Influenza B virus particles, colorized orange and pink, seen through a scanning electron microscope.
Image Credit: NIAID/NIH
Scientific Frontline: Extended "At a Glance" Summary: The Viral ORFeome
The Core Concept: The viral ORFeome is a comprehensive genetic library containing 13,000 physical DNA sequences that encode approximately 9,000 proteins from 513 different viruses, enabling scientists to study thousands of viral proteins simultaneously.
Key Distinction/Mechanism: Unlike previous viral libraries that were limited to a single virus or family (usually restricted to 100 or 200 sequences), the viral ORFeome scales up analysis using genetic barcoding. Researchers can safely insert thousands of noninfectious viral DNA constructs into cell cultures at once, using unique ID tags to track which specific proteins disrupt cellular functions, block interferon, or evade immune responses.
Major Frameworks/Components:
- Open Reading Frames (ORFs): Engineered DNA sequences designed to instruct host cells to produce specific viral proteins without synthesizing or replicating the entire virus.
- Genetic Barcodes: Unique identifier tags attached to each ORF, allowing researchers to conduct and track large-scale, multiplexed genetic screens in a single experiment.
- Ubiquitin Proteasome System: The cellular garbage-disposal machinery frequently hijacked by viral proteins (such as the NSP1 protein from rotavirus) to degrade host defenses and remain undetected.
- Unified Workflow: A flexible, biosafety-compliant design that allows biologists outside of specialized virology fields to integrate the library into common laboratory test models.
Branch of Science: Virology, Molecular Biology, Genetics, and Immunology.
Future Application: The viral ORFeome is designed to accelerate the development of vaccines, targeted therapeutics, and broad-spectrum antiviral drugs by revealing shared mechanisms among diverse viruses, ultimately bolstering global pandemic preparedness.
Why It Matters: By transforming virology from a single-pathogen focus to a broad, comparative science, this tool exposes unseen vulnerabilities and common evolutionary strategies across the viral world, providing critical insights for defending humanity against emerging infectious diseases.
To prevent viruses from sickening or killing humans—whether in an individual case of hepatitis B or a COVID-19 pandemic—it is crucial to understand how the proteins they produce initiate changes in the body that allow them to flourish. A new tool has 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 of its kind and enables the analysis of thousands of viral proteins in a single experiment. Its design also expands access to biologists who did not train in virology.
“This library reveals how viruses manipulate human cells on a scale that simply was not 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 the immune response. In a second paper, published July 9 in Science, Elledge and colleagues delve into new insights the tool has revealed regarding how viruses hijack cells’ garbage-disposal systems to evade immune attack.
The Largest Library of Viral Proteins
It is estimated that nearly 300 types of viruses can harm humans. However, most of them—along with the proteins they produce—are not well studied. Scientists tend to focus on a small subset of these viruses, often because they are medically significant (such as influenza) or serve as effective models for understanding larger groups of viruses (such as rabies serving as a stand-in for all rhabdoviruses).
The ORFeome, along with similar ORF libraries, is 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 approximately 100 to 200 sequences each, the authors noted. The new ORFeome contains about 13,000 physical DNA sequences, or constructs, that code for approximately 9,000 proteins from 513 different viruses, ranging from the Andes hantavirus to the Ebola virus and the 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 are 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 cannot rebuild any of the original viruses, replicate themselves, or infect cells. The team also followed strict federal guidelines when working with a biotechnology 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 worse, and then identify those by their barcodes when the experiment is finished," said O’Leary. "It has not 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 are not virologists can use it," said Glassman. "A big advantage is that we have a unified resource that is 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 had not been studied before. Some had been studied but were not known to have these particular functions.
The team also discovered that some viral proteins perform actions scientists would not 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 does not realize they are 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 target host proteins and documented the parts of the protease they act on, as well as the host-cell proteins they destroy. In doing so, the team discovered new strategies viruses employ.
"They are 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, repurposed host proteins to make a ubiquitin-modifying complex rarely observed in host cells.
Uncovering things that viruses make or do that host cells do not 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.
Reference material: What Is: The Virome
Funding: 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.
Disclosure: 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.
Published in journal:
- Science
- Cell
- Cell
Title:
- Virome-wide ubiquitin ligase discovery reveals diverse mechanisms of immune evasion
- Systematic discovery of pathogen effector functions across human pathogens and pathways
- A viral ORFeome library for systems-level genetic dissection of host-pathogen interactions
Authors:
- Caleb R. Glassman, Kheewoong Baek, Gaopeng Hou, Qiru Zeng, Christopher Nardone, Kate B. Juergens, Eric Fujimura, Colin N. O’Leary, Mamie Z. Li, Joao A. Paulo, Eric S. Fischer, Siyuan Ding, J. Wade Harper, Stephen J. Elledge
- Tomas Pachano, He Leng (冷赫), Guillaume Dugied, Travis Tribble, Vincent Loubiere, Yeojin Lee, Felix Rauh, Victor Manon, Kevin Yuan, Jocelyn Nurtanto, Alexander Schleiffer, Veronika Young, Benjamin Weller, Eleanor A. Lyons, Matthew R. Hass, Leah C. Kottyan, Matthew T. Weirauch, Juan I. Fuxman Bass, Hayley J. Newton, Alexander W. Ensminger, Pascal Falter-Braun, Jue Chen, Daniel Schramek, Alexander Stark, Mikko Taipale
- Eric Fujimura, Colin N. O’Leary, Mamie Z. Li, Rachel A. Roberts, Caleb R. Glassman, Joao A. Paulo, Hanjie Jiang, Nouran S. Abdelfattah, Eric C. Wooten, Zachary Mirman, J. Wade Harper, Philip A. Cole, Stephen J. Elledge
Source/Credit: Harvard Medical School | Stephanie Dutchen
Edited by: Scientific Frontline
Reference Number: vi071026_01
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