Abstracts
Probing the structure and function of long viral RNAs
Presenting author: Redmond Smyth
CNRS, Institut de Biologie Moléculaire et Cellulaire (IBMC), 1 rue Laurent Fries, 67404 Illkirch, France, r.smyth@ibmc-cnrs.unistra.fr
Authors: Li J.¹, Gribling-Burrer A.-S.¹, Brunotte L.², Smyth R.¹
¹Institute of Molecular and Cellular Biology, Strasbourg, France
²Institute for Molecular Virology, University of Münster, Münster, Germany
Viral RNA populations are highly heterogeneous, comprising full-length genomes together with alternatively processed as well as defective RNA species. Although these RNA isoforms may share extensive sequence identity, they can nevertheless have distinct biological functions, potentially arising from differences in RNA structure and associated protein interactions. However, conventional short-read RNA structure probing largely averages structural information across molecules and therefore cannot readily resolve such isoform-specific conformations.
We are developing a suite of long-read approaches that exploit nanopore sequencing to investigate the structure and function of individual long viral RNAs. Nano-DMS-MaP and Nano-SHAPE-MaP probe viral RNA structure, Nano-PAR-CL maps RNA–protein interactions, and nanopore direct RNA sequencing quantifies native RNA isoforms. Together, these complementary approaches provide a framework for linking RNA sequence, structure, protein association, and functional state on individual RNA molecules.
We have applied this strategy to influenza A virus, whose eight negative-sense RNA genome segments are assembled into viral ribonucleoprotein complexes (vRNPs) and frequently give rise to defective viral genomes (DVGs) containing large internal deletions. These DVGs are generated when the viral polymerase disengages from one region of the template and resumes synthesis at a downstream site, producing shortened viral RNAs that can both interfere with viral replication and can trigger excessive inflammatory immune response. Direct RNA sequencing of cells infected with different influenza strains revealed reproducible, segment-specific hotspots and cold-spots of DVG formation. Using long-read RNA structural probing, we find that DVG hotspots correlate with both local RNA structural features, deposition of the viral nucleoprotein, and the global organization of the vRNP. This suggests that DVG formation is not determined solely by primary sequence, but may also be constrained by how the viral RNA is folded and spatially organized within the ribonucleoprotein complex.