The role of cryptic influenza virus proteins in replication and pathogenesis

Productive infections by RNA viruses require faithful replication of full-length genomes. 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 influenza 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 or by triggering innate immune responses. Here, we show this understanding is incomplete. We discovered a new inhibitory mechanism mediated by a previously unknown class of viral proteins. Specifically, 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 protein truncations with novel C-termini translated from alternative reading frames. We show that DPRs are common features of influenza virus infections in culture and in humans. Preliminary results provide strong support and mechanistic data for DPR-mediated interference of wild-type virus replication. Yet, as we only recently discovered DPRs, we have limited knowledge on the scope and diversity of DPRs made during infection, the different activities they might possess, and their cumulative impact on disease. As all RNA viruses make DelVGs, many with the potential to express DPRs, this leaves a large gap in our knowledge across RNA virology. We address this with three Aims. In Aim 1, we characterize the interference activity of DPRs we have already discovered and establish their mechanism of action. Aim 2 defines the full repertoire of DPRs from primary influenza virus isolates, and tests almost all possible DPR types by screening a synthetic DPR library. Aim 3 then investigates the in vivo inhibition by DPRs and establishes their contribution to interference activities that suppresses wild-type virus replication and disease. These self-reinforcing Aims will provide a comprehensive view of DPR formation, mode of action, and impact on infection and pathology. Moreover, the detailed knowledge gained here will be critical in achieving the long-sought goal of developing DelVG-containing viruses as antiviral therapeutics. Project Number: 1R01AI187065-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Andrew Mehle | Institution: UNIVERSITY OF WISCONSIN-MADISON, MADISON, WI | Award Amount: $3,085,097 | Activity Code: R01 | Study Section: Molecular and Cellular Biology of Virus Infection Study Section[MCV] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI18706501A1