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Adding Function to Structure
When Molecular Biology Professor John Johnson started working with cowpea mosaic virus (CPMV) in 1978, he was aggressively pursuing what was then one of the cutting-edge problems in structural biology—solving the complete structure of an intact virus. In 1986, when Johnson published the first complete structure of CPMV, it was one of the first such structures solved. Johnson was concerned then with the relationship of structure to function. How is the viral genome packaged inside the viral capsid (shell), and how does that shed light on how the virus works? Now, decades later, Johnson knows the structure of CPMV very well, and he is asking how the virus can be made to work for us. In recent years, he has collaborated with Dr. Tianwei Lin, Assistant Professor of Molecular Biology, and Dr. George Lomonossoff of the John Innes Institute in England to change the genetic makeup of the virus to modify the capsid proteins and change a few amino acids on the outside of the virus. More recently, Johnson has collaborated with two other TSRI researchers, M.G. Finn of the Department of Chemistry and The Skaggs Institute for Chemical Biology and Marianne Manchester of the Department of Cell Biology. These researchers have been able to attach a wide range of molecules to the CPMV surface, essentially enhancing the virus with the properties of those molecules. This has led to a program, which Johnson, Lin and Finn are pursuing, in molecular electronics—aiming to create logic elements out of viral particles. And, with Manchester, they have been experimenting with adding proteins and peptides to the virus surface to create viral warheads that can attack infectious agents, like measles. "We never in our wildest dreams imagined that [the virus] would have these kinds of applications when we started working on it," says Johnson. Anatomy of a Cowpea VirusCowpea mosaic virus withers and stunts the leaves and pods of the Vigna unguiculata plant—an important crop and source of protein in many parts of the world. Like most plant viruses, CPMV is delivered by insects into plant cells, and like most plant viruses, CPMV has little need for its viral envelope to facilitate entry into cells. All these envelopes are, basically, are a rigid, stable container—shells. The shell of a CPMV particle is some 30 nanometers in diameter and is formed by 60 identical copies of a viral protein surrounding a single strand of viral RNA. These 60 copies constitute 60 equivalent sites for attaching molecules through molecular genetics. With molecular genetics Johnson and Lin have developed a general technique for inserting particular amino acids of interest onto the surface of the virus by making relatively conservative mutations in a loop of viral protein on the outside of the virus. The loop can tolerate different amino acid sequences without altering the basic structure of the virus. In fact, by replacing a few amino acids like threonine and serine with cysteines, the researchers have been able to make minimal variations to the capsid architecture while putting these highly reactive groups on the surface of the virus. These cysteine-containing groups can then be used to attach other molecules. All of Johnson's years working on the structure of CPMV help him direct the mutations to specific sites on the viral surface. "We know what we are changing," says Johnson. In what he calls a wonderful "Scrippsian story," Finn describes the beginning of his collaboration with Johnson as the day they sat down a few years ago to look at Johnson's crystal structures. "I was agog," says Finn. "And as soon as I got it into my head that the viruses were obtainable in gram quantities and the crystal structures were known, I immediately began to think of them as molecules." Molecules to a chemist are also molecular subunits—scaffolds upon which higher order molecules can be built—and treating the virus particles as molecular subunits meant that these viruses could be used to build higher order structures. Finn immediately proposed that he and Johnson collaborate. "My laboratory is fortunate enough to have some funds from the Skaggs Institute for Chemical Biology, so we had some resources available that we could put to this immediately," says Finn. "That was crucial." In a recent study by the two laboratories, Qian Wang, Tianwei Lin, Liang Tang, Johnson, and Finn reported the first results showing that CPMV particles can be used as chemical scaffolds. Through chemical manipulations, the team attached fluorescent dyes and clusters of gold molecules to the cysteine residues because the dyes and the gold clusters could be easily imaged. The study was a proof-of-principle—an aperitif for the more hearty applications that they are working on at the moment. A particularly tantalizing one is to build circuits of conducting molecules on the surfaces of the viruses to form a component of a molecular-scale computer—a new type of "nanowire." Molecular Electronics"The ultimate goal in this part of the program," says Finn, "is to create virus particles that have a function that is useful in electronic or computational applications." The primary advantage of a viral wire would be one of scale, potentially reducing the size of logic elements by orders of magnitude. Another potential advantage would be cost. Because the materials are biological, they could possibly be constructed through self-assembly. The home run, Finn says, would be to engineer a virus particle to be a logic element in a circuit—in other words, to lay down conducting material on the surface of the virus in a pattern that allows one to probe at one end of the virus and get an answer at the other end. But, he adds, they are nowhere near there yet. Johnson and Finn are currently working on the preliminary problem of mastering control over the conductive properties of the virion. Viruses are natural insulators, and the researchers are attempting to turn them into not just conductors, but conductors that can be asymmetrically patterned and connected. Crystallizing the particles could potentially give larger circuitry. The crucial first step will be to see if the researchers can make contact points on the surfaces of the CPMV particles with elemental gold and then connect these gold contact points with conducting organic molecules in order to make molecular circuits. Another possible application the researchers are pursuing is blocking viral infection. Attacking a Virus with a VirusManchester, like Finn and Johnson, comes to the collaboration from a diverse past and sees in CPMV a potential fountainhead of applications that address her interests, which range from understanding how viruses attach to and enter cells to developing new antiviral agents and vaccines. Manchester is particularly interested in the measles virus. Measles is a highly infectious virus that causes a maculopapular rash, fevers, diarrhea, and, in one to two cases out of a thousand, death. Measles is also highly contagious, and until the advent of mandatory vaccination programs in the United States, there were an estimated three to four million cases annually. Some 90 percent of the U.S. population had had measles by the age of 15. "It's basically the most contagious infectious agent there is," says Manchester. There has been a