# IDENTIFICATION AND CHARACTERIZATION OF RNA STRUCTURES IN THE GENOME OF INFLUENZA VIRUS > **NIH NIH R01** · WASHINGTON UNIVERSITY · 2020 · $521,901 ## Abstract PROJECT SUMMARY Influenza poses a significant health risk worldwide. It is estimated that between 250,000 and 500,000 people die each year from the virus. This number can increase dramatically during an influenza pandemic, such as the 1918 Spanish influenza pandemic, when more than 25 million people died. The genome of influenza A virus consists of eight segments of single-stranded, negative-sense RNA that are encapsidated as individual ribonucleoprotein complexes (RNPs). Each RNP contains one strand of RNA, a viral polymerase complex and multiple copies of the viral nucleoprotein (NP). The segmented nature of influenza virus allows for reassortment or mixing of gene-segment and this is a major contributor to the emergence of pandemic influenza viruses. Image analysis of individual RNP complexes show a double-helical conformation of two strands of NP connected by a small loop and the polymerase proteins at the termini of the complex. The organization of the viral RNA in this structure is not known, which limits our ability to understand and to target RNA dependent processes that contribute to the generation of pandemic influenza viruses. To address this gap, we applied a new technology, cross-linking and immunoprecipitation coupled with next-generation sequencing (CLIP-seq), to discern the organization of the RNA in native RNP complexes of IAV. Analysis of the immunoprecipitated viral RNA revealed non-uniform binding of the NP to the genome. Specifically, we identified 34 regions that range in size between 14-85 nucleotides that are significantly underrepresented in the NP-bound RNA fraction. Nearly 50% of the low-NP binding regions are predicted to form stable secondary structures, including RNA hairpins and RNA pseudoknot structures. This led us to hypothesize that the RNA structural features, that are poorly bound by NP, are pivotal for gene-segment interactions and are the primary features controlling IAV segment packaging at the molecular level. In support of this hypothesis we found that the introduction of synonymous structural mutations that disrupt the predicted RNA structures attenuated the resulting viruses without exception. In contrast, synonymous mutations that did not alter the predicted RNA structure, or mutations in control regions had no impact on the growth of the virus. The attenuation in low-NP binding mutant viruses was due to a defect in genome packaging. The goal of this application is to determine the mechanism by which low-NP binding regions affect IAV replication, genome packaging, and virus reassortment. ## Key facts - **NIH application ID:** 9962289 - **Project number:** 5R01AI139251-03 - **Recipient organization:** WASHINGTON UNIVERSITY - **Principal Investigator:** Adrianus CM Boon - **Activity code:** R01 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2020 - **Award amount:** $521,901 - **Award type:** 5 - **Project period:** 2018-07-13 → 2022-06-30 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/9962289 ## Citation > US National Institutes of Health, RePORTER application 9962289, IDENTIFICATION AND CHARACTERIZATION OF RNA STRUCTURES IN THE GENOME OF INFLUENZA VIRUS (5R01AI139251-03). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/9962289. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*