Northwestern Medicine scientists have identified a component of the herpesvirus that “hijacks” machinery inside human cells, allowing the virus to rapidly and successfully invade the nervous system upon initial exposure.
Led by Gregory Smith, associate professor in immunology and microbiology at Northwestern University Feinberg School of Medicine, researchers found that viral protein 1-2, or VP1/2, allows the herpesvirus to interact with cellular motors, known as dynein. Once the protein has overtaken this motor, the virus can speed along intercellular highways, or microtubules, to move unobstructed from the tips of nerves in skin to the nuclei of neurons within the nervous system.
This is the first time researchers have shown a viral protein directly engaging and subverting the cellular motor; most other viruses passively hitch a ride into the nervous system.
“This protein not only grabs the wheel, it steps on the gas,” says Smith. “Overtaking the cellular motor to invade the nervous system is a complicated accomplishment that most viruses are incapable of achieving. Yet the herpesvirus uses one protein, no others required, to transport its genetic information over long distances without stopping.”
Herpesvirus is widespread in humans and affects more than 90 percent of adults in the United States. It is associated with several types of recurring diseases, including cold sores, genital herpes, chicken pox, and shingles. The virus can live dormant in humans for a lifetime, and most infected people do not know they are disease carriers. The virus can occasionally turn deadly, resulting in encephalitis in some.
Until now, scientists knew that herpesviruses travel quickly to reach neurons located deep inside the body, but the mechanism by which they advance remained a mystery.
Smith’s team conducted a variety of experiments with VP1/2 to demonstrate its important role in transporting the virus, including artificial activation and genetic mutation of the protein. The team studied the herpesvirus in animals, and also in human and animal cells in culture under high-resolution microscopy. In one experiment, scientists mutated the virus with a slower form of the protein dyed red, and raced it against a healthy virus dyed green. They observed that the healthy virus outran the mutated version down nerves to the neuron body to insert DNA and establish infection.
“Remarkably, this viral protein can be artificially activated, and in these conditions it zips around within cells in the absence of any virus. It is striking to watch,” Smith says.
He says that understanding how the viruses move within people, especially from the skin to the nervous system, can help better prevent the virus from spreading.
Additionally, Smith says, “By learning how the virus infects our nervous system, we can mimic this process to treat unrelated neurologic diseases. Even now, laboratories are working on how to use herpesviruses to deliver genes into the nervous system and kill cancer cells.”
Smith’s team will next work to better understand how the protein functions. He notes that many researchers use viruses to learn how neurons are connected to the brain.
“Some of our mutants will advance brain mapping studies by resolving these connections more clearly than was previously possible,” he says.
Sofia V. Zaichick, Kevin P. Bohannon, Ami Hughes, Patricia J. Sollars, Gary E. Pickard, Gregory A. Smith. The Herpesvirus VP1/2 Protein Is an Effector of Dynein-Mediated Capsid Transport and Neuroinvasion. Cell Host & Microbe, 2013; 13 (2): 193 DOI: 10.1016/j.chom.2013.01.009
In separate papers published online in Nature, two research teams report identifying a critical protein that Ebola virus exploits to cause deadly infections. The protein target is an essential element through which the virus enters living cells to cause disease.
The first study was led by four senior scientists: Sean Whelan, associate professor of microbiology and immunobiology at Harvard Medical School; Kartik Chandran, assistant professor at Albert Einstein College of Medicine; John Dye at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) and Thijn Brummelkamp, originally at the Whitehead Institute for Biomedical Research and now at the Netherlands Cancer Institute. The second study was led by James Cunningham, a Harvard Medical School associate professor of medicine at Brigham and Women’s Hospital, and also co-authored by Chandran.
“This research identifies a critical cellular protein that the Ebola virus needs to cause infection and disease,” explained Whelan, who is also co-director of the HMS Program in Virology. “The discovery also improves chances that drugs can be developed that directly combat Ebola infections.”
Both papers are published in the August 24 online issue of Nature.
The African Ebola virus — and its cousin, Marburg virus — are known as the filoviruses. Widely considered one of the most dangerous infections known, Ebola was first identified in 1976 in Africa near the Ebola River, an area in Sudan and the Democratic Republic of the Congo. Infections cause severe hemorrhage, multiple organ failure and death. No one quite knows how the virus is spread, and there are no available vaccines or anti-viral drugs that can fight the infections.
