Viruses are one one-hundreth the size of a standard bacterium. In fact, hundreds and thousands of bacteria could fit on the period at the end of this sentence. And with the ability to invade, replicate, and destroy their host, it is little wonder that they have fascinated researchers since their initial discovery.
But for all the fascination surrounding viruses, they have proven difficult to detect using traditional microscopy techniques; in order to truly visualize a virus and understand its biology, one must be able to see the proteins that form the virus. Different microscopy techniques are able to view viruses, but at limited resolutions that only show the basic outlines of the virus without any details. “[With previous techniques,] you can see the house; but if you cannot see the bricks, then you cannot see how the house is built,” says Z. Hong Zhou, professor of microbiology, immunology and molecular genetics at the University of California, Los Angeles (UCLA) and director of the Electron Imaging Center for NanoMachines (EICN) at the UCLA California NanoSystems Institute.
Recently, several new imaging techniques have been developed that allow researchers to get a better look at the secrets held by single viruses. These new microscopy techniques focus on imaging viral particles, detailing their structure, and observing their interactions with other molecules. With higher resolution and precise wave diffraction measurements, several teams have been able to finally visualize the major protein ”brick” walls, the minor proteins that act as ”mortar” between the ”bricks,” as well as resolve the images to accurately calibrate the size and mass of these tiny structures.
In the eye of the beholder
Since viruses are so small, traditional detection techniques that suspend particles of interest in liquid have difficultly differentiating between the target viral signals and background particles or contaminants in the solution. Established single virus imaging techniques rely on fluorescent probes or other tools that chemically bind to the target molecules to boost their signal, but these tactics alter the intrinsic physical properties of the viruses. Tao’s goal over the past decade has been to overcome such limitations to enable true single virus imaging.
Those years of hard work have paid off. Tao and his lab had previously shown that SPR can measure local electrochemical activity by detecting variations in current density signals as they pass over a chip. The activity, generated as chemical species are created or consumed on the electrode chip surface, correlates to an optical signal with high spatial resolution and sensitivity. In their recent study, published at the Proceedings of the National Academy of Sciences, they describe the use of SPR microscopy to detect high-resolution, label-free imaging of single viruses (1).
Using SPR microscopy to image the small viral particles, the research team was able to maximize signal and minimize background noise without affecting the physical properties of the virus. The imaging technique only captures signals from viruses specifically-bound to the SPR chip, thereby eliminating non-specific signals and allowing researchers to focus solely on the region of the chip that holds these tiny molecules. The chemical composition of the SPR chip can be modified; by controlling the binding surface of the SPR microscopy set up, researchers can adjust what molecules can or cannot bind to the imaging plane and observe affinity interactions of particular molecules across a variety of surfaces.
Since SPR chips can be covered with virtually any material, they can be adapted for clinical research by growing biologically relevant cell cultures on the chips to test the viruses’ affinity for the cells. Tao’s lab has already used a derivative of their published SPR imaging method to produce proof-of-concept data for such clinical applications. “We have a pretty high sensitivity and spatial resolution to look at small objects, and we’re trying to look at even smaller objects, like proteins,” says Tao. “But on the larger scale, we’re looking at cells and drug-cell interactions.”
The development of a label-free technique that does not affect the essential properties of the object has allowed Tao’s lab to categorize affinity interactions and determine viral mass. Specifically, his lab showed that SPR can be used to precisely discriminate two similarly-massed viruses and accurately track the kinetics of single molecules. Additionally, the SPR technique images viruses in an aqueous solution, as opposed to a non-biological medium, which allows researchers to determine the latent in vivo behavior of these molecules.
To test their methods, Tao’s lab observed the binding properties of influenza virus and compared it to those of human cytomegalovirus (HCMV). Although these clinically relevant viruses share a similar size, they exhibit different antibody binding affinities; their similarities and differences were a perfect combination to test the precision of the SPR detection and measurement techniques. The SPR images allowed Tao’s lab to differentiate between the viruses and calibrate their masses in agreement with literature values. Additionally, they were able to image and compare the binding interactions and kinetics of the two viruses. The SPRM images are coded along a color spectrum that ranges from blue to yellow to white as density increases. Bound viral particles emit diffraction patterns like bright yellow flowers on a rich blue background and are easy to track.
Tao’s technique captures unaltered images of small viruses, and he revels in the future possibilities of SPR imaging. “That’s the power of high-resolution imaging: if you look at the image and you can see it clearly, you can see how a single molecule appears and the beauty of it.”
Wu’s research in the UCLA Interdepartmental Program of Molecular Cellular and Integrative Physiology has focused on adapting viruses as cancer therapy vectors, but her goals required a better image of adenovirus before she could modify and test it. So she contacted her UCLA colleague Zhou, the 2004 recipient of the American Microscopy Society Barton Award, for microscopy aid.
Zhou’s laboratory uses cryo-electron microscopy (cryo-EM) to determine the assembly and structure of biological specimens. The group has had success using cryo-EM to determine the atomic structure of other viruses and identified previously unknown protein interactions within the virus. Cryo-EM is a derivative of the more common scanning electron microscopy (SEM), which uses a beam of electrons that interacts with the atoms on a sample’s surface to obtain information about the sample, but cryo-EM has the additional benefit of imaging the particle of interest in an aqueous solution. By freezing the sample in water, the physical and chemical properties of the sample remain unchanged, and the particle can be imaged to show both its structure and its interactions with other biological surfaces.
