Scientists Solve First Flavivirus Structure; Feat Provides Insights Into Dengue, West Nile, Yellow Fever, Others

Purdue University researchers, working with scientists at the California Institute of Technology, have solved the three-dimensional structure of the dengue virus, which is transmitted by mosquitoes and causes more than 50 million cases of infection and 24,000 deaths worldwide each year.

The findings, published in Friday's (3/8) issue of the scientific journal Cell, provide the first detailed view of a flavivirus and offer structural information that can be used to unravel the processes that lead to viral infection.

The flavivirus family includes a number of dangerous insect-borne diseases such as dengue, West Nile, yellow fever, tick-borne encephalitis and St. Louis encephalitis. Together these viruses cause millions of cases of human illness each year. Several viruses from this family also are among a select group of viruses being studied to counteract potential bioterrorist attacks.

The discovery may help scientists develop antiviral compounds and other strategies to target dengue and other diseases caused by flaviviruses, says Richard Kuhn, associate professor of biological sciences and lead author of the study.

"This is an extremely important family of human pathogens, including West Nile, that is now present in the United States," Kuhn says. "By studying the structure of the virus, we can gain insights into the chemical and biological activity that occurs when the virus infects a human cell, and develop experiments to identify and target those activities."

Because all flavivirus are closely related, Kuhn says studying the structure of the dengue virus will reveal strategies that can be used to study other viruses in the family.

"We have every expectation that the West Nile virus and others from this family will be very similar in structure to this one," he says.

Purdue researcher Michael Rossmann, a co-author of the study who in 1985 became the first scientist to solve the structure of a human virus, called the achievement a milestone in viral studies.

"The flavivirus family contains a number of medically important viruses that have proved to be difficult to control and study," he says. "Our findings provide new molecular insights into the process of infection used by these viruses and a structural basis for targeting those activities."

Dengue fever is a severe, flu-like illness that causes high fever, rash and extreme pain in the head, muscles and joints. Dengue haemorrhagic fever is a potentially lethal complication that can cause internal bleeding, vomiting, severe abdominal pain and death.

The dengue virus is transmitted to humans through the bites of infected mosquitoes and is most often found in tropical and sub-tropical regions of the world. Last year, 74 cases of dengue infection were reported in the state of Hawaii, and health agencies have reported an increased risk for persons living along the Texas-Mexico border. In recent weeks, an outbreak of the infection has left tens of thousands of people ill in Rio de Janeiro, Brazil.

Though vaccines have been developed for dengue, control of the virus by vaccination has proved to be elusive. The disease may be caused by any one of four different strains of the dengue virus, and vaccinating against only one or two of the viruses can increase the risk of more serious forms of the disease.

Rossmann says mapping the structure of the dengue virus posed a number of technical challenges for the research group. Flaviviruses have been especially difficult to study in part because their properties make it difficult to produce the large amounts of undamaged particles needed for the high-resolution techniques used to study virus structures.

"To do these experiments, we need large amounts of undamaged virus, and the flaviviruses are difficult to culture," he says.

In addition, the flaviviruses can pose serious health risks to scientists who study them. Few scientific laboratories work with viruses from this family, and those that do either use a vaccine strain or an attenuated strain, modified to reduce the virulence of the virus. These precautions eliminate much of the risk, Rossmann says.

In their studies at Purdue, researchers used a dengue strain originally sequenced by scientists at the California Institute of Technology. Under the guidance of James Strauss, who is the Ethel Wilson and Robert Bowles Professor of Biology at Caltech, the group developed a strain of the virus that could be manipulated safely.

Studies of the dengue structure were done using the techniques of cryo-electron microscopy and three-dimensional image reconstruction. These techniques — which use hundreds of highly detailed, two-dimensional images and powerful computer programs developed by Tim Baker at Purdue — allowed the group to develop a three-dimensional view of the virus. A program developed by Rossmann was then used to interpret the three-dimensional images in terms of the viral molecular components.

The image shows how the major protein — called "E" for envelope protein — organizes itself to form a protective shell around the virus. The protein shell is made up of 60 subunits to form a 20-sided sphere. The shell serves as a cage for the genetic material inside, sheltering it from harm until it is released inside a host cell. While simple viruses consist of only a protein shell and genetic information, more complex viruses, such as flaviviruses, also contain a lipid bilayer that sits between the protein shell and viral genome.

Kuhn says the three-dimensional reconstruction reveals several unexpected features in the dengue virus architecture.

"The organization of the shell protein is very different from what we've seen before and what we might have predicted for this type of virus," he says. "For example, the protein completely covers the surface of the virus. Generally, when a virus contains a lipid bilayer beneath, we can see some of the lipid on the surface of the virus. In this case, the lipid bilayer is completely concealed."

The virus surface also is unusually smooth, he says, noting that the dengue virus resembles a golf ball, with small indentations stamped upon the shell where the protein structures come together.

"Most viruses have a number of spikes and projections on their surface, and these are used by the virus to interact with host cells," Kuhn says. "Even among the enveloped viruses, such as HIV or influenza, such spikes are common."

The structure of the dengue virus also suggests it employs a cell entry pathway similar to another group of viruses known as togaviruses, Kuhn says.

The Purdue team is now pursuing experiments to determine how the virus puts itself together and how it carries out the fusion process with the host cell.

The studies at Purdue were funded by the National Institutes of Health.