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Research Project: Viral Ecology of Henipaviruses in Endemic Settings and Intervention Strategies to Prevent their Spread to Domestic Animals

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Title: Pan-viral ORFs discovery using massively parallel ribosome profiling

Author
item WEINGARTEN-GABBA, SHIRA - Broad Institute Of Mit/harvard
item SABETI, PARDIS - Broad Institute Of Mit/harvard
item BAUER, MATTHEW - University Of California (UCLA)
item STANTON, ALEXANDRA - Broad Institute Of Mit/harvard
item KLAEGER, SUSAN - Broad Institute Of Mit/harvard
item VERZANI, EVA - Broad Institute Of Mit/harvard
item LÓPEZ, DANIEL - Veterinary Laboratory Of Central Kinshasa
item Hensley, Lisa
item CLAUSER, KARL - Broad Institute Of Mit/harvard
item CARR, STEVEN - Broad Institute Of Mit/harvard
item ABELIN, JENNIFER - Broad Institute Of Mit/harvard
item RICE, CHARLES - Rockefeller University

Submitted to: Science
Publication Type: Pre-print Publication
Publication Acceptance Date: 3/17/2025
Publication Date: 6/12/2025
Citation: Weingarten-Gabba, S., Sabeti, P.C., Bauer, M.R., Stanton, A.C., Klaeger, S., Verzani, E.K., López, D., Hensley, L.E., Clauser, K.R., Carr, S.A., Abelin, J.G., Rice, C.M. 2025. Pan-viral ORFs discovery using massively parallel ribosome profiling. Science. 388(6752):1218-1224. https://doi.org/10.1126/science.ado6670.
DOI: https://doi.org/10.1126/science.ado6670

Interpretive Summary: Previously something was called a protein based on strict criteria driven by our understanding of biochemistry and cellular biology. As new scientific techniques have been developed the scientific community have recognized that there were many proteins that did not fall within the existing criteria. These proteins can affect many aspects of biology. This paper describes a strategy to identify proteins that are missed using previous criteria and provides a tool that does not require the use of high containment laboratories.

Technical Abstract: Unveiling the complete proteome of viruses is crucial to our understanding of the viral life cycle and interaction with the host. We developed Massively Parallel Ribosome Profiling (MPRP) to experimentally determine open reading frames (ORFs) in 20,170 designed oligonucleotides across 679 human-associated viral genomes. We identified 5,381 ORFs, including 4,208 noncanonical ORFs, and show successful detection of both annotated coding sequences (CDSs) and reported non-canonical ORFs. By examining immunopeptidome datasets of infected cells, we found class I human leukocyte antigen (HLA-I) peptides originating from non-canonical ORFs identified through MPRP. By inspecting ribosome occupancies on the 5’untranslated regions(5'UTR) and CDS regions of annotated viral genes, we identified hundreds of upstream ORFs (uORFs) that negatively regulate the synthesis of canonical viral proteins. The unprecedented source of viral ORFs across a wide range of viral families, including highly pathogenic viruses, expands the repertoire of vaccine targets and exposes new cis-regulatory sequences in viral genomes.