Zhou and Wu were able to push the cryo-EM resolution to under 4Å, at which point they were able to visualize, for the first time, the amino acid side chains in the major proteins and get a clearer understanding of how the proteins came together within the virus. They recently published their findings in the journal Science (2).
"Technologically speaking, this is a major development for science,” says Zhou. “It opens the door to look at many other things, not only viruses, but protein complexes or even cells in their latent form; since you don’t have the gold crystals of standard electron microscopy, you can look at anything and image it in water.”
With cryo-EM imaging, the research team was able to identify glue proteins that are sandwiched between major proteins, acting as mortar between the protein bricks that create the viral architecture. Assays of protein-protein interactions demonstrated that the glue proteins serve as the principal chemical link between the major proteins and the minor proteins.
Additionally, Zhou and Wu discovered another type of minor protein that acts as a longer-range stabilizing force. “In traditional architecture, you build a house with bricks. But more recently, there’s a second kind of architecture where you use ropes; the Golden Gate Bridge is a good example. You use bricks to build it, but you use cables to reinforce it.” Zhou and Wu classified these minor proteins, usually twisted together over long distances, as rope proteins. Like the glue proteins, they aid in protein-protein connectivity and mediate chemical interactions.
Knowing the amino acid composition of the bricks, mortar, and the ropes that hold the virus together, the UCLA team will be able to modify adenoviruses in new ways. Zhou and Wu are already experimenting to increase the carrying capacity of adenovirus by altering the number and length of specific minor proteins. “Adenovirus is basically an eighteen-wheeler, carrying the good genes to the region of interest,” says Zhou. “You can engineer a virus to carry genes only to cancer cells and improve the delivery of gene therapy.”
With a little help from my friends
While adenovirus may be considered a “good virus,” it still initiates an immune response in most patients undergoing adenovirus gene therapy. The release of antibodies in an immune response truncates the action of the vector, limiting its effectiveness. To create a recombinant adenovirus that will maintain its clinically-useful qualities while removing its barriers to gene therapy, Nemerow and Reddy wanted a complete image of the virus.
Nemerow and Reddy decided to use X-ray crystallography to image adenovirus. However, X-ray crystallography—a technique that uses the diffraction of X-ray beams off a crystallized sample to determine the 3D atomic arrangement of the sample—provided the two with multiple challenges. Before they could use the technique, the team first had to obtain diffractable crystals of the virus, a difficult task due to the adenovirus’ structural barriers to crystallization. The major icosahedral proteins that make up adenovirus have long, thin, fibrous proteins emanating from the vertices that prevent the close packing of particles necessary to form crystals. After four years, Nemerow and Reddy crystallized a recombinant virus that had shorter fibers but still maintained overall viral stability. It then took another eight years for them to produce crystals with a high enough quality for imaging, and for x-ray techniques to reach a resolution high enough to observe individual amino acids. After taking millions of images, the two self-proclaimed “millionaires” had enough views of the virus to piece together its structure.
With a full picture of the atomic bricks of the virus, Nemerow and Vijay were able to confirm several suspicions about host-virus interactions and test some of their hypotheses. In particular, they were able to observe certain conformational changes in their crystals that could play a role in cell entry. “The vertex region is a pretty dynamic region of the capsule,” Reddy says. “It’s like a fuse of the virus. Once it comes off, the virus gets activated.” Further research into these changes could influence how gene transfer is conducted in clinical applications.
Nemerow says that their continued work with adenovirus may help to combat issues of pathogenic and immune response in gene therapy patients. “We now are getting hints about where those antibodies bind on the virus, and having the crystal structure of the virus might allow us to design a vector to evade antibody detection.”
It is clear that the way in which small viral imaging and detection is performed is changing rapidly. Developments in microscopy techniques have allowed researchers to peek into the ”windows” of these miniscule protein ”houses” and learn the secrets of assembly, cell entry, and gene delivery in the hopes of creating more efficient recombinant viral vectors to combat cancer. “If you have bricks to build things, it’s rather easy to do so,” says Zhao. “These discoveries are significant because they really tell how things are put together and how to take advantage of them.”
1. Wang, S., X. Shan, U. Patel, X. Huang, J. Lu, J. Li, and N. Tao. 2010. Label-free imaging, detection, and mass measurement of single viruses by surface plasmon resonance. Proceedings of the National Academy of Sciences. doi: 10.1073/pnas.1005264107 Epub 2010 26 August.
2. Liu, H., L. Jin, S.B.S. Koh, I. Atanasov, S. Schein, L. Wu, and Z.H. Zhou. 2010. Atomic structure of human adenovirus by cryo-EM reveals interactions among protein networks. Science. 329: 1038-1043. Epub 2010 27 August.
3. Reddy, V.S., S.K. Natchiar, P.L. Stewart, and G.R. Nemerow. 2010. Crystal structure of human adenovirus at 3.5Å resolution. Science 329: 1071-1075. Epub 2010 27 August.