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Vaccinations have revolutionized healthcare, but designing them remains complex, requiring time and creativity, especially for vaccines against viruses, which are difficult to study.
“Each virus demands a specific culturing system, some viruses cannot be cultured in the lab, and highly pathogenic viruses require high-containment facilities,” wrote the authors of a new study in Science from the Broad Institute of MIT and Harvard
“Most viruses are genetically diverse, necessitating a method that can evaluate multiple variants in parallel,” they added.
To address this challenge, the researchers developed a technique called Massively Parallel Ribosome Profiling (MPRP), which enables rapid identification of viral proteins without the need to grow live viruses.
Their method and findings are described in a paper entitled, “Pan-viral ORFs discovery using massively parallel ribosome profiling.”
“Defining viral proteomes is crucial to understanding viral life cycles and immune recognition, but the landscape of translated regions remains unknown for most viruses,” the authors wrote. The viral proteome is difficult to quantify, despite advances in sequencing technology.
Using their high-throughput system, the researchers synthesized thousands of short fragments from viral genomes, cloned them into plasmids, and transfected them into human cells. This allowed them to safely study viral translation events in parallel across a vast range of viruses.
By sequencing ribosome-bound mRNA fragments, the team was able to quickly pinpoint where ribosomes were actively translating RNA into proteins, providing direct evidence of which viral open reading frames (ORFs) were being used by the cell. They identified 4,208 previously unannotated ORFs across 679 human-associated viral genomes.
Many of these newly identified peptides originate from noncanonical ORFs—those that don’t start with the typical ATG codon or encode very short proteins.
In one example, the team found viral peptides from noncanonical ORFs presented on class-I human leukocyte antigen molecules in infected cells, which are essential for immune system recognition. Further, they identified hundreds of upstream ORFs that may be involved in the initiation of the viral protein translation.
These novel proteins not only improve understanding of viral-immune system interactions but may be targets for new vaccine design or antiviral therapies.
Rapidly profiling ORFs across hundreds of viruses without handling live pathogens could accelerate vaccine development, particularly for emerging viruses. MPRP is a scalable, virus-agnostic platform that could be used to investigate other classes of viruses or to study viral translation under different cellular conditions.
While the use of MPRP has yielded thousands of candidate ORFs, many of these regions remain unclassified and have yet to be fully explored.
The authors concluded that their “study yields thousands of candidates for unexplored ORFs across hundreds of human viruses, which can enhance our understanding of viral biology and contribute to vaccine development.”
