Mar 31 2023 Investigating RNA–RNA interactions through computational and biophysical analysis

Unraveling the intricate RNA-RNA interactions in Japanese encephalitis virus (JEV) opens new possibilities for understanding viral replication and designing antiviral therapeutics, as our recent study reveals the importance of a conserved cyclization sequence in driving these interactions

In this study, we have utilized a unique combination of computational and biophysical techniques to investigate RNA-RNA interactions in Japanese encephalitis virus (JEV). We identified a specific JEV isolate with a hypothesized high-affinity interaction, and then employed various biophysical characterization methods to validate and characterize this interaction. Through size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) and analytical ultracentrifugation (AUC), we demonstrated the direct interaction between the 5' and 3' terminal regions (TRs) of JEV with nanomolar affinity and 1:1 stoichiometry.

Our computational analysis supported a kinetic favorability of the cyclization sequence interaction and its competitive advantage over potential homo-dimer RNA interactions. This provides valuable insights into the replication pathway/mechanism of flavivirus NS5 in replicating the viral genome. Our study also presents a low-resolution ab initio model that offers a potential architectural arrangement of this RNA-RNA interaction in solution. These findings lay a solid foundation for the development of pharmaceutical therapies targeting viral replication through interruption of the cyclization sequence, and also shed light on the fundamental understanding of RNA-RNA interactions in flaviviruses and other viral systems.

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Abstract

Numerous viruses utilize essential long-range RNA–RNA genome interactions, specifically flaviviruses. Using Japanese encephalitis virus (JEV) as a model system, we computationally predicted and then biophysically validated and characterized its long-range RNA–RNA genomic interaction. Using multiple RNA computation assessment programs, we determine the primary RNA–RNA interacting site among JEV isolates and numerous related viruses. Following in vitro transcription of RNA, we provide, for the first time, characterization of an RNA–RNA interaction using size-exclusion chromatography coupled with multi-angle light scattering and analytical ultracentrifugation. Next, we demonstrate that the 5′ and 3′ terminal regions of JEV interact with nM affinity using microscale thermophoresis, and this affinity is significantly reduced when the conserved cyclization sequence is not present. Furthermore, we perform computational kinetic analyses validating the cyclization sequence as the primary driver of this RNA–RNA interaction. Finally, we examined the 3D structure of the interaction using small-angle X-ray scattering, revealing a flexible yet stable interaction. This pathway can be adapted and utilized to study various viral and human long-non-coding RNA–RNA interactions and determine their binding affinities, a critical pharmacological property of designing potential therapeutics.

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Citation

Investigating RNA-RNA interactions through computational and biophysical analysis
Tyler Mrozowich, Sean Park, Maria Waldl, Amy Henrickson, Scott Tersteeg, Corey R. Nelson, Anneke De Klerk, Borries Demeler, Ivo L. Hofacker, Michael T. Wolfinger, Trushar R. Patel
Nucleic Acids Res. gkad223 (2023) | doi:10.1093/nar/gkad223 | PDF | Supplement

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