MICROBIOLOGY

Lethal New World Viruses Put Iron Grip on Cells

Seize Iron Receptor for Infection Leading to Hemorrhagic Fever

Scientists first isolated the Machupo virus in 1963, during an epidemic smoldering in a tropical savannah in eastern Bolivia. The clinical symptoms matched an outbreak a few years earlier in neighboring Argentina by another deadly and previously unknown virus. It took a year for researchers to figure out how to stop new infections at their source: by trapping and killing common village mice whose saliva, urine, or feces transmitted virus particles to humans in the air and on food. Since then, two related viruses have emerged—Guanarito in Venezuela and Sabiá in Brazil. Altogether, the four viruses have killed thousands. There are no vaccines or proven treatments, except for the Argentine Junin virus (an experimental vaccine and, for treatment, the plasma of recovering patients). Until now, no one knew exactly how the viruses infect people.

Photo by Graham Ramsay

A family of often deadly hemorrhagic fever viruses found in the Americas invades cells through the well-known revolving molecular door that admits the essential nutrient iron, says a new study by (from left) Michael Farzan, Hyeryun Choe, Jonathan Abraham, Sheli Radoshitzky, and colleagues.

Recently, in the course of studying microbes with bioterrorism potential, HMS researchers have discovered that Machupo and its three deadly viral cousins enter cells through the well-known molecular revolving door for the essential nutrient iron.

The study suggests that iron deficiency, which can triple or quadruple the number of iron receptors on cells, may make people more susceptible to infection or the disease more severe.

The findings, reported on Feb. 7 in Nature online, also point to an immediate therapeutic possibility—receptor antibodies that are being tested for an entirely different medical purpose might treat or prevent infection.

Quick Hit
The story is remarkable, in part, for the deceptive ease and speed of the receptor discovery and for the way it links to other HMS scientists’ hard-won expertise on the iron transport receptor and iron metabolism.

In a larger context, the discovery also ushers in the possibility of new insights into the ecology and evolution of the viruses, their rodent hosts, the molecular mechanisms of human infection, and the potential for other outbreaks and emerging diseases brought on by forces as disparate as biological warfare, global climate change, and genetic mutations.

“We were lucky that it was easy this time,” said Hyeryun Choe, HMS assistant professor of pediatrics at Children’s Hospital Boston. Choe’s team studies three families of the most deadly hemorrhagic fever viruses in the world: arenaviruses, including those in this study; filoviruses, including Ebola and Marburg; and bunyaviruses, including hantaviruses. All are ranked as top-priority pathogens on the National Institute of Allergy and Infectious Diseases’s biodefense research agenda. “The Machupo virus entry protein is very small, easy to handle, and seemed to have a high affinity to the receptor,” she said.

Technically, the researchers work with pseudoviruses. These virionlike particles display the entry proteins, but lack the virulent portions that first provoke fever, muscle aches, and fatigue, often progressing to bleeding under the skin and from body orifices and internal organs, followed by shock, coma, seizures, and nervous system malfunction.
Despite Choe’s disclaimer, luck had little to do with it. Choe and her collaborator (and husband) Michael Farzan, HMS assistant professor of microbiology and molecular genetics at the New England Primate Research Center, have accumulated much knowledge and experience from other viruses they have studied over the years. Their groups identified a key HIV-1 coreceptor (CCR5) 10 years ago. Three years ago, they reported the SARS coronavirus receptor ACE2 (see Focus, Dec. 12, 2003).

For almost every virus, the full entry protein is loaded with camouflage carbohydrates and protein pieces to hide the critical receptor-binding domain from the immune system. “We learned from HIV and SARS that if you chop off the viral entry protein here and there, you can make them bind the receptor better,” Choe said. “That’s the trick we played.”

They call the method “pull down.” They slice and dice the entry protein into short pieces based on their best guesses of what makes up the essential part of the binding domain. They attach those pieces to tiny beads and mix them with cells known to be infected by the virus. Then they lyse the cells and wash away everything except the beads and the entry protein and whatever is tightly bound to it. Mass spectrometry and a computer-based protein sequence match and identify the bound proteins.

