Introduction: Flaviviruses subvert host mRNA decay pathway during an infection to generate a functional non-coding RNA, known as the Flaviviral Subgenomic RNA (sfRNA), produced by incomplete exonuclease (XRN1) digestion of the Flaviviral genomic 3’UTR. We recently demonstrated that the Zika Virus (ZIKV) sfRNA binds and stabilizes the viral non-structural protein 5 (NS5), allowing for intracellular accumulation of NS5 and potent antagonism of the innate antiviral response. Herein we aim to characterize the interaction between ZIKV sfRNA and NS5 and elucidate the mechanism underlying protein stabilization, and to investigate whether the sfRNA-NS5 interaction is an evolutionarily conserved mechanism similarly employed by other Flaviviruses.
Methods: Two label-free techniques were employed to study the binding interaction between ZIKV sfRNA and NS5, Electrophoretic Mobility Shift Assay (EMSA) and Isothermal Titration Calorimetry (ITC), with both techniques utilizing in vitro transcribed RNA and purified NS5 proteins. In addition, sfRNA-deficient West Nile (WNV) and Yellow Fever (YFV) viruses were generated as loss of function models to investigate sfRNA function in NS5 stabilization and antagonism of interferon signalling.
Results: EMSA revealed interactions between ZIKV NS5 and sfRNA, localized to highly structured segments of sfRNA which form pseudoknots, also designated as XRN1-resistant RNAs (xrRNAs). ZIKV xrRNA1 and xrRNA2 were observed to bind with NS5, resulting in RNA-protein complexes with reduced electrophoretic mobility. These results were further validated through ITC, where the binding affinity and thermodynamic parameters of the xrRNA-NS5 interaction were characterized. Furthermore, sfRNA-deficient mutant WNV and YFV exhibited attenuation in antagonism of interferon signalling, as observed from their limited ability to inhibit STAT1 phosphorylation.
Conclusion: In this study, the ZIKV sfRNA-NS5 binding interaction is detailed and characterized for the first time. Further investigation is still required to determine whether sfRNA expressed by WNV and YFV interacts with NS5 with similar protein-stabilizing mechanisms.