To prevent viruses from making us sick or dying, whether it’s in individual cases of hepatitis B or the coronavirus pandemic, it’s important to understand how the proteins that viruses produce cause changes in our bodies that allow them to multiply.
The new tools will greatly expand the scale at which researchers can study these proteins, and are expected to accelerate fundamental discoveries in virology, inform the development of new vaccines and treatments, and help protect humanity from emerging pathogens.
The tool is called viral ORFeome and was described on July 2 as follows: cellis the largest of its kind, capable of analyzing thousands of viral proteins in a single experiment. Its design also expands access to biologists who are not trained in virology.
“This library reveals how viruses manipulate human cells on a scale never before possible,” said lead author Stephen Elledge, the Gregor Mendel Professor of Genetics and Medicine at Harvard Medical School and Brigham and Women’s Hospital, who led the team that created the tool.
We believe this will move 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. ”
Stephen Elledge, Gregor Mendel Professor of Genetics and Medicine at Harvard Medical School and Brigham and Women’s Hospital
of cell The paper shows how the research team has already used ORFeome to identify hundreds of viral proteins that interfere with the immune response. In a second paper published on July 9, scienceElledge and colleagues delve into new insights revealed about how viruses hijack cells’ garbage disposal systems to evade immune attack.
Largest library of viral proteins ever
It is estimated that there are nearly 300 types of viruses that can harm humans. However, most of them, along with the proteins they produce, have not been well studied. Scientists tend to focus on a small subset of these viruses. Often because they are medically important (e.g., influenza) or because they serve as good models for understanding larger groups of viruses (e.g., rabies as a surrogate for all rhabdoviruses).
ORFeome and other ORF libraries like it are named after open reading frames, a technical term for DNA sequences that code for proteins. Previous viral ORF libraries by other groups have focused on individual viruses or virus families, each containing perhaps 100 to 200 sequences, the authors said. The new ORFeome contains approximately 13,000 physical DNA sequences or constructs that encode approximately 9,000 proteins from 513 viruses, ranging from Andean hantavirus to Ebola virus to Zika virus.
“Most viruses have never been studied in detail, but evolution has already performed countless experiments for us, and 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.
Viruses included in the library were selected because they are known to infect humans or are closely related species that can infect animals and pose a threat to humans.
(ORFeome itself is harmless; the proteins it contains cannot reassemble the original virus, replicate itself, or infect cells.) The team also followed strict federal guidelines when working with biotech companies to synthesize the DNA sequences.
“This is a biosafe way to study viral proteins individually, rather than studying the whole virus,” said Caleb Glassman, an HMS medical research fellow at the Brigham and Women in the Elder Research Institute and co-author of the paper. cell Paper and its first author science paper. )
A new ORF library by another team was reported in the same issue. cell The inclusion of nearly 4,000 sequences from viruses, bacteria, and parasites, called the eORFeome, takes the field a step in a different direction.
Researchers can take every one of the 13,000 DNA constructs in Elledge et al.’s viral ORFeome and insert it into cell culture, where each cell receives instructions to make a single viral protein. Researchers can investigate which proteins influence cellular functions, such as camouflaging cells from immune attack, interfering with metabolism, or prioritizing virus replication, and how they do so.
“With this library, we can start looking at how viruses interact with all sorts of different cellular processes,” said Colin O’Leary, an HMS medical research fellow at the Brigham and Women in the College and co-author of the paper. cell papers and their co-authors science paper.
The results could point researchers to human or viral proteins, genes or processes that can be disabled or enhanced to fight infection, whether through vaccines or antiviral drugs. If the tool reveals that multiple viruses employ the same specific tactics, it could help develop treatments to prevent multiple diseases, O’Leary said.
Genetic barcoding and other benefits
The research team attached a unique ID tag, known as a genetic barcode, to each ORF, allowing researchers to study all 13,000 ORFs at once and track each one.
“You can insert sequences into a population of cells, ask questions such as which sequences make the cells grow better or worse, and when the experiment is finished you can identify the sequences with a barcode,” O’Leary said. “It has never been possible to perform genetic screens like this using viral proteins.”
The team plans to make ORFeome freely available to scientists. Elledge et al. designed it to be flexible so that researchers can apply it to different model systems and types of tests.
“We designed it to be usable by biologists who are not virologists,” Glassman said. “The big advantage is that we have integrated resources that are compatible with common laboratory workflows.”
Revealing the invisible features of virology
To demonstrate the tool’s functionality, the team conducted a genetic screen in three cell types, looking for viral proteins that affect cell growth. It prevents cells from presenting antigens on their surface that trigger an attack by the immune system. Or it blocks the effects of interferon, a substance that gives nearby cells increased defenses against infection.
They discovered more than 700 viral proteins that contribute to at least one of these effects. Many of these proteins have not been previously studied. Although some had been studied, they were not known to have these specific functions.
The research team also discovered that some viral proteins perform actions that scientists had not predicted based on their structure or genetic sequence, O’Leary said. This demonstrates the value of ORF libraries representing actual viral proteins compared to libraries containing sequences computationally predicted to have specific functions, the authors said.
in science In this paper, the research team focused on identifying viral proteins that activate the cell’s garbage disposal machinery, known as the ubiquitin proteasome, to remove host cell proteins that might get in the way of the virus.
“The virus has to act very quickly so that the cell doesn’t know it’s there,” Glassman explains. “They can connect to the ubiquitin proteasome system to degrade certain proteins, copy themselves and hide from the immune system.”
Glassman and colleagues created a list of viral proteins that remove host genes and documented the parts of proteases they act on and the host cell proteins they destroy. In doing so, the team discovered new strategies employed by the virus.
“They use the ubiquitin-proteasome system in diverse and innovative ways, but they tend to target early steps in host pathways that sense and block infection,” Glassman said.
For example, they discovered that a protein called NSP1, made by rotaviruses that cause intestinal disease, remixes host genes to create ubiquitin-modified complexes that are rarely observed in host cells.
The authors said that uncovering what viruses, rather than host cells, make and do opens opportunities to design drugs that block virus activity while preserving normal function.
The team looks forward to discovering more about the potential of ORFeome. Proteins may be added as new viruses emerge, a phenomenon that recent history has amply demonstrated.
When O’Leary began his doctoral work through the virology program at Harvard University’s Kenneth C. Griffin School of Arts and Sciences, there was an Ebola outbreak in West Africa and a Zika outbreak in Latin America.
“I became convinced that viruses are very important to study and understand,” O’Leary said.
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