hnRNPA1 Remodels SARS-CoV-2 Genomic G-Quadruplexes through Its UP1 Domain

Abstract

The single-stranded RNA genome of SARS-CoV-2 contains several predicted G-quadruplex (G4) forming sequences. Pharmacological stabilisation of these structures has been shown to suppress viral replication, suggesting that their resolution may be required for efficient viral replication. However, the roles of host proteins in stabilization or destabilization of G4 structures have remained largely unexplored. Human hnRNPA1, one of the most abundant RNA-binding proteins in eukaryotic cells, is a G-quadruplex unfolding protein and has independently been identified as a SARS-CoV-2 genome-binding protein. However, whether it acts on the G-quadruplexes within the SARS-CoV-2 genome has not been examined. Here, we screened eight putative G-quadruplex forming sequences conserved across SARS-related coronaviruses and identified two sequences that fold into G4 structures, confirmed by circular dichroism (CD) spectroscopy and thioflavin T (ThT) fluorescence-based experiments. Furthermore, we showed that the UP1 and UP1+RGG domains of hnRNPA1 specifically bind and unfold these two viral RNA G-quadruplexes. Both UP1 and UP1+RGG unfold viral RNA G-quadruplexes to similar extent, indicating that recognition and unfolding of these viral G-quadruplexes are mediated mainly by the UP1 domain of hnRNPA1. Together, these findings identify hnRNPA1 as a host factor capable of binding and remodelling SARS-CoV-2 genomic RNA G-quadruplexes and suggest a mechanism by which the virus may exploit an abundant host protein to resolve G4 structures within its genome.