Biologists visualize protein interaction that may initiate viral infection



This composite image depicts a "snapshot" of a Velcro-like protein on a cell's surface just after it attached to the dengue virus, a linkup thought to initiate the early stages of infection.(Graphic/Department of Biological Sciences, Purdue University)
Click here for a high resolution image.

Biologists at Purdue University have taken a "snapshot" of a Velcro-like protein on a cell's surface just after it attached to the dengue virus, a linkup thought to initiate the early stages of infection.

The virus, which is spread by mosquitoes, infects more than 50 million people annually, killing about 24,000 each year, primarily in tropical regions.

During the earliest stages of infection, the dengue virus attaches to the "carbohydrate recognition domain," or CRD, of a key binding protein called DC-SIGN, located on a host cell's surface.

Using a powerful imaging tool called cryo-electron microscopy, the biologists took a picture of the virus attached to the CRD shortly after the two joined together. It is the first time scientists have visualized the virus and CRD binding.

"We formed the virus-CRD complex, took a snapshot and determined its structure," said Michael Rossmann, the Hanley Distinguished Professor of Biological Sciences in Purdue's College of Science. "Ultimately, researchers might want to find ways to treat or prevent viral infections, but in order to do that we first have to learn how viruses work and how they initiate infection."

The findings are detailed in a research paper to appear on Feb. 10 in the journal Cell. The research was carried out by Elena Pokidysheva and Ying Zhang, post-doctoral research associates working with Rossmann and Richard J. Kuhn, a professor and head of Purdue's Department of Biological Sciences.

Researchers from the Howard Hughes Medical Institute at Columbia University provided a cloned gene that enabled the Purdue scientists to produce the CRD.

The CRD is part of a protein receptor molecule called DC-SIGN - or dendritic cell-specific ICAM3 grabbing non-integrin. ICAM stands for intercellular adhesion molecule, a family of cell proteins that viruses bind to, and the number 3 defines a specific protein.

"The binding occurs on dendritic cells, which are usually one of the first lines of defense in the immune system," Kuhn said. "The first step in a virus infecting a cell is usually the attachment of the virus to the receptor. That's essentially what we are looking at, except in this case, instead of having the receptor, which is normally bound or attached to the cell, we have just a portion of the receptor, the CRD, which we produced separately."

Dengue belongs to a family of viruses known as flaviviruses, which includes a number of dangerous insect-borne diseases such as West Nile, yellow fever and St. Louis encephalitis. These diseases, however, use different biological mechanisms than dengue to infect host cells. Dengue is prevalent in Southeast Asia, Central America and South America. Mosquitoes transmit the virus to people, setting in motion the infection process.

"We and others think that this CRD acts sort of like Velcro to get the virus to stick to the surface of the cell, although this has not been proven," Kuhn said. "Once the virus and protein receptor are linked, perhaps the virus then moves across the cell surface to find a second protein, attaching to that receptor and entering the cell.

"One of the things that this study shows is that only a very small portion of the cell's surface is occupied by the DC-SIGN molecule, which means a significant amount of space is still available for that other receptor protein that people don't know about yet."

Zhang said that the initial binding of the CRD and the virus might result in a "signaling event between the DC-SIGN molecule and the other primary receptor, leading to activating the other protein and promoting the cell for infection."

The virus has a diameter of 50 nanometers, or billionths of a meter, and the CRD is 3 nanometers wide.

In cryo-electron microscopy, specimens are first frozen before they are studied with an electron microscope. The method enables scientists to study details as small as 8 angstroms, or .8 nanometers, resolution high enough to see groups of atoms. An angstrom is one ten-billionth of a meter, or roughly a millionth as wide as a human hair.

Zhang discovered that the CRD attaches to a structure on the virus surface that contains two carbohydrates a distance of 18 angstroms apart. This feature apparently is essential for the binding to take place, she said.

"Why doesn't the binding happen at other sugar-binding sites?" she asked. "The answer is that we need two carbohydrate sites that are 18 angstroms apart. There are no other sites that are 18 angstroms apart."

Each virus particle contains 60 of the features, each having two carbohydrates 18 angstroms apart, representing 60 potential binding sites for the CRD.

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The research has been funded primarily through a grant from the National Institutes of Health.

The paper was co-authored by Pokidysheva; Zhang; Anthony J. Battisti, a graduate student; Carol M. Bator-Kelly, a technical assistant; Paul R. Chipman, an electron microscopist; and post-doctoral researcher Chuan Xiao, all at Purdue University; G. Glenn Gregorio, a graduate student at the Howard Hughes Medical Institute at Columbia University; Wayne A. Hendrickson, a researcher at the Howard Hughes institute and a professor of biochemistry and molecular biophysics at Columbia University's College of Physicians and Surgeons; as well as by Kuhn and Rossmann.

Writer: Emil Venere, (765) 494-4709, venere@purdue.edu

Sources: Michael Rossmann, (765) 494-4911, mgr@indiana.bio.purdue.edu

Richard J. Kuhn, (765) 494-1164, kuhnr@purdue.edu

Elena Pokidysheva, (765) 494-4908,epokidys@purdue.edu

Ying Zhang, (765) 494-4925, yzhang@purdue.edu

Related Web sites:
Michael Rossmann: http://www.biology.purdue.edu/people/faculty/rossmann/index.htm

Richard Kuhn: http://www.biology.purdue.edu/people/faculty/kuhn/index.htm

IMAGE CAPTION:
This composite image depicts a "snapshot" of a Velcro-like protein on a cell's surface just after it attached to the dengue virus, a linkup thought to initiate the early stages of infection. The virus, which is spread by mosquitoes, infects more than 50 million people annually, killing about 24,000 each year, primarily in tropical regions. During the earliest stages of infection, the dengue virus attaches to a location on a host cell's surface called the CRD, or carbohydrate recognition domain, which is part of a receptor molecule called DC-SIGN - or dendritic cell-specific ICAM3 grabbing non-integrin. Here, the DC-SIGN molecule is represented as a string-like green structure connected to the CRD, which is binding to a virus particle, pictured as a spherical green object. Using a powerful imaging tool called cryo-electron microscopy, biologists at Purdue University took electron microscope pictures of the virus attached to the CRD shortly after the two joined together. It is the first time scientists have visualized the virus and CRD binding. (Graphic/Department of Biological Sciences, Purdue University)

A publication-quality graphic is available at http://news.uns.purdue.edu/images/+2006/rossmann-dengue.jpg


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