Through conducting a genome-wide genetic screen in human cells aimed at identifying molecules essential for Ebola’s virulence, Whelan and his colleagues homed in on Niemann-Pick C1 (NPC1).
NPC1 has been well known in the biomedical literature. Primarily associated with cholesterol metabolism, this protein, when mutated, causes a rare genetic disorder in children, Niemann-Pick disease.
Using cells derived from these patients, the group found that this mutant form of NPC1 also completely blocks infection by the Ebola virus. They also demonstrated that mice carrying a mutation in the NPC1 gene resisted Ebola infection. Similar resistance was found in cultured cells in which the normal molecular structure of the Niemann-Pick protein has been altered.
In other words, targeting NPC1 has real therapeutic potential. While such a treatment may also block the cholesterol transport pathway, short-term treatment would likely be tolerated.
Indeed in the accompanying paper, Cunningham’s group describes such a potential inhibitor.
Cunningham and his group at Brigham and Women’s Hospital investigated Ebola by using a robotic method developed by their colleagues at the National Small Molecule Screening Laboratory at Harvard Medical School to screen tens of thousands of compounds. The team identified a novel small molecule that inhibits Ebola virus entry into cells by more than 99 percent.
The team then used the inhibitor as a probe to investigate the Ebola infection pathway and found that the inhibitor targeted NPC1.
For Cunningham and Chandran, this finding builds on a 2005 paper of theirs for which Whelan was also a collaborator. In that study, he and his group discovered how Ebola exploits a protein called cathepsin B. This new study completes the puzzle. It now seems that cathepsin B interacts with Ebola in a way that preps it to subsequently bind with NPC1.
“It is interesting that NPC1 is critical for the uptake of cholesterol into cells, which is an indication of how the virus exploits normal cell processes to grow and spread,” said Cunningham. “Small molecules that target NPC1 and inhibit Ebola virus infection have the potential to be developed into anti-viral drugs.”
The paper coauthored by Whelan was funded by the U.S. National Institute of Allergy and Infectious Diseases and the National Human Genome Research Institute, the U.S. Army, and the Burroughs Wellcome Foundation. Cunningham’s work was funded by the New England Regional Center of Excellence for Biodefense and Emerging Infectious Diseases at Harvard Medical School.
- Jan E. Carette, Matthijs Raaben, Anthony C. Wong, Andrew S. Herbert, Gregor Obernosterer, Nirupama Mulherkar, Ana I. Kuehne, Philip J. Kranzusch, April M. Griffin, Gordon Ruthel, Paola Dal Cin, John M. Dye, Sean P. Whelan, Kartik Chandran, Thijn R. Brummelkamp. Ebola virus entry requires the cholesterol transporter Niemann–Pick C1. Nature, 2011; DOI: 10.1038/nature10348
- Marceline Côté, John Misasi, Tao Ren, Anna Bruchez, Kyungae Lee, Claire Marie Filone, Lisa Hensley, Qi Li, Daniel Ory, Kartik Chandran, James Cunningham. Small molecule inhibitors reveal Niemann–Pick C1 is essential for Ebola virus infection. Nature, 2011; DOI: 10.1038/nature10380
The Ebola virus causes a hemorrhagic fever that can be deadly. (up)
Ebola is one virus you never want to catch. Ever.
After some aches and a fever, many infected people develop uncontrolled bleeding. The mortality rates from Ebola infection can run as high as 90 percent.
There’s no cure for Ebola. But a group of scientists is exploring whether some drugs already approved to treat cancer might help tame the virus.
Sounds wild. But there’s a reason — and now some evidence — to think it might work.
To reproduce, the Ebola virus needs the help of cells it invades. And a couple of cancer drugs tweak a human protein that new copies of the virus use to leave their host cells so they can infect others.
The tested drugs — Gleevec and Tasigna, both sold by Novartis — are called tyrosine kinase inhibitors. Tyrosine kinases are enzymes that put a phosphate group on a particular amino acid. Amino acids, as you might remember from high school biology, are the building blocks of proteins.
When a phosphate group gets attached to the right tyrosine block on the right protein, it changes the shape and function of the protein. And that might change everything when it comes to Ebola.
