First-ever images of developing dengue virus obtained at Purdue

June 18, 2003

WEST LAFAYETTE, Ind. - High-quality images of a virus still forming in its cellular host shed light on how viruses reproduce, knowledge that could prove important to the development of antiviral drugs.

A team including Purdue University's Michael Rossmann and Richard Kuhn has solved the structure of the immature dengue virus, which is related to West Nile virus and yellow fever. Dengue is a mosquito-borne pathogen that kills more than 24,000 people in the world annually. The pair solved the structure of the mature dengue virus particle last year (see related story), and Rossmann said the new findings were a significant step toward unraveling the behavior of viruses.

"We're beginning to dissect the individual steps in a virus' life cycle," said Rossmann, who is Henley Distinguished Professor of Biological Sciences in Purdue's School of Science. "We hope to learn a great deal more about viral development so that approaches to preventing infection become conceivable."

The study, a collaboration among Rossmann, Kuhn and Tim Baker at Purdue and James Straus at the California Institute of Technology, appears in the June 2 issue of EMBO.

The research group used an advanced imaging technique, known as cryoelectron microscopy, to take 3-D pictures of the dengue particle - the term experts use to denote a single virus. While viruses are not considered to be "alive" by the standards we apply to plants and animals, the team's images have revealed that particles go through a complex developmental process.

"We have discovered that an astonishing structural change occurs between the immature and mature dengue shells," said Kuhn, also professor of biology. "We don't yet know how it all happens - but even though we have only seen two points along the viral assembly line so far, we can tell it's quite a dynamic metamorphosis."

Compared to the mature dengue particle, for example, the immature form is 15 percent greater in diameter.

"The immature particle is covered with 60 three-pronged protein spikes, called trimers, that jut from its surface," Kuhn said. "In contrast, the mature particle is a nearly smooth sphere, like a golf ball. Somewhere in the assembly process, these trimers flatten out, making the surface appear more even." The proteins are important because each contains a short amino acid sequence called a fusion peptide that the virus needs to attach itself to a potential host. Without this fusion peptide, the virus cannot successfully invade a cell.

"If you compare a virus to a pirate ship, these peptides are the grappling hooks by which they attach themselves to their prey," Kuhn said. "A particle can only inject its genetic material into a cell after it has bonded with its surface. Fusion peptides allow the virus to prepare for boarding, so to speak."

The peptides need to be protected until the virus is ready to bond with a cell, so in the immature particle, each peptide is covered with a special cap that protects it until the time is right.

"We would like to know more about how a virus changes," Rossmann said. "Our imaging techniques are now giving us vastly greater perspective on how a particle becomes a successful invader. Now we want to know how it marshals its offenses and defenses."

It is in examining the changes a virus undergoes- for example, in the case of dengue, how it uncaps its fusion peptides to become an infectious agent - that the team hopes to find clues to stopping the developmental process in its tracks.

"Any knowledge of the steps in a virus' assembly process provides a potential target for an antiviral agent," Rossmann said. "If you are trying to assemble something, introducing a foreign body into the process could gum up the works."

But Kuhn said much more work needs to be done before such medicines will appear in your drugstore, as the full picture of viral assembly remains unclear.

"This is only one step in the viral maturation process," Kuhn said. "We still need other scenes from its cycle of existence - snapshots of it fusing with a cell, for example, and of it entering - to have complete understanding."

The team's next step will be to confirm its findings, which Kuhn considers critical. The metamorphosis the dengue particle undergoes is so radical, he said, that there is a possibility the immature form the team has seen is not actually a step in dengue's development. For the moment, however, the results are encouraging enough to pursue the research further.

"Knowledge of how a virus assembles itself can reveal its vulnerabilities," Kuhn said. "This is what our research techniques allow us to explore - and perhaps exploit."

This research was funded in part by the Allergy and Infectious Diseases Institute at the National Institutes of Health.

Rossmann and Kuhn are associated with Purdue's Markey Center for Structural Biology, which consists of laboratories that use a combination of cryoelectron microscopy, crystallography and molecular biology to elucidate the processes of viral entry, replication and pathogenesis.
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cb/Rossmann.immature

Writer: Chad Boutin, 765-494-2081, cboutin@purdue.edu

Sources: Michael Rossmann, 765-494-4911, mrossmann@purdue.edu

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

Related Web sites:

EMBO Journal: http://emboj.oupjournals.org/

March 2002 Purdue news release on dengue virus structure: http://news.uns.purdue.edu/UNS/html4ever/020307.Kuhn.dengue.html

PHOTO CAPTION:

The structure of the mature dengue virus particle was determined last year by Michael Rossmann and Richard Kuhn's team. The virus' surface is unusually smooth and its membrane is completely enclosed by a protein shell. The protein is color-coded blue, green and yellow to show the three specific domains of the protein. The protein shell serves as a cage for the genetic material inside. (Purdue University computer illustration)

A publication-quality graphic is available at ftp://ftp.purdue.edu/pub/uns/rossmann.dengue.jpeg.

PHOTO CAPTION:

This image shows the immature dengue particle. Notable are the 60 protein "spikes" which jut from the surface, making the immature particle far less smooth than the mature form. One spike is circled for reference. Knowledge of how the immature particle develops into the mature, infectious dengue virus could be important to developing antiviral medicines. (Purdue University computer illustration)

A publication-quality graphic is available at ftp://ftp.purdue.edu/pub/uns/rossmann.immature.jpeg.

ABSTRACT

Structures of Immature Flavivirus Particles

By Ying Zhang, Jeroen Corver, Paul R. Chipman, Wei Zhang, Sergei V. Pletnev, Dagmar Sedlak, Timothy S. Baker, James H. Strauss, Richard J. Kuhn and Michael G. Rossmann

Structures of prM-containing dengue and yellow fever virus particles were determined to 16 and 25 angstrom resolution, respectively, by cryoelectron microscopy and image resolution techniques. The closely similar structures show 60 icosahedrally organized trimeric spikes on the particle surface. Each spike consists of three prM:E heterodimers, where E is an envelope glycoprotein and prM is the precursor to the membrane protein M. The pre-peptide components of the prM proteins in each spike cover the fusion peptides at the distal ends of the E glycoproteins in a manner similar to the organization of the glycoproteins in alphavirus spikes. Each heterodimer is associated with an E and prM transmembrane density. These transmembrane densities represent either an EE or prMprM antiparallel coiled coil by which each membrane spans the protein twice, leaving the C-terminus of each protein on the exterior of the viral membrane, consistent with the predicted membrane-spanning domains of the unprocessed protein.

STORY AND PHOTO CAN BE FOUND AT:http://news.uns.purdue.edu/html4ever/030618.Rossmann.immature.html

NOTE TO JOURNALISTS: Publication-quality graphics are available at ftp://ftp.purdue.edu/pub/uns/rossmann.dengue.jpeg and ftp://ftp.purdue.edu/pub/uns/rossmann.immature.jpeg.

Purdue University

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