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. We have developed massively parallel ribosome profiling (MPRP) to determine open reading frames (ORFs) across tens of thousands of designed oligonucleotides. MPRP identified 4208 unannotated ORFs in 679 human-associated viral genomes. We found viral peptides originating from detected noncanonical ORFs presented on class-I human leukocyte antigen (HLA-I) in infected cells and hundreds of upstream ORFs that likely modulate translation initiation of viral proteins. The discovery of viral ORFs across a wide range of viral families—including highly pathogenic viruses—expands the repertoire of vaccine targets and reveals potential cis-regulatory sequences. Ribo-seq of human lung A549 cells and human kidney HEK293T trasnfected with a synthetic pan-viral library