At a glance:
- Researchers have built a tool to safely study, at scale, the proteins made by hundreds of viruses that can cause disease in humans.
- The work enables deeper investigation into how viruses disrupt cells, promising to help scientists develop treatments and vaccines and prepare for emerging pathogens.
- The tool has already revealed previously unknown ways that viral proteins hijack cells' garbage-disposal machinery and evade the immune system.
To prevent viruses from sickening or killing us — whether it's an individual case of hepatitis B or a COVID pandemic — it's crucial to understand how the proteins they make initiate changes in our bodies that allow them to flourish.
A new tool has just vastly broadened the scale at which researchers can study these proteins, promising to speed basic discoveries in virology, inform the development of new vaccines and treatments, and help humanity protect against emerging pathogens.
The tool, called a viral ORFeome and described July 2 in Cell , is the largest yet of its kind and enables analysis of many thousands of viral proteins in a single experiment. Its design also expands access to biologists who didn't train in virology.
"This library reveals how viruses manipulate human cells on a scale that simply wasn't possible before," said senior author Stephen Elledge , the Gregor Mendel Professor of Genetics and of Medicine at Harvard Medical School and Brigham and Women's Hospital, whose team led the creation of the tool.
"We believe it changes virology from studying one virus at a time to discovering the common strategies and surprising innovations that viruses have evolved, providing a powerful new foundation for understanding emerging viral threats," he said.
The Cell paper shows how the team has already used the ORFeome to identify hundreds of viral proteins that interfere with immune response. In a second paper, published July 9 in Science , Elledge and colleagues delve into new insights it has revealed into how viruses hijack cells' garbage-disposal systems to evade immune attack.
The largest-yet library of viral proteins
It's estimated that nearly 300 kinds of viruses can harm humans. But most of them — along with the proteins they produce — aren't well studied. Scientists tend to focus on a small subset of these viruses, often because they're medically significant (like influenza) or serve as good models for understanding larger groups of viruses (like rabies as a stand-in for all rhabdoviruses).
The ORFeome and other ORF libraries like it are named after open reading frames, the technical term for DNA sequences that encode proteins. Previous viral ORF libraries from other groups focused on individual viruses or virus families and contained perhaps 100 or 200 sequences each, the authors said. The new ORFeome contains about 13,000 physical DNA sequences, or constructs, that code for about 9,000 proteins from 513 different viruses, from the Andes hantavirus to Ebola virus to Zika virus.
"Most viruses have never been studied in detail, yet evolution has already performed countless experiments for us. This library gives us a way to read the results of those experiments across the viral world," said Elledge, who is also a Howard Hughes Medical Institute Investigator.
The viruses included in the library were selected because they're known to infect humans or because they are close relatives that infect animals and could become a threat to humans.
(The ORFeome itself is harmless; the proteins it contains can't rebuild any of the original viruses, replicate themselves, or infect cells. The team also followed strict federal guidelines when working with a biotech company to synthesize the DNA sequences.
"This is a biosafe way to study viral proteins individually instead of studying a whole virus," said Caleb Glassman , HMS research fellow in medicine at Brigham and Women's in the Elledge Lab, co-author of the Cell paper, and first author of the Science paper.)
A new ORF library from a separate team, reported in the same issue of Cell and dubbed the eORFeome, takes the field a step in another direction by including nearly 4,000 sequences from viruses, bacteria, and parasites.
Researchers can take anywhere between one and all 13,000 of the DNA constructs in Elledge and colleagues' viral ORFeome and insert them into cell cultures such that each cell receives instructions for making a single viral protein. Researchers can then investigate which proteins affect a cellular function — such as camouflaging the cell from immune attack, disrupting metabolism, or prioritizing viral replication — and how they do it.
"Once you have this library, you can start looking at how viruses interact with all kinds of different cell processes," said Colin O'Leary , HMS research fellow in medicine at Brigham and Women's in the Elledge Lab, co-first author of the Cell paper, and co-author of the Science paper.
The results could point researchers to human or viral proteins, genes, or processes that could be disabled — or augmented — to fight infection, whether through a vaccine or an antiviral drug. If the tool reveals that multiple viruses employ the same specific tactic, that could aid in the development of therapies that protect against more than one disease, O'Leary said.
Genetic barcoding and other advantages
The team attached a unique ID tag known as a genetic barcode to each ORF, allowing researchers to conduct studies of all 13,000 ORFs at once and keep track of each one.
"We can insert the sequences into a population of cells, ask questions like which ones cause the cells to grow better or less, and then identify those by their barcodes when the experiment is finished," said O'Leary. "It hasn't been possible before to do genetic screens like this with viral proteins."
The team will make the ORFeome freely available for scientists to use. Elledge and colleagues gave it a flexible design so researchers can apply it to different model systems and types of tests.
"We designed it so biologists who aren't virologists can use it," said Glassman. "A big advantage is that we have a unified resource that's compatible with common laboratory workflows."
