Antoine van Oijen used his physics background to design a novel system for observing a flu virus in the process of fusing with a cellular membrane, paving the way for screens of drugs which can prevent viral infection.
Anna Kushnir
Imagine the strides in research a lab could make if a physicist, a microbiologist, a biochemist, and a virologist joined forces and worked together daily to solve some of the longest standing and most interesting problems in science today. Now imagine the pace and progress of that lab if all those scientists are combined into one person. Antoine van Oijen is all of those scientists fused into one talented Harvard Medical School (HMS) researcher.
Toward the end of his Ph.D. studies in soft condensed matter physics at Leiden University in the Netherlands, van Oijen’s work veered toward applying physical techniques to solving biological problems. While he admits his knowledge of biology was shaky at the time, he was hooked. He went on to a post doctoral position in single molecule biophysics, the study of the activities and interactions of single molecules, under the guidance of X. Sunney Xie at Harvard University.
Focusing on single molecules permits quantification of kinetics and characterization of transient intermediates, which cannot be resolved using assays which average the activities of a population of molecules, as would be found in the context of a complex cell. Despite his immersion in biology in the course of his post doc, van Oijen says he had reservations about accepting an assistant professorship in the department of biological chemistry and molecular pharmacology (BCMP) at HMS, as he was worried about finding a common language with biologists. Luckily, he was proven wrong. “This [medical school] is just the best environment to be a physicist,” he now says.
At HMS, van Oijen embarked on a collaboration with Stephen Harrison, a protein crystallographer. They discussed the possibility of developing an assay which would apply single molecule techniques to studying the process of viral entry into host cells, since, van Oijen says, “understanding how fusion proteins mediate viral fusion hasn’t really benefited as much from single molecule approaches as a lot of other fields… so there clearly was a gap there and something we thought we could contribute.” The project flourished with the arrival of an enterprising graduate student, Dan Floyd, intent on developing such an assay using influenza virus.
Influenza, the causative agent of the dreaded flu, has been studied by generations of researchers, making it an excellent model system for study of viral entry. The entry of lipid envelope-enclosed influenza virus particles is known to be mediated by the viral fusion protein, hemagglutinin (HA). HA binds to its cellular receptor, and is activated for fusion by a low pH-triggered conformational change. Through a series of conformational changes, HA forces the viral membrane into close proximity with the cellular membrane. Fusion of the outer leaflets of the membrane bilayers, a fusion intermediate known as hemifusion, is followed by pore formation and expansion, which allows the contents of the viral particle to enter the cell and thus initiate infection.
The system developed by Dan Floyd in van Oijen’s lab, published on September 30 in the Proceedings of the National Academy of Sciences, allowed for the calculation of the time it takes an individual influenza particle to fuse with a target membrane, the number of intermediate steps in the fusion event, and determination of which of the steps are rate limiting. It also allowed for a calculation-based estimate of how many HA molecules are necessary for complete fusion to occur (van Oijen et al. contend it is three), a question that has been something of a holy grail in the viral fusion field. There is no doubt that van Oijen’s background in mathematics and physics aided in carrying out the complicated data analyses which allowed for mathematical modeling of the process of viral fusion.
The single particle fusion system consists of a chamber containing an artificial membrane studded with the influenza virus receptor, mounted under a microscope and camera. The camera records the spread and activation of fluorescent dyes, contained in specially labeled influenza virus particles. The viral lipid envelope is labeled with a lipid-soluble green fluorescent dye, while the genome-containing interior is loaded with a water-soluble, red dye. The introduction of low pH media into the chamber triggers the fusion of the influenza particles with the artificial lipid membrane.
The camera detects the precise moment at which a decrease in pH (marked by a change in a pH-sensitive fluorescent dye in the media) triggers HA to adopt a fusion-competent conformation. This is followed by the spread of green lipid dye, marking hemifusion, and the release of red water-soluble content dye, indicating the completion of fusion. Van Oijen and colleagues then calculate fusion kinetics by focusing on the fluorescent dye spread from individual viral particles, an approach which yields high precision and temporal resolution.
One of the many advances of this system is the successful simulation of a cellular membrane. In a cell, the membrane is fluid, with cholesterol and lipids moving freely. To date, most approaches taken to simulate a membrane have relied on fixing the membrane components to a substrate, thus limiting the potential motion of the lipids. In Floyd’s system, however, lipids float atop a hydrated cushion of high molecular weight polymers. Since the lipids are not adhered or bonded to the cushion, they are able to move much like they would in a cell’s membrane.
While the fusion system itself is a fascinating advance and ranks highly on the “science is cool” scale, a practical question remains – how will this system lead to developments that affect us? How do these findings impact the search for effective antiviral drugs targeting influenza and other viruses? Van Oijen says that his laboratory’s future goals include adapting the current fusion system to a high throughput screen for antivirals that inhibit viral entry. The chamber housing the artificial membrane can be extended and partitioned, allowing the flow of different drug-containing media over the virus particles within the same chamber. The drugs which inhibit mixing of the lipid and aqueous dyes – indicating they block viral entry – will be further tested for antiviral effects. Van Oijen hopes to take this system even further and screen for viral entry inhibitors of other enveloped viruses, such as HIV. Don Coen, a professor in the BCMP department, thinks this technique has promise. “These processes are already targets for FDA-approved antiviral drugs, so these approaches may, at the least, permit a better understanding of drug mechanisms and, possibly, suggest approaches for new therapeutics,“ he said.
The fusion (pun intended) of van Oijen’s physics and biology education led to the development of a technique that required the expertise of both fields. As the boundaries between scientific disciplines blur and evaporate, we should see more novel solutions to old-standing problems.