Nucleoside Analogs and Perylene Derivatives Modulate Phase Separation of SARS-CoV-2 N Protein and Genomic RNA In Vitro

Abstract

The life cycle of severe acute respiratory syndrome coronavirus 2 includes several steps that are supposedly mediated by liquid–liquid phase separation (LLPS) of the viral nucleocapsid protein (N) and genomic RNA. To facilitate the rational design of LLPS-targeting therapeutics, we modeled N-RNA biomolecular condensates in vitro and analyzed their sensitivity to several small-molecule antivirals. The model condensates were obtained and visualized under physiological conditions using an optimized RNA sequence enriched with N-binding motifs. The antivirals were selected based on their presumed ability to compete with RNA for specific N sites or interfere with non-specific pi–pi/cation–pi interactions. The set of antivirals included fleximers, 5′-norcarbocyclic nucleoside analogs, and perylene-harboring nucleoside analogs as well as non-nucleoside amphiphilic and hydrophobic perylene derivatives. Most of these antivirals enhanced the formation of N-RNA condensates. Hydrophobic perylene derivatives and 5′-norcarbocyclic derivatives caused up to 50-fold and 15-fold enhancement, respectively. Molecular modeling data argue that hydrophobic compounds do not hamper specific N-RNA interactions and may promote non-specific ones. These findings shed light on the determinants of potent small-molecule modulators of viral LLPS.

Exploring the conformational dynamics of the SARS-CoV-2 SL4 hairpin by combining optical tweezers and base analogues

“…Increasing attention regarding the infection cycle of SARS-CoV-2 is drawn to the intracellular liquid-liquid phase separation of the gRNA driven by the N protein. 3,7,41,42 This genome packaging mechanism enables highly specific recognition and selection of the SARS-CoV-2 genetic material from an abundance of viral and cellular RNAs present in infected cells. The SL4 hairpin of the SARS-CoV-2 gRNA 5′-end examined herein has been proposed as one of the N proteinbinding sites in the initiation phase of gRNA condensation 3,4 and a promising therapeutic target, structurally conserved among SARS-CoV-2 variants and other Betacoronaviruses.…”