“Proteins are like little machines,” says Emory University’s Dan Kalman, one of the researchers. “As with a machine, they can be turned or turned off. The switch for turning things on or off is a modification. And one of those modifications is a phosphate group.”
In some cancers, the tyrosine kinases help trigger the uncontrolled division of cells. Gleevec and Tasigna help stop that.
When it comes to Ebola, the researchers think drugs like these could turn off a transport protein and could keep new viruses bottled up inside cells.
The Ebola lab work using collections of human cells was published in the latest issue of Science Translational Medicine. It showed that the drugs dramatically decrease the ability of Ebola to replicate. “The effect was quite pronounced,” Kalman told Shots.
And, if the theory holds, such a reduction might be enough to allow an infected person’s immune system to mop up the Ebola viruses.
“Ebola is a very nasty infection,” Kalman says. “The whole concept of containing the disease in a local group before it spreads all over the planet is something clearly we want to do.”
The next step will be to see if the drugs can make a difference in animal experiments.
A team of researchers led by Kasturi Haldar and Souvik Bhattacharjee of the University of Notre Dame’s Center for Rare and Neglected Diseases has made a fundamental discovery in understanding how malaria parasites cause deadly disease.
The researchers show how parasites target proteins to the surface of the red blood cell that enables sticking to and blocking blood vessels. Strategies that prevent this host-targeting process will block disease.
The research findings appear in the Jan. 20 edition of the journal Cell, the leading journal in the life sciences. The study was supported by the National Institutes of Health.
Malaria is a blood disease that kills nearly 1-3 million people each year. It is caused by a parasite that infects red cells in the blood. Once inside the cell, the parasite exports proteins beyond its own plasma membrane border into the blood cell. These proteins function as adhesins that help the infected red blood cells stick to the walls of blood vessels in the brain and cause cerebral malaria, a deadly form of the disease that kills over half a million children each year.
In all cells, proteins are made in a specialized cell compartment called the endoplasmic reticulum (ER) from where they are delivered to other parts of the cell. Haldar and Bhattacharjee and collaborators Robert Stahelin at the Indiana University School of Medicine – South Bend (who also is an adjunct faculty member in Notre Dame’s Department of Chemistry and Biochemistry), and David and Kaye Speicher at the University of Pennsylvania’s Wistar Institute discovered that for host-targeted malaria proteins the very first step is binding to the lipid phosphatidylinositol 3-phosphate, PI(3)P, in the ER.
This was surprising for two reasons. Previous studies suggested an enzyme called Plasmepsin V that released the proteins into the ER was also the export mechanism. However, Haldar, Bhattacharjee and colleagues discovered that binding to PI(3)P lipid which occurs first is the gate keeper to control export and that export can occur without Plasmepsin V action. Further, in higher eukaryotic cells (such as in humans), the lipid PI(3)P is not usually found within the ER membrane but rather is exposed to the cellular cytoplasm.
Haldar and Bhattacharjee are experts in malaria parasite biology and pathogenesis. Stahelin is an expert in PI(3)P lipid biology, and David and Kaye Speicher are experts in proteomics and a method called mass spectrometry.
A biology lab at Washington University has just cracked the structure and function of a protein that plays a key role in the life of a parasite that killed 655,000 people in 2010.
A cartoon based on the electron density map makes it easier to see the protein’s structure and figure out how it works. The enzyme’s job is to add a methyl group — three times — to a starting molecule as part of a process for making cell membranes. In this cartoon, the phosphate is a stand-in for the starting molecule and the green molecule is the one that donates a methyl group. Both are positioned in the active site of the enzyme, the pocket where the chemistry takes place. (Credit: Jez)
The protein is an enzyme that Plasmodium falciparum, the protozoan that causes the most lethal form of malaria, uses to make cell membrane.
The protozoan cannot survive without this enzyme, but even though the enzyme has many lookalikes in other organisms, people do not make it. Together these characteristics make the enzyme an ideal target for new antimalarial drugs.
The research was published in the January 6 issue of the Journal of Biological Chemistry (JBC) as “Paper of the Week” for that issue.
The work also will be featured in ASBMB Today (the newsletter of the American Society for Biological Molecular Biology, which publishes JBC).
Sweating the cold room
The protein’s structure might have remained an enigma, had it not been the “unreasonable optimism” of Joseph Jez, PhD, associate professor of biology in Arts & Sciences, which carried his team through a six-year-long obstacle course of failures and setbacks.
