Phase separation underlies interactions of the SARS-CoV-2 nucleocapsid protein with nucleoli.

Abstract

RNA viruses hijack host cell functions to promote viral replication. During infection, viral components enter membrane-bound and membrane-less cellular organelles, including the phase-separated nucleolus, where they perturb nucleolar proteins to aid virus replication. Recently, viruses have been shown to utilize the process of liquid-liquid phase separation (LLPS) to package their genome and promote viral RNA synthesis. The viral nucleocapsid (N) protein is critically involved in several aspects of virus replication. Importantly, the nucleolar phosphoprotein, Nucleophosmin (NPM1), with roles in ribosome biogenesis, was reported to interact with the N protein of several different RNA viruses and facilitate viral replication. However, the impact of this interaction and SARS CoV-2 N protein (NCoV-2) nucleolar localization on ribosome biogenesis and viral life cycle remains poorly understood. Our in vitro studies show that NPM1 undergoes heterotypic phase separation with NCoV-2, causing the formation of liquid-like droplets or condensates with restricted NPM1 mobility. Sequence analysis of NCoV-2 revealed enrichment of positively charged (Arg and Lys) amino acids, an important “sequence feature” that governs nuclear and nucleolar localization. Importantly, our cellular studies reveal the colocalization of NCoV-2 with NPM1 in the nucleoli of Vero E6 cells. These results suggest that there is a direct interaction between NPM1 and NCoV-2. Strikingly, NCoV-2 displaces NPM1 from reconstituted, granular component-like condensates and from nucleoli. Based on our collective findings, we propose that displacement of regulatory proteins (e.g., NPM1) from nucleoli by NCoV-2 through interactions of its Arg residues with ribosomal RNA (rRNA), may promote protein synthesis and the viral life cycle. These mechanistic insights will provide new avenues for the development of novel antiviral therapies for inhibition of nucleolar localization of NCoV-2.