Erica Saphire, 2003 Career Award in the Biomedical Sciences
2005 - Long before modern media turned the Ebola virus into a household name, the virus may have been the cause behind the plauge in Athens in 430 B.C. some scientists suspect. The Greek historian Thucydides recorded his experiences, not only as an observer but also as a sufferer.
“As a rule, however, there was no ostensible cause,” he wrote. “But people in good health were all of a sudden attacked by violent heats in the head, and redness and inflammation in the eyes, the inward parts, such as the throat or tongue, becoming bloody and emitting an unnatural and fetid breath.”
Despite its similarities to the hemorrhagic fever symptoms described in the ancient text, Ebola has only become known to modern science since the mid-1970s. Named after a river in the Democratic Republic of the Congo (formerly Zaire) in Africa, where it was first recognized, its activity was mostly confined to remote regions of Africa.
“I find it absolutely shocking that in 2005 we still can’t really cure a viral infection,” Dr. Erica Ollmann Saphire, a Burroughs Wellcome Fund 2003 Career Awards in the Biomedical Sciences recipient, said. “We just give the patients some sort of support for their symptoms and hope their immune systems can take care of the rest.”
Dr. Saphire, a former rugby player at Rice University and manager for the United States Women’s Rugby Team, is an assistant professor in the Department of Immunology at the Scripps Research Institute in La Jolla, Ca.
The Ebola virus, Saphire noted, has had as many outbreaks in the past five years as it had in the preceding 30. The disease has a 50 to 90 percent human fatality rate. The increased incidence may be due largely to the removal of the rain forest, encroachment on wild areas, and increased human contact with primates in previously remote regions.
As a structural biologist, Saphire studies the virus at a molecular level. Her laboratory specializes in crystallizing the viral proteins that play key roles in the pathogenesis of viral hemorrhagic fevers using X-ray crystallography, an experimental technique that utilizes the diffraction of X-ray light off of crystals.
Saphire hopes that studying the structures of these proteins will provide information crucial to the development and design of vaccines and inhibitors against the virus. These structures may potentially provide insightful information on other viral threats. Part of the problem of finding a cure for Ebola is that the virus has a few tricks up its sleeve, she said.
“You have to admit Ebola is a pretty spectacular virus,” she said. “There’s some biological mechanism, some scientific reason at the molecular level, why this thing is so incredibly pathogenic and why there is very little we can do about it.”
One of Ebola virus' clever mechanisms is its ability to encode two difference glycoproteins--proteins containing covalently specific groups of covalently linked carbohydrates--from the same gene.
The proteins, sGP and GP, have largely the same N-terminal sequence of 295 amino acids, but a transcriptional editing event causes them to have different C-terminal sequences. It’s this slight difference at the C terminus that enables the viral proteins to make two entirely different structures because of unique patterns of disulfide bonding. Importantly, this alteration causes the two proteins to fold differently, allowing the single coding gene to play more than one role in pathogenesis.
“If you’re a biologist, you learn on your mother’s knee that one sequence causes one structure,” she said. “Here it looks like we have the same sequence that might cause two different structures. Certainly, it looks like the immune system sees these proteins in very different ways.”
Ebola also suppresses the innate immunity, the most ancient part of the immune system and the first line of defense against invading pathogens. VP35, another Ebola virus protein, stops the initiation of some of the pathways that might upregulate the adaptive immune response. It is also part of the machinery by which Ebola builds up more copies of itself by constructing the core framework of the virus.
The virus' quick action hinders response from the more specifically targeted immune system. The virus can go from contacting the body to full blown infection in less than two weeks, in some case only taking a couple of days.
“It’s a bit hard to get together a good adaptive immune response in that time frame,” Saphire said.
In order to create a replica of the glycoproteins' structure so that she can understand how the immune systems "sees" them, Saphire and her lab make recombinant proteins in a variety of cell lines. The expression cells manufacture a large quantity of the protein and by using chromatography the protein is purified.
Once the high purity level is achieved, crystallization is attempted by removing water vapor at a very slow rate. The protein molecules will orient themselves in a certain way to try to make the most stable contacts that will form the crystal. Saphire likens this process to what happens to “really old maple syrup.”
A single molecule of protein will not cause X-ray light to scatter in a detectable way. However, a million or so protein molecules, all oriented in the same pattern, increases the scatter of diffracted light. The diffractions of light beams are collected and mathematically refocused using specialized computer software.
Attempting to describe crystallography takes some of the complexity and tedium out of a very difficult process, which is still very much trial and error. Some crystals will grow overnight and others take years to grow, making the experiments and the search for results very taxing on researchers.
“It’s a huge relief to know that I have the chance to try something risky that hasn’t been done before and that’s going to make a big impact on medical science,” she said about the BWF award. “It’s extremely technically challenging. It might take me a little while to figure out how to get it to work. However, when it does work it’s going to open up all kinds of new avenues for research.”
By Russ Campbell, communications officer.