A team of researchers at The Scripps Research Institute (TSRI; La Jolla, CA) has solved the structure of the viral machinery that the Lassa virus uses to enter human cells and cause Lassa fever (an acute viral illness that occurs in West Africa). X-ray laser beams from the Stanford Synchrotron Radiation Lightsource (SSRL) at the Department of Energy (DOE)'s SLAC National Accelerator Laboratory (Menlo Park, CA) allowed them to show a key piece of the viral structure, called the surface glycoprotein, for any member of the deadly arenavirus family, and the new structure provides a blueprint to design a Lassa virus vaccine.
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X-ray data for this study was collected at SLAC and the DOE's Argonne National Laboratory (Argonne, IL). For the SLAC experiments, the researchers used a station at SSRL, a DOE Office of Science User Facility that has a strong program in biological x-ray crystallography. In this method, scientists prompt biological molecules to align and form a crystal, which they then study with powerful x-rays. The way the x-rays scatter off the crystal reveals the structure of the molecules inside—in 3D and with atomic detail.
The effort began with TSRI staff scientist Kathryn Hastie, the lead author of a paper describing the work. In 2007, then a graduate student in the lab of Erica Ollmann Saphire, a professor of Immunology and Microbial Science from TSRI and senior author of the new study, she told her thesis committee she wanted to solve the structure of the assembled arenavirus glycoprotein, hoping to create a map of the target on the virus where antibodies need to attack—a key step in developing a vaccine.
Such maps can be obtained with x-ray crystallography, but the method depends on having a stable protein. Yet, all the Lassa virus glycoprotein wanted to do was "fall apart." Glycoproteins are made up of smaller subunits, while other viruses have bonds that hold the subunits together "like a staple," Hastie says. Arenaviruses don't have that staple, so the subunits just floated away from each other whenever Hastie tried to work with them.
Another challenge was to recreate part of the viral lifecycle in the lab—a stage when Lassa's glycoprotein gets clipped into two subunits. "We had to figure out how to get the subunits to be sufficiently clipped, which is necessary to make the biologically functional assembly, and also where to put an engineered staple to make sure they stayed together," Hastie says.
By creating mutant versions of important parts of the molecule, Hastie engineered a version of the Lassa virus surface glycoprotein that didn't fall apart. She then used this model glycoprotein as a sort of magnet to find antibodies in patient samples that could bind with the glycoprotein to neutralize the virus.
With this latest study, she solved the structure of the Lassa virus glycoprotein, bound to a neutralizing antibody from a human survivor. Her structure showed that the glycoprotein has two parts: she compared the shape to an ice cream cone and a scoop of ice cream, where a subunit called GP2 forms the cone and the GP1 subunit sits on top. They work together when they encounter a host cell. GP1 binds to a host cell receptor, and GP2 starts the fusion process to enter that cell.
The new structure also showed a long structure hanging off the side of GP1, like a drip of melting ice cream running down the cone. This "drip" holds the two subunits together in their pre-fusion state.
Zooming in even closer, Hastie discovered that three of the GP1-GP2 pairs come together like a tripod. This arrangement appears to be unique to Lassa virus. Other viruses, such as influenza and HIV, also have three-part proteins (called trimers) at this site, but their subunits come together to form a pole, not a tripod. The structure is also important because it can be used as a model to conquer related viruses throughout the Americas, Europe, and Africa for which no equivalent structure yet exists.
This tripod arrangement offers a path for vaccine design. The scientists found that 90% of the effective antibodies in Lassa patients targeted the spot where the three GP subunits came together. These antibodies locked the subunits together, preventing the virus from gearing up to enter a host cell.
A future vaccine would likely have the greatest chance of success if it could trigger the body to produce antibodies to target the same site. The next step is to test a vaccine that will prompt the immune system to target Lassa's glycoprotein.
Full details of the work appear in the journal Science; for more information, please visit http://dx.doi.org/10.1126/science.aam7260.