“What my lab does is crystallize proteins so that we can see what they look like in three dimensions,” Jez says. “The idea is that if we know a protein’s structure, it will be easier to design chemicals that would target the protein’s active site and shut it down,” Jez says.
The latest discovery is the culmination of a project that began years before when Jez was working at the Danforth Plant Science Center in St. Louis and collaborating with scientists at the local biotech startup Divergence. “At the time, C. elegans had just been sequenced and the Divergence scientists were looking at using it as an easy model to work out the biochemistry of parasitic nematodes,” Jez says.
C. elegans is a free-living nematode, or microscopic roundworm, but many nematodes are parasitic and cause disease in plants, livestock and people.
During this project, Lavanya Palavalli, a summer intern working with Jez, crystallized the C. elegans version of the enzyme. The job of the enzyme, phosphoethanolamine methyltransferase, thankfully abbreviated to PMT, is to add methyl groups to a starting molecule, phosophoethanolamine.
“When Soon Goo Lee later took up the project,” says Jez, “the plan was to try to grow better crystals of the C. elegans protein, ones good enough to get readable X-ray diffraction patterns.
Two years later, the crystals were looking better but still not good enough.
So Jez suggested that Lee go after homologous (look-alike) proteins in other organisms. “Even though the proteins are homologous, each has a different amino acid sequence and so will behave differently in the crystallizations,” Jez says. “Lee went from working with two C. elegans proteins to three plant proteins, two other nematode proteins and then the Plasmodium protein,” Jez says.
“He took all six of those PMT versions into the crystallization trials to maximize his odds,” Jez says.
“To crystallize a protein,” Jez says, “we put a solution of a salt or something else that might work as a desiccant in the bottom of a small well. And then we put a drop of our liquid protein on a microscope cover slip and flip it over the top of the well, so the drop of protein is hanging upside down in the well.”
“What we’re trying to do is to slowly withdraw water from the protein. It’s exactly like making rock candy, only in that case, the string hanging into the jar of sugar solution helps to withdraw water,” he says.
The difference is that sugar wants to form crystals and proteins are reluctant to do so.
“There are 24 wells to a tray, and we usually screen 500 wells per protein at first,” Jez says. “Lee had eight proteins and so his first pass was to screen 4,000 conditions. And then he had to try different combinations of ligands to the proteins and crystallize those. This is why it took a few years to finally get where he needed to go.”
The scientists need crystals — preferably nice, big ones — to stick in the path of an X-ray beam at Argonne National Laboratory in Chicago. (If the crystal is a good one, and all the atoms are lined up in a repeating array, the scattered X-rays will produce a clear pattern of spots.)
Embedded in that pattern is the mathematical information needed to back-calculate to the position of the atoms in the protein, a process a bit like throwing a handful of pebbles in a lake and then calculating where they landed by the pattern of waves arriving at the shoreline.
Lee got the PMT from Haemonchus contortus to crystallize first, but there were technical issues with the diffraction pattern that would have made solving it technically and computationally very demanding.
“When the Plasmodium enzyme finally crystallized, Soon got four crystals kind of stacked on top of each other and each of them was paper thin,” Jez says.
“I never thought it would work, but we took them to Argonne anyway and he actually did surgery under the microscope and cracked off a little tiny piece of it.”
To everyone’s surprise, he got a clean diffraction pattern from the crystal. “Because the Plasmodium enzyme was the smallest one and the easiest to work on, we pushed that one first,” Jez says.
The moment of truth
“Once we had a Plasmodium crystal that was diffracting really well, we could try back-calculating to see whether we could extract the atom positions from the data,” Jez says.
After the computer finished its calculations, Lee clicked a mouse button to see the results, which would reveal whether his years of work finally would pay off.
When Lee clicked the mouse, he got an electron density map in exceptionally sharp focus.
“When you see a map like that, it’s like suddenly the wind has kicked up and you’re sailing free,” Jez says, “because there’s this moment, like, before you click that button, no one has ever seen how this protein is put together in three dimensions. You’re the first person to ever see it.
“The irony of it is we got such good quality diffraction pattern and electron density maps off such an ugly crystal,” he says.
Lock and load
“Once you have the electron density map, the task is to build a structure that matches the amino acid sequence of the protein,” Jez says.
