Human Frontier - 10th Anniversary Brochure
A yellow virus revealed by its symmetry
The tick-borne encephalitis virus has a very regular structure. Thanks to its symmetric shape, five groups of researchers are studying how it penetrates the cells it infects. The project is especially interesting because the virus belongs to a family which includes the agents of dengue, Japanese encephalitis and yellow fever.
These viruses are known collectively as flaviviruses (from the Latin flavus: yellow), because one of their most prominent members—the yellow fever virus—produces jaundice in patients. Flaviviruses are responsible for tens of thousands of deaths each year, throughout several continents.
Distribution map of the diseases caused by flaviviruses. The family of enveloped viruses is particularly feared owing to the sudden and very widespread epidemics it could bring about in the future ("emerging diseases"). In addition to tick-borne encephalitis (red), Japanese encephalitis (blue) and yellow fever (yellow) shown on the map, mosquito-borne dengue is found practically throughout the tropical belt.
"We have brought together five research groups with complementary skills in an effort to understand how a flavivirus penetrates the cell it infects", says Stephen Fuller (European Molecular Biology Laboratory, Heidelberg, Germany), leader of the Human Frontier project Molecular organization and structural rearrangements in flaviviruses. "Flaviviruses are enveloped viruses like influenza and HIV. Their genetic material is enclosed in a protein capsid, which itself is wrapped in a membrane similar to the one that surrounds a human cell. The fusion of the viral and cellular membranes is a key stage in enveloped virus infection. With the tick-borne encephalitis flavivirus, more simply known as TBE, we have a rare opportunity to visualize the process in the whole virus, whereas until now it was only possible to observe the behavior of isolated viral elements."
Made in Austria • As a precaution, the researchers on this HFSP project started by vaccinating themselves against TBE. The vaccines were supplied by the Institute of Virology of the University of Vienna, where Prof. Franz Heinz is working. Austria is one of many Eurasian countries affected by this all too often fatal form of encephalitis, which is transmitted by tick bites.
The tick Ixodes ricinus Unlike yellow fever vaccine, which remains effective for several years, TBE vaccine has to be renewed after a year, and then at least every three years. In Austria and neighbouring Central European countries—at the western edge of the area affected by the virus—vaccination campaigns are regularly organized among high risk persons, especially those working in forests. Unfortunately, owing to lack of resources this is not the case in regions further east, such as Siberia. The scientists are hoping to come up with a vaccine which remains effective for a longer time.
Professor Heinz’ lab also supplies his colleagues with non-infective virus particles known as RSPs (recombinant subviral particles), that have no capsids and no RNA. "With RSPs, you can work in a normal laboratory", explains Fuller. "Working safely with infectious TBE viruses requires strict containment. There is no high performance microscope available in the world under suitable containment conditions. These RSPs are of course smaller than the full viruses, but they still have the basic characteristics we want, namely the outer membrane with its two kinds of embedded proteins."
All identical • However, even a complete virus is far too small to be visualized directly in any detail. Fortunately, unlike some enveloped viruses like HIV, which exhibit an irregular and variable morphology, the TBE particles are regular and identical. All of them have icosahedral symmetry, like soccer balls and many other viruses, including those which cause polio and the common cold.
In order to understand the analytical technique used by researchers on this project, you have to imagine dozens of identical soccer balls scattered around a soccer field and photographed from a helicopter. You do not know the precise structure of the ball in advance. What is more, all the balls are very small on the photograph and lie around on the ground in different orientations. And yet it is possible to describe the objects, because it is as if you were looking at the same soccer ball from several angles.
The knowledge that the objects have icosahedral symmetry is used to determine the orientations of the individual particles. These orientations are then refined by comparing the data between the particles. Ultimately, all this information is combined to determine the structure of an "average object" in three dimensions.
Vitrified viruses • Instead of a soccer pitch, researchers use a very thin film of water, which is vitrified by cooling to -180°C so rapidly that ice crystals cannot form. The vitrified water is a glass-like state, which maintains properties similar to water at room temperature and avoids the deformation of the TBE viral particles that occurs upon freezing. "The other advantage is that there is no need to stain or to fix the samples for observation", adds Yoshiaki Kimura (Biomolecular Research Institute, Osaka, Japan). "Under the electron microscope, viruses look as natural as possible."
Like Fuller’s group, Kimura’s researchers are able to examine the vitrified samples using electron microscopy. Their microscope is one of the most advanced in the world and maintains the sample at liquid helium temperature (-269°C) and so minimizes the damage due to the electron beam and movements which occur in more conventional microscopes. This is why some flavivirus images are taken in Japan (they come in the form of 9 x 13 cm negatives with extremely fine resolution), before being sent on to Fuller’s laboratory in Germany. There they are put through a high-resolution scanner to be digitized.
The rest of the work is done by computer. Each of the particles is analyzed individually and the best are combined to determine the three-dimensional structure. "The best 74 particles from several hundred processed were combined to produce a structure with a resolution of 19 Ångström", says Fuller. "We should be able to produce a higher resolution structure by analyzing several thousands of particles, which remains a formidable computational task!"
The E-protein • The computer-assisted reconstruction of the TBE virus was facilitated by prior knowledge of the so-called E-protein, which seems to play a central role in mediating the fusion of the cellular and viral membranes. The E-proteins on the TBE virus are arranged in pairs lying flat along the surface of the membrane, resembling opened pairs of spectacles that extend through the membrane towards the capsid.
In fact, two laboratories that are also involved in this HFSP project had already isolated and studied the main part of this E-protein, but in a crystallized form. These scientists, led by Prof. Stephen Harrison (the Children’s Hospital, Boston, USA) and Felix Rey (CNRS, Paris XI University, France) are now studying other viral proteins, in the agents of dengue and yellow fever.
By comparing the E-proteins observed in the crystals to those in the vitrified particles, the researchers realized that the latter were slightly distorted. They are now exploring the possibility that this flexibility is related to conformational changes, which occur during the membrane fusion leading to flavivirus infection of human cells.
| < Prev | Home | Next > |