commercially available vaccine for measles in use since 1963, and, though effective, this vaccine is expensive and must be kept refrigerated for the duration of its one-year shelf life. This is problematic in tropical climates like southern Asia and sub-Saharan Africa, which continue to support endemic measles infection and millions of cases a year. The viral receptors that facilitate the entry of measles into cells are known, and one of these receptors, called CD46, is of particular interest to Manchester. "We have done a lot of studies to characterize the binding of the virus to the outer part of the receptor," says Manchester. CD46 is expressed on virtually all cells in the body, and the measles virus has a hemagglutinin glycoprotein that binds to a single, broad surface on one side of CD46. The area of CD46 that measles binds to is quite large, and this has allowed Manchester to make a series of peptides that correspond to the different regions to which measles binds and test the peptides for efficacy against measles infection. "We asked whether they could prevent the virus from infecting by competing for binding," says Manchester. "And they did." Since the peptide bound to the virus, preventing the virus from binding to CD46, Manchester and Johnson wondered what would happen if they could introduce these peptides onto the surface of the cow pea mosaic virus using the same general technique. Could the peptide expressed on the surface of the virus attack the measles just as the free peptide had? It could. "What we did was to take this peptide that we knew could inhibit the binding and infection of measles and display it on the surface of the plant virus," says Manchester. What Manchester and Johnson found was that not only were they able to protect cells from measles infection, but they were able to do so with at least 100-fold greater efficacy than with the peptide alone, and Manchester was able to prevent infection in vivo. The secret of the CPMV's success, they believe, is its polyvalency—the fact that it displays multiple copies of the anti-measles peptide. "When the [cowpea mosaic] virus comes in contact with the measles virus, it's not just bringing one copy, but 60 copies," says Johnson. Moreover, the hemagglutinin molecule to which the peptides bind is a trimer and so binding is favorable. And they could potentially attach more than one peptide to the cow pea mosaic virus and target measles with even higher efficacy. The same approach might be used to create a vaccine in the traditional sense by putting antigen molecules from measles or some other virus that would stimulate an immune response to block an infection from a later exposure. One of the great advantages of using such an approach is its frugality—the virus does the work of making the peptide. This is an advantage for achieving Manchester's ultimate goal of making new vaccines, since one of the main criteria for any globally effective vaccine is its price. Another preferential feature for a vaccine's success is its bioavailability, since delivering a vaccine to remote regions is made much more difficult if specialized equipment or training is required. And Manchester says that the modified CPMV looks reasonably orally bioavailable, based on some preliminary studies she completed with Finn. Finn attached fluorescent dyes to the viral particles to see where they go in vivo and how long they last in tissues, and Manchester found that, indeed, the CPMV molecules are distributed throughout the organism. Now she and Finn are trying to make further improvements to this distribution by attaching polyethylene glycol molecules to the virions to dampen the immune response to them. "If you coat a protein with polyethylene glycol, it tends to dramatically reduce its visibility to the immune system," says Johnson. Bringing their Collaboration to the New CenterFinn, Johnson, Lin and Manchester are all part of the new Center for Integrative Molecular Biosciences (CIMBio) faculty and have laboratory space in the newly constructed CarrAmerica B building. CIMBio was designed to be the most advanced biological microscopy center in the world and to provide an environment in which the expertise and resources of many research groups could be combined. Finn and Johnson provide a service to the center by providing tailored CPMV for the molecular microscopes and for the new automated processes. Since the structure is so well determined, it makes a good test bed to determine how well the electron microscopes are doing. Finn will also conduct an independent program on basic research into new labels for electron microscopy. In EM, heavy atom labels are routinely attached to the particular molecules of interest in order to image these molecules. Currently, there are only a few commercially available labels. "That's just not versatile enough for the kinds of applications this center is going to deal with," says Finn, adding that the result of his research into new labels will be a specialized chemistry service for researchers using the CIMBio facilities. "We want biologists and biochemists to come to us with a problem, 'Here's a protein I need to label with a heavy atom residue' or 'I tried what's available and it doesn't work so well.' That's partly what we're here to do." For the most part, though, Johnson, Lin, Finn, and Manchester are collaborating with each other to find and test uses for the modified CPMV particles. And they have many possibilities. They have already successfully attached biotin (Vitamin B), sugars, and organic chemicals to the viral surface, and they can immobilize large molecules on the surface—whole proteins even. "We can attach anything we want to the surface of the virus," says Johnson. One possible attachment are molecules that can be used to image cancers or other biological states in living cells by labeling CPMV with an anti-tumor agent or some molecule that targets a particular biology of interest along with radiolabels or some other sensing agent that could be visible under magnetic resonance or microscope imaging. Finn, Johnson, and Lin found that cysteines could also be double-labeled by placing cysteine on both the inside and outside of the virus shell and that a pattern of attachment sites could then be created that would allow for novel chemistry. Catalysis could potentially be carried out with the virus particles. The temperature and pH stability, solubility, and the chromatographic properties of the virus can be altered at will, by adding the right molecules. And the virus particles can self-organize into network arrays in a crystal, which may make it a useful building block for various applications in nanotechnology, the field that seeks to build functional material on the nanometer scale (roughly one to one hundred billionths of a meter). "You can, in principle, determine the type of assembly you get by programming the building blocks," says Finn. And in collaboration with Manchester, Finn and Johnson are excited about the possibility of testing CPMV as a polyvalent delivery vehicle. Since there are 60 attachment sites, the virus will present multiple copies of the attached molecules wherever it goes. "This is something uniquely Scripps," says Johnson. "We have three different departments, three different backgrounds, and yet here we are."
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