“The first thing you do is put in the amino acid backbones and connect them together to form a chain. It’s like having a long thread, each inch of which is an amino acid, and your job is to take that thread and move it in three dimensions through that electron density map.”
The next step is to add the side chains that make one amino acid different from another, Jez says. “The amino acid sequence is known,” he says. “Your goal is to match the way you string together the amino acids in the electron density map to that sequence.”
“Once you have the overall structure, you can start to figure out how the enzyme works. The PMT enzyme is trying to join two molecules,” Jez says. “To do that, it has to lock them in place so that the chemistry can happen, and then it has to let go of them.
“We think the protein has a lid that opens and closes,” he says. “The active site stays open until the substrates enter, and then the lid clamps down, and when it clamps down it actually puts the substrates together.”
Calling Bill and Melinda Gates
Not only do infections by Plasmodium falciparum cause the most severe form of malaria, about 40 percent of the human population lives in areas where the parasite is endemic. Moreover, drugs that used to be effective against malaria are beginning to fail, in part because widespread drug counterfeiting has led to resistance.
New anti-malarial drugs are desperately needed, and the PMT protein is an ideal target. If PMT is disabled, the protozoan can’t make cell membranes and it dies. Moreover, a drug that would kill Plasmodium might have minimal side effects on patients.
Although the process of identifying compounds that would target PMT is in the early stages, a handful of anti-parasitical compounds used to treat diseases are known to block PMT as well.
As for Lee, he has had a hard go of it, but now things are breaking his way. Plasmodium PMT is giving up its secrets, and the plant and nematode PMTs are coming along as well.
When he clicked the mouse button and a clean electron density map came up, he says, it was like seeing “the light at the end of a five-year-long tunnel.”
S. G. Lee, Y. Kim, T. D. Alpert, A. Nagata, J. M. Jez.Structure and Reaction Mechanism of Phosphoethanolamine Methyltransferase from the Malaria Parasite Plasmodium falciparum: AN ANTIPARASITIC DRUG TARGET. Journal of Biological Chemistry, 2011; 287 (2): 1426 DOI:10.1074/jbc.M111.315267
The same menace that spreads malaria — the mosquito bite — could help wipe out the deadly disease, according to researchers working on a new vaccine at Tulane University.
(left) Nirbhay Kumar, professor of tropical medicine at Tulane University, is working on a vaccine that aims to wipe out malaria using the same menace that spreads it — the mosquito bite. (Credit: Image courtesy of Tulane University)
The PATH Malaria Vaccine Initiative (MVI), established in 1999 through a grant from the Bill & Melinda Gates Foundation, announced February 15 a collaboration with Tulane University School of Public Health and Tropical Medicine and India’s Gennova Biopharmaceuticals Ltd. to produce and test a novel vaccine that aims to inoculate mosquitoes when they bite people.
The vaccine would work by triggering an immune response in people so they produce antibodies that target a protein the malaria parasite needs to reproduce within a mosquito.
Malaria, which kills nearly 800,000 people every year worldwide, is caused by a microscopic parasite that alternates between human and mosquito hosts at various stages of its lifecycle. Once a mosquito bites a vaccinated person, the antibodies would neutralize the protein essential for malaria parasite’s reproduction, effectively blocking the parasite’s — and the mosquito’s — ability to infect others.
The vaccine relies on a protein — known as Pfs48/45 — which is very difficult to synthetically produce, says Nirbhay Kumar, professor of tropical medicine at Tulane.
“With MVI’s support we can now work with Gennova to produce sufficient quantity of the protein and develop a variety of vaccine formulations that can be tested in animals to determine which one give us the strongest immune response,” Kumar says.
Such transmission blocking vaccines, though not yet widely tested in humans, are attracting widespread interest due to their potential to be used in conjunction with more traditional malaria vaccines and other interventions — such as malaria drugs and bed nets — to make gradual elimination and even eradication of the disease a reality.
“We’re investing in developing transmission blocking malaria vaccines to support two long-term goals: introducing an 80 percent efficacious malaria vaccine by the year 2025 and eventually eradicating malaria altogether,” says Dr. Christian Loucq, director of MVI. “A vaccine that breaks the cycle of malaria transmission will be important to our success.”
The above story is reprinted (with editorial adaptations by ScienceDaily staff) from materials provided by Tulane University.