RNA-dependent RNA polymerase of predominant human norovirus forms liquid-liquid phase condensates as viral replication factories

Abstract

Biomolecular condensates are membrane-less cellular foci formed via liquid-liquid-phase separation (LLPS) of specific biological macromolecules to provide specialized compartments for regulating cellular functions. Many viral proteins undergo LLPS to form such condensates to support viral replication and evade host antiviral responses, and thus, these condensates are potential targets for designing antivirals. Human noroviruses (HuNoV) cause epidemic and sporadic gastroenteritis worldwide and are of significant health and economic burden. Here, we show that the RNA-dependent-RNA polymerase (RdRp) of the pandemic GII.4 HuNoV, which is essential for viral replication, forms distinct condensates capable of recruiting other viral replication components. Confocal microscopy and light scattering experiments show that RdRp phase separates into dynamic liquid-like condensates at physiological conditions. These condensates exhibit all the signature features of LLPS, including fluorescence recovery after photo-bleaching, droplet-fusion, surface wetting, and dripping in vitro and in live cells. More importantly, within these condensates, the RdRp exhibits a significant time-dependent increase in its enzymatic activity and recruits other components, such as RNA and the viral genome-linked protein (VPg), which are essential for viral replication. Such condensates, recognized by anti-RdRp antibodies, are observed in HuNoV-infected human intestinal enteroid cultures. Together, our studies demonstrate a hitherto unsuspected activity of HuNoV RdRp to form LLPS, which we suggest provides distinct cellular sites for efficient viral replication and its regulation.