High-throughput single-virion DNA-PAINT reveals structural diversity, cooperativity, and flexibility during selective packaging in influenza

Journal article

Influenza A, a negative-sense RNA virus, has a genome that consists of eight single-stranded RNA segments. Influenza co-infections can result in reassortant viruses that contain gene segments from multiple strains, causing pandemic outbreaks with severe consequences for human health. The outcome of reassortment is likely influenced by a selective sequence-specific genome packaging mechanism. To uncover the contributions of individual segment pairings to selective packaging, we set out to statistically analyse packaging defects and inter-segment distances in individual A/Puerto Rico/8/34 (H1N1) (PR8) virus particles. To enable such analysis, we developed a multiplexed DNA-PAINT approach capable of assessing the segment stoichiometry of >10 000 individual virus particles in one experiment; our approach can also spatially resolve the individual segments inside complete virus particles with a localization precision of ∼10 nm. Our results show the influenza genome can be assembled through multiple pathways in a redundant and cooperative process guided by preferentially interacting segment pairs and aided by synergistic effects that enhance genome assembly, driving it to completion. Our structural evidence indicates that the interaction strength of segment pairs affects the spatial configuration of the gene segments, which appears to be preserved in mature virions. As our method quantified the interactions of whole influenza segments instead of identifying individual sequence-based interactions, our results can serve as a template to quantify the contributions of individual sequence motifs to selective packaging.

Authors: Hepp, C; Zhao, Q; Robb, N; Fodor, E; Kapanidis, AN

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Oxford University Press
• Journal: Nucleic Acids Research
• Volume: 53
• Issue: 19
• Pages: gkaf1020
• Publication date: 2025-10-01
• Acceptance date: 2025-09-09
• DOI: 10.1093/nar/gkaf1020
• EISSN: 1362-4962
• ISSN: 0305-1048
• Pmid: 41099695
• Language: English
• Keywords: Virion; Virus Assembly; Viral Genome Packaging; Influenza A Virus, H1N1 Subtype; DNA, Viral; Humans; Genome, Viral; RNA, Viral