Revealing unseen functions in virology
To demonstrate the tool's capabilities, the team conducted genetic screens in three cell types, looking for viral proteins that affect cell proliferation; stop cells from presenting antigens on their surfaces that trigger the immune system to attack; or block the effects of interferon, a substance that prompts nearby cells to raise defenses against infection.
They found more than 700 viral proteins that contribute to at least one of those actions. Many of those proteins hadn't been studied before. Some had been studied but weren't known to have these particular functions.
The team also discovered that some viral proteins perform actions scientists wouldn't have predicted based on their structures and genetic sequences, O'Leary said. This demonstrates the value of ORF libraries representing actual viral proteins compared to libraries that contain sequences computationally predicted to have certain functions, the authors said.
In the Science paper, the team focused on identifying viral proteins that activate a cellular garbage disposal known as the ubiquitin proteasome to get rid of host-cell proteins that could hinder the virus.
"Viruses have to act super quickly to ensure the cell doesn't realize they're there," Glassman explained. "They plug into the ubiquitin proteasome system to degrade certain proteins so they can go about copying themselves and hiding from the immune system."
Glassman and colleagues built a list of viral proteins that remove host genes and documented the parts of the protease they act on as well as the host-cell proteins they trash. In doing so, the team discovered new strategies viruses employ.
"They're using the ubiquitin proteasome system in diverse and innovative ways while tending to target early steps in host pathways that sense and block infection," Glassman said.
For example, they found that a protein called NSP1, made by a rotavirus that causes intestinal illness, remixed host genes to make a ubiquitin-modifying complex rarely observed in host cells.
Uncovering things that viruses make or do that host cells don't opens opportunities to design drugs that hinder viral activity while sparing normal function, the authors said.
The team looks forward to more discoveries the ORFeome has the potential to power. Proteins can be added as new viruses emerge — a phenomenon that recent history has demonstrated all too well.
When O'Leary began his PhD studies in the Harvard Kenneth C. Griffin Graduate School of Arts and Sciences through the Program in Virology , there was an Ebola epidemic in West Africa and a Zika outbreak in South and Central America.
"It convinced me that viruses are very important to study and understand," O'Leary said.
Authorship, funding, disclosures
O'Leary is co-first author of the Cell paper with Eric Fujimura, a former Harvard Griffin GSAS student in the Chemical Biology PhD Program in the Elledge Lab, who died in 2021 at age 28. Additional authors include Mamie Z. Li, Rachel A. Roberts, Joao A. Paulo, Hanjie Jiang, Nouran S. Abdelfattah, Eric C. Wooten, Zachary Mirman, J. Wade Harper , and Philip A. Cole . The authors dedicate the paper to the memory of Fujimura, "without whom this work would not have been possible."
Elledge is senior author of the Science study. Additional authors include Kheewoong Baek, Gaopeng Hou, Qiru Zeng, Christopher Nardone, Kate B. Juergens, Fujimura, Li, Paulo, Eric S. Fischer , Siyuan Ding, and Harper.
The Cell work was supported in part by grants from the Gates Foundation and the National Institutes of Health (AG11085 and CA74305) and HHMI. The Science work was funded by grants from the Gates Foundation and the NIH (AG11085, R01AI150796, and R01CA262188). Glassman is a fellow of the Jane Coffin Childs Memorial Fund for Medical Research and HHMI. Mirman is supported by NIH/NCI grant K00CA245720. Baek is a Meghan E. Raveis Fellow of the Damon Runyon Cancer Research Foundation. The authors also thank the HMS Center for Macromolecular Interactions (CMI) , the Microscopy Resources on the North Quad (MicRoN) core, and the Harvard Cryo-EM Center for Structural Biology .
Elledge is a founder of TScan Therapeutics, Maze Therapeutics, Infinity Bio, Inc., and Mirimus; serves on the scientific advisory board of TScan Therapeutics and Infinity Bio; and is a member of the advisory board for Cell. Harper is a co-founder of Caraway Therapeutics, a subsidiary of Merck & Co., Inc., and a member of the scientific advisory board for Lyterian Therapeutics. Fischer is a founder, scientific advisory board member, and equity holder of Civetta Therapeutics, Proximity Therapeutics, Neomorph, Inc. (also board of directors), Stelexis Biosciences, Inc., Anvia Therapeutics, Inc. (also board of directors), CPD4, Inc. (also board of directors), and Nias Bio, Inc.; an equity holder in Avilar Therapeutics, Ajax Therapeutics (also scientific advisory board), Photys Therapeutics (also scientific advisory board), and Light Horse Therapeutics; and a consultant to Novartis, EcoR1 Capital, and Deerfield Management. The Fischer Lab has received or currently receives research funding from Deerfield, Novartis, Ajax, Interline Therapeutics, Bayer, and Astellas.