So how easy was it? HMS first-year MD–PhD student Jonathan Abraham pulled down transferrin receptor 1 (TfR1), using the Machupo virus entry protein, while he was on a summer rotation in the Choe lab. A week later, he started medical school. Once the high-affinity version of the entry protein was made, “it worked on the first try,” he said. Abraham expects more of a struggle with his thesis project: solving the X-ray crystal structure of the entry protein bound to the receptor.

Co-first author Sheli Radoshitzky, a graduate student in the collaborating Farzan lab, verified that the three other New World arenaviruses known to cause hemorrhagic fever in the Americas also show a strong affinity for the same receptor, in contrast to two Old World arenaviruses from Africa, including Lassa, neither of which favored the receptor.

“It’s not surprising that the New World and the Old World arenaviruses have evolved to use different receptors,” said biologist and pathologist Terry Yates, vice president for research at the University of New Mexico. “Even though the viruses use rodents in the same family, the rodents have been geographically isolated from each other for 20 million years and have co-evolved with the viruses.” Yates believes rodents brought hantaviruses and arenaviruses with them when they crossed into the Americas over the Bering Strait land bridge and that many undiscovered viruses lurk within different rodent populations, such as the recently discovered Whitewater Arroyo virus in wood rats in New Mexico.

The Bugs’ MO
Iron metabolism expertise in the Children’s Hospital research group of Nancy Andrews, who is the Leland Fikes professor of pediatrics and dean for basic sciences and graduate studies at HMS, sped up the next round of experiments.

“I’m not an immunologist, but I think it’s an ingenious way for the virus to invade a cell,” said Paul Schmidt, a postdoctoral research fellow in the Andrews lab. With or without an iron complex, Schmidt said, the receptor continually cycles in and out of cells. Iron levels regulate the receptor numbers, liberally peppering the cell surface when levels are low and depleting their ranks when iron levels rise. Every cell has receptors, because every cell needs iron. Fast-dividing cells, including macrophages and activated lymphocytes involved in fighting the viral infection, as well as cancer cells, have receptors in particular abundance.

The New World arenaviruses seem to use a different part of the receptor than the iron complex to enter cells, Radoshitzky and Abraham found, but the viruses infect cells more easily when more receptors are made available by iron depletion. One of several antitumor antibodies targeting TfR1 blocked replication of the New World viruses—Machupo, Junin, Guanarito, and Sabiá—but not the two Old World viral controls. The findings need to be verified in animal studies, said Choe, who is also following up evidence of a co-receptor involved in one or more of the viruses.

Image courtesy of Stephen Harrison

The butterfly effect. New World hemorrhagic fever viruses enter human cells through the transferrin receptor 1 (above), whose structure was first solved by researchers in the lab of Stephen Harrison, a Howard Hughes investigator and HMS professor of biological chemistry and molecular pharmacology. Thomas Walz, HMS assistant professor of cell biology, and his colleagues showed that the iron-loaded transferrin binds to the bottom lobes of the butterfly-shaped dimer receptor (see Focus, March 5, 2004). The arenaviruses probably bind to the top and do not seem to interfere with iron transport.

Researchers at the U.S. Centers for Disease Control and Prevention found the same results when they repeated the experiments with the full viruses in cell cultures at the CDC’s biosafety level 4 containment facility near Atlanta.
In the absence of a structural picture of the receptor–entry protein complex, Radoshitzky is conducting mutagenesis studies for insights into how different features of the receptor affect the viral–entry protein binding. Farzan’s lab is also studying TfR1 of wild rodents, the natural reservoir, to learn why some viruses can jump from animals to humans, but others cannot. All the information will help scientists screen for more antiviral compounds that inhibit the crucial molecular interactions.

The research is supported in part because of bioterrorism concerns, but other experts see much broader implications in the work.

“They have found something important,” said Karl Johnson, who is writing a book about his discovery of Machupo virus in Bolivia. “My next question is, why, with 20-some New World arenaviruses, are there only four that are human pathogens? If it turns out there are differences in the hosts or receptors that pathogenic and nonpathogenic viruses use, it would mean that every time a new one is found, you could rapidly say whether or not to worry about it.”

—Carol Cruzan Morton

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