Todays press announcement was released by the Broad Institute of MIT and Harvard.
While incidences of Ebola virus are infrequent, the illness is severe and frequently deadly, offering limited therapeutic options. Instead of directly attacking the virus, a potentially effective therapeutic strategy involves disrupting the proteins in human host cells that the virus depends on. However, identifying these regulators of viral infection through current techniques has proven challenging, particularly for highly dangerous viruses like Ebola, which necessitate stringent high-containment biosafety measures.
Researchers from the Broad Institute and the National Emerging Infectious Diseases Laboratories (NEIDL) at Boston University have now utilized an image-based screening approach created at the Broad to discover human genes that, when silenced, hinder the Ebola virus’s capacity to infect. This technique, known as optical pooled screening (OPS), allowed the scientists to examine, in approximately 40 million CRISPR-altered human cells, how the silencing of each gene in the human genome influences virus replication.
By employing machine-learning-driven analyses of images of altered cells, they identified various host proteins that play roles in different phases of Ebola infection, which, when suppressed, incapacitated the virus’s ability to replicate. These viral regulators may serve as potential targets for future therapeutic interventions to alleviate the severity of the disease in individuals already infected with the virus. The method may also be applied to investigate the function of various proteins during infections caused by other pathogens, paving the way for new treatments for difficult-to-manage infections.
The findings are published in Nature Microbiology.
“This research highlights the capability of OPS to examine the reliance of hazardous viruses like Ebola on host factors throughout the viral life cycle and to uncover new pathways for enhancing human health,” stated co-senior author Paul Blainey, a core faculty member at the Broad and a professor in the Department of Biological Engineering at MIT.
Previously, researchers in the Blainey lab developed the optical pooled screening technique as a method to merge the advantages of high-content imaging, which can illustrate a variety of intricate changes in substantial numbers of cells simultaneously, with those of pooled perturbational screens, which reveal how genetic components influence these alterations. In this investigation, they collaborated with Robert Davey’s lab at BU to employ optical pooled screening for the Ebola virus.
The team utilized CRISPR to deactivate individual genes in the human genome sequentially across nearly 40 million human cells, subsequently infecting each cell with the Ebola virus. They then stabilized those cells in laboratory plates and rendered them inactive, allowing the remaining procedures to be conducted outside of the high-containment facility.
Following the capture of images of the cells, they assessed overall viral protein and RNA in each cell using the CellProfiler image analysis software, and to extract even more insights from the images, they consulted AI. With support from team members in the Eric and Wendy Schmidt Center at the Broad, led by study co-author and core faculty member Caroline Uhler, they employed a deep learning model to automatically ascertain the stage of Ebola infection for each single cell. This model could distinguish subtle variations between infection stages in a high-throughput manner that was not achievable with prior techniques.
“This work represents the most in-depth exploration to date of how the Ebola virus alters the cellular environment to induce disease, and the first genuine insight into the timing of that reprogramming,” remarked co-senior author Robert Davey, director of the National Emerging Infectious Diseases Laboratories at Boston University and a professor of microbiology at the BU Chobanian and Avedisian School of Medicine. “AI provided us with an unparalleled capacity to accomplish this on a large scale.”
By sequencing segments of the CRISPR guide RNA from all 40 million cells individually, the researchers identified which human gene had been silenced in each cell, thereby indicating which host proteins (and potential viral regulators) were affected. The analysis uncovered hundreds of host proteins that, when silenced, modified the overall level of infection, including several essential for viral entry into the cell.
Deactivating other genes increased the quantity of virus contained within inclusion bodies, structures that form in human cells to serve as viral manufacturing sites, and halted the infection from advancing further. Some of these human genes, such as UQCRB, highlighted a previously unrecognized function for mitochondria in the process of Ebola virus infection that could be therapeutically targeted. Notably, treating cells with a small molecule inhibitor of UQCRB decreased Ebola infection without affecting the health of the cells.
Other genes, when silenced, modified the equilibrium between viral RNA and protein. For instance, altering a gene named STRAP led to a rise in viral RNA compared to protein levels. The researchers are currently conducting further laboratory studies to gain deeper insights into the role of STRAP and other proteins in Ebola infection and their potential for therapeutic targeting.
In a series of follow-up screens, the scientists evaluated certain highlighted genes’ involvement in infections with similar filoviruses. Silencing some of these genes disrupted replication of Sudan and Marburg viruses, which have high mortality rates and lack approved treatments, suggesting that one treatment might be effective against several related viruses.
The methodology employed in this study could also be utilized to investigate other pathogens and emerging infectious diseases, searching for novel therapeutic strategies.
“With our approach, we can concurrently measure numerous features and unveil new insights into the interplay between virus and host, in ways that are unattainable through alternative screening techniques,” stated co-first author Rebecca Carlson, a former graduate researcher in the labs of Blainey and Nir Hacohen at the Broad who co-led the work along with co-first author J.J. Patten at Boston University.
This research received funding from the Broad Institute, the National Human Genome Research Institute, the Burroughs Wellcome Fund, the Fannie and John Hertz Foundation, the National Science Foundation, the George F. Carrier Postdoctoral Fellowship, the Eric and Wendy Schmidt Center at the Broad Institute, the National Institutes of Health, and the Office of Naval Research.