Ranum, J.N., Ledwith, M.P., Alnaji, F.G., Diefenbacher, M., Orton, R., Sloan, E., Güereca, M., Feltman, E.M., Smollett, K., da Silva Filipe, A., Conley, M., Russell, A.B., Brooke, C.B., Hutchinson, E., Mehle, A., 2024. Nucleic Acids Research 52, 3199–3212. https://doi.org/10.1093/nar/gkae133

Productive infections by RNA viruses require faithful replication of the entire genome. Yet many RNA viruses also produce deletion-containing viral genomes (DelVGs), aberrant replication products with large internal deletions. DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes. The DelVG RNA itself is hypothesized to confer this interfering activity. DelVGs antagonize replication by out-competing the full-length genome and triggering innate immune responses. Here, we identify an additionally inhibitory mechanism mediated by a new class of viral proteins encoded by DelVGs. We identified hundreds of cryptic viral proteins translated from DelVGs. These DelVG-encoded proteins (DPRs) include canonical viral proteins with large internal deletions, as well as proteins with novel C-termini translated from alternative reading frames. Many DPRs retain functional domains shared with their full-length counterparts, suggesting they may have activity during infection. Mechanistic studies of DPRs derived from the influenza virus protein PB2 showed that they poison replication of wild-type virus by acting as dominant-negative inhibitors of the viral polymerase. These findings reveal that DelVGs have a dual inhibitory mechanism, acting at both the RNA and protein level. They further show that DPRs have the potential to dramatically expand the functional proteomes of diverse RNA viruses.