Since the first outbreak of Ebola virus in 1976, the frequency and scale of the deadly disease’s outbreaks have increased. The 2014-2015 outbreak in West Africa caused 11,000 deaths, precipitated panic at airports and emergency rooms worldwide, and renewed the urgency to find cures.
Kartik Chandran’s group has zeroed in on what they believe to be one of the most vulnerable areas for drug targeting—viral entry into the host cell.
“We’re really interested in the initial step of Ebola virus infection—from the virus sticking to the surface of the cell all the way to entering the cytoplasm of the cell where all the goodies are that viruses need to replicate themselves,” says Chandran, a virologist at Albert Einstein College of Medicine in Bronx, New York.
Like other viruses in the Filoviridae family (filoviruses), Ebola virus is an enveloped RNA virus that uses a glycoprotein ‘spike’ molecule (GP) to navigate its way into host cells. The virus attaches to a cell and then coaxes the cell to engulf it and deliver it to a membrane compartment called the endosome, the first of a series of compartments that lead to lysosomes, the recycling yards of the cell.
Once there, GP binds to host cellular factors to bring the endosomal membrane and viral membranes close enough to meld together, ultimately dumping the virus’ RNA genome into the cell’s cytoplasm where it can replicate. However, exactly how GP and its host partners cause the energetically unfavorable proccess of fusion to occur is unclear.
“All filoviruses, including Sudan and Marburg viruses, exploit the same host factors [to get inside the cell],” explains Chandran. “If we can block those interfaces, we might be able to develop a treatment that works against all of these viruses.”
He and his team, led by postdoctoral fellow and virologist Jennifer Spence, set out to clarify how GP works with cellular factors to initiate and complete fusion. They published their findings this week in mBio. “We wanted to visualize the entire process in live cells—from single virus particles binding to cells, to their being trafficked inside the cell and fusing inside endosomes,” says Spence.
To do so, the team engineered the relatively benign vesicular stomatitis virus (VSV) to carry the Ebola GP protein, and labeled the viral membranes with a lipophilic, self-quenching dye. When the virus was mixed with human cells in a lab dish, Spence could follow fusion events in real time under the microscope. As viral membranes initially fused with the cell’s unlabeled endosomal membrane, the dye’s local concentration dropped and it de-quenched, emitting a flash of fluorescence.
As Spence explains, researchers already knew that Ebola virus GP first has to be cleaved by cellular proteases called cathepsins, and then must bind to a cellular protein called NPC1 for infection to occur. “But we didn’t know the precise role of GP-NPC1 binding in the Ebola entry process.”
The first thing Spence did was show that even the initial step of fusion, lipid mixing, could not take place if the human cells she used did not contain the NPC1 protein. It also wouldn’t work if the Ebola GP molecule had three mutations in it that blocked its ability to bind NPC1. This meant that the GP-NPC1 binding step is necessary for Ebola fusion to be triggered. Importantly, the team also demonstrated that the ZMapp™ antibody cocktail used to treat some Ebola survivors last year was indeed working by blocking GP’s role in fusion.
Next, Spence also looked at mutations in the domain of GP that mediates fusion, showing that one mutation allowed lipid mixing, but not full fusion, and another prevented lipids from mixing at all.
Finally, the team showed that even after the initial lipid mixing stage, the host cathepsin proteases were still needed for full fusion and viral infection to occur. Chandran speculates that these enzymes’ actions are needed again for the most energetically costly step when GP “jackknifes on itself” smushing and holding the cellular and viral membranes together to form a stable fusion pore that is big enough for infection to happen (see animation).
By watching individual virus particles fusing in real-time, Spence and Chandran also learned that very few particles actually make it all the way to the infection stage to deliver viral genes to the cell’s innards. “The propensity for viruses to fuse is pretty low,” says Chandran. “About only 5-10% of particles actually initiate fusion and only 1-2% of them are going to infect the cell.”
Ultimately, Chandran and Spence would like to rebuild the entire Ebola virus entry process from start to finish in their real-time laboratory system. “This is a very useful system for understanding the role of cellular factors during viral fusion, and for testing antiviral drugs and antibodies,” says Chandran.
He notes that drug or vaccine developers could then focus on disrupting those interactions between cellular and viral components: “Preventing the virus from binding NPC1 would be a great way to block Ebola virus infection.”
For more on the Ebola research in Chandran’s laboratory, see this movie made by Albert Einstein College of Medicine.
How the Ebola virus glycoprotein is thought to mediate fusion between cellular and viral membranes. Animation provided by Erica Ollman Saphire's group at The Scripps Research Institute in La Jolla, California.