The surfaces of human cells are chock-full of proteins that help cells communicate with one another. Unfortunately, many viruses coopt these proteins, latching on in order to enter cells. By revealing which proteins are receptors for which viruses, researchers may be able to develop more targeted therapies and offer a valuable tool for designing new vaccines.
But linking a virus to a specific receptor is no simple feat. “The receptors that mediate infection remain unknown for most pathogens,” says biochemist Nadia Martinez-Martin of Genentech in South San Francisco, California. Now, Martinez-Martin and Genentech colleagues, working with researchers from Swiss research institutions, have built a library of cell surface proteins that offers a powerful new tool for matching pathogens to receptors.
Their work, published in Cell, revealed a long sought-after receptor for cytomegalovirus, a herpes virus that infects more than 70 percent of people and can cause congenital birth defects. But the technology promises to reveal more. “We have a method to understand the cellular receptors for mostly any pathogen that you can think of,” says Martinez-Martin.
The team already knew that a protein complex known as “trimer” on the cytomegalovirus latches onto a specific receptor protein on fibroblasts, cells that make up connective tissue. The trouble is, the virus infects a range of cells, especially epithelial cells. To stop the attack, researchers wanted to understand how the virus invades this other cell type. They knew that a protein complex known as a “pentamer” on the virus was responsible, but they didn’t know which receptor protein on the epithelial cells it was drawn to. It was a “missing piece” that both academic and biopharmaceutical institutions had spent decades searching for, says senior author Claudio Ciferri, a structural biologist at Genentech.
Finding this missing piece was historically difficult, in part, because one of the primary tools—a method called affinity purification-mass spectrometry—worked best when the interaction between proteins was very stable. The interaction between a cell surface protein and a virus is often weak.
The team began by harnessing a technique known as AVEXIS that involves bunching multiple copies of viral proteins together. This “bouquet” of viral proteins, as Martinez-Martin describes it, makes the interaction with the cell surface receptors more stable.
The researchers then used a high-throughput technique to test around 2,000 protein interactions at once. They synthesized the DNA sequences for nearly every human gene known to encode for single-transmembrane cell surface proteins, a group of proteins that pass through a cell’s membrane once and are receptors for many pathogens. They expressed these nearly 1,300 DNA sequences in human cell lines. The cells secreted the receptors coded by these gene sequences. The researchers then captured the proteins and adhered them to wells within a microplate; each well contained one individual receptor type.
When they introduced the cytomegalovirus trimer to this cell surface protein library, they found, as expected, a strong connection to the receptor for fibroblast cells. When they introduced the cytomegalovirus pentamer, they finally found that missing piece—a strong connection with a cell surface protein known as “neuropilin-2.”
Scientists had already determined the individual structures of the virus’s pentamer protein complex and neuropilin-2 using an imaging technique known as x-ray crystallography. The team used a form of electron microscopy to reveal how the two proteins fit together.
“Now, therapeutically, it would be possible to attack two ends, one end on the virus and one end on the cells,” says Ciferri. But the broader importance of the method, Ciferri says, is that it allows researchers studying both bacterial and viral pathogens to “find a receptor that until now was completely unknown and then try to understand what needs to be done to block it.”
This work is “a real tour de force of extracellular virus receptor discovery,” says biochemist Gavin Wright of the Wellcome Sanger Institute in England, who developed the AVEXIS technique used by this team, but was not involved in the study. “Hopefully they can use the same methods to find more host virus receptors.”
This screen should work for many viruses, says virologist Judith White of the University of Virginia, who was not involved in the study. “It really is a big advance,” she notes. But she adds that the technology wouldn’t work for all pathogens. The Ebola virus, her group’s focus, uses a multi-membrane spanning protein receptor that is actually inside of the cell. The virus connects rather loosely to a range of molecules on the surfaces of different types of cells before getting taken into the cell and latching onto a key receptor.
The Genentech team says that they focus on proteins on the cell surface because this is where the majority of pathogens initially target receptors. But they are working to expand their library of cell surface proteins to include multi-transmembrane proteins that wind back and forth through the cell membrane multiple times. They hope that this broader effort will reveal receptors for an even wider range of pathogens, though the receptors for crafty pathogens like Ebola may escape detection.