A U.S. research team has discovered a protein called Niemann-Pick C1 (NPC1), which they found was responsible for allowing Ebola virus to enter and replicate within cells.
Researchers from Whitehead Institute, Harvard Medical School, Albert Einstein College of Medicine and U.S. Army Medical Research Institute of Infectious Diseases have joined forces to better understand Ebola virus in order to eventually lead to the development of a vaccine that prevents Ebola hemorrhagic fever (EHF).
Ebola virus is classified as a category A bioterrorism agent by the U.S. Centers for Disease Control and Prevention (CDC). While Ebola virus outbreaks are rare, they can cause EHF and are extremely dangerous to humans. Those with EHF have symptoms such as fever, diarrhea, vomiting, intense weakness, joint and muscle pain, and sometimes external or internal bleeding due to the disintegration of blood vessels.
While the symptoms of EHF can be treated, there is currently no vaccine to prevent it. According to the World Health Organization (WHO), there have been 1,850 EHF cases with 1,200 deaths since the virus was found in 1976.
Thijn Brummelkamp, former Whitehead Fellow and current leader at the Netherlands Cancer Institute (NKI) said, “Right now, people make therapeutics to inactivate the pathogen itself…But the problem is that pathogens can quickly change and escape detection and elimination by the immune system. Here we get a good idea of the host genes that are needed for the pathogen to enter the cell for replication. Perhaps by generating therapeutics against those host factors, we would have a more stable target for antiviral drugs.”
The team of researchers discovered how Ebola virus gained entry into a host cell by using an unusual human cell line. All humans receive one copy of each chromosome from each parent, but cell lines have a single set instead with only one copy of each individual gene. Researchers wanted to use gene disruption, which “knocks out” a gene function in host cells one-by-one, to silence one copy of a gene.
The effects of the active Ebola virus were tested on mice who had a knocked-out copy of the NPC1 gene and mice with two functioning NPC1 genes. Those with two functioning NPC1 genes were affected by the virus very quickly while those with a knocked-out NPC1 gene were significantly protected.
Kartik Chandran, a professor of microbiology and immunology at Albert Einstein, is a senior author of the study. “The critical step that we were studying is what we call viral entry," Chandran explained. "And it’s basically the step that results in the virus getting into the cytoplasm where the [genetic] goodies are for making copies of itself.” Researchers looked at normal cell proteins that the Ebola virus might be hijacking, in effect, to get inside and infect mammalian cells.
Chandran says that in experiments with both human cells and in mice, the Ebola virus was unable to gain a toehold in cells that were missing the NPC1 protein. “You couldn’t infect the cells with Ebola. And there are also mice that, like the human[s], don’t have the protein and develop Neimann-Pick disease," Chandran said.
Chandran says the disease progresses over time. But to prevent or treat an Ebola infection, he thinks it might be possible to design a small molecule that interferes with production of the Neimann-Pick protein in cells temporarily -- too briefly to cause problems with elevated cholesterol. Chandran says such a compound would not have to totally block production of the NPC1 protein.
“You know the [Ebola] virus, it’s like ‘shock and awe.’ It’s like over within a week," noted Chandran. "I mean the virus grows very quickly and it kills off the very cells you need to mount your immune response. If we could stop that from happening or slow it down enough, we might give the person a chance.”
Two articles by Chandran and colleagues on the biochemical keys involved in Ebola virus infections are published in the journal Nature.
In the other study, hematologist James Cunningham of Harvard Medical School and his colleagues tested tens of thousands of compounds. The researchers used a robotic device to scan lab plates containing cells exposed to Ebola while various compounds were added. One called benzylpiperazine adamantane diamide 3.0 seemed to protect against the virus. The researchers made roughly 100 variations on the 3.0 compound, Cunningham says, and came up with several that inhibited the virus’s infectivity. These compounds, the researchers found, interfere with NPC1.
“These are important new findings,” says virologist Judith White of the University of Virginia School of Medicine. “What’s really powerful is that these two very different approaches converged on the same molecule. That makes it very convincing.”
NPC1 now becomes the second human protein implicated in Ebola virus infection. Chandran, Cunningham and others reported in 2005 that an enzyme called cathepsin B shears off part of Ebola’s protein coat as part of the infection process. Chandran says the next step will be to test the NPC1-neutralizing compounds against Ebola in other animals, preferably monkeys.
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