by verifying the mechanism by which this protein could modulate movement.
Viruses do not have cells of their own so they require a host cell for their replication and movement. They literally must overpower the plant's RNA defense system, all the while accumulating and moving their "virus replication complexes" - usually composed of a ball of RNA and proteins - through the cell. As viruses move cell to cell, they repeat the process thousands of times.
Nelson's group helped determine that many viruses move through the cell on a road made up of protein (actin) building blocks (like bricks that together form a road). As it turned out, the TMV protein (the 126 kDa protein) which was responsible for modulating disease also attached to the actin road.
In addition, Nelson's group determined that a plant protein called myosin, was also required for moving virus replication complexes in the cell. Myosin is the protein that connects the virus replication complex to the actin "road" (microfilament). It is attached to the complex and walks it along the microfilament to the other side of the cell like a child walking on a sidewalk holding a balloon.
Nelson recently determined that a specific myosin (of the 17 known plant myosins) is responsible for moving TMV through the cells in the model plant Nicotiana benthamiana. Additionally, he learned this particular myosin did not assist other viruses. "This tells us that different viruses use different myosins to move across the cell, something like using different brands of cars to travel on highways," Nelson said. "If you can identify the specific myosin the virus requires for movement and control its presence, you should be able to keep that virus from spreading."
Nelson is now looking at two other steps in the process. Once a virus moves across a cell, there is a complex series of chemical interactions that takes place to allow the virus to leave the cell. "It's as though the virus is paying a toll to get out," Nelson said. "We are investigating if we can prevent it from arriving at or getting through this toll gate, which would prevent it from exiting the cell and spreading through the plant."
The group is also exploring the content of the virus replication complex, hoping that its makeup reveals another way of halting virus movement. "By understanding how viruses interact with their host, move and accumulate, we can begin to design ways to make plants resistant to viral infections," he said. "These processes could be combined with other proven strategies to create a potentially impenetrable barrier to viral infection."
Silence of the genes
A serendipitous outcome of Nelson's work with the 126 kDa protein was that it could be used to improve a method to study gene function. Called "virus-induced gene silencing" (VIGS), Nelson's group takes a portion of a gene of interest and inserts it into a virus. The virus will then replicate and accumulate it. The researcher then puts the gene fragment back into the plant by inoculating the plant with the virus. The plant's defenses respond and destroy the virus, but in the process the plant is tricked into "knocking down" or destroying the function of the gene whose fragment was present in the virus.
Researchers can then see what function the target gene controlled. "Maybe the plant's flower turns from purple to white," Nelson said. "In that case, the target gene was necessary for pigment production."
The expression of the 126 kDa protein at low levels actually enhances VIGS. Thus the study of a single viral protein has resulted in multiple findings, helping to define how viruses accumulate through the control of plant defense systems, move within the plant and alter host gene expression.
Currently, the Nelson laboratory is working on a project for the Department of Energy involving ethanol production from cellulosic material such as switchgrass. Scientists are interested in modifying plant cell walls to improve access to sugars for fermentation to ethanol. Many candidate genes have been suggested to be involved in plant cell wall formation, but most have yet to be functionally characterized, and doing this by traditional transformation methods will take many years. "Virus-induced gene silencing has advanced the study of gene function light years," said Richard Dixon, senior vice president and director of the Plant Biology Division. "It provides a rapid method for understanding gene function, which is one of the most vital branches of plant science research today. This, in turn, opens up endless possibilities for plant improvement - a key to sustaining agriculture in the coming generations."
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