Multivalent Interactions Between the Picornavirus 3C(D) Main Protease and RNA Oligonucleotides Induce Liquid–Liquid Phase Separation

The picornavirus 3CD protein is a precursor to the 3C main protease and the 3D RNA-dependent RNA polymerase. In addition to its functions in proteolytic processing of the virus polyprotein and cleavage of key host factors, the 3C domain interacts with cis-acting replication elements (CREs) within the viral genome to regulate replication and translation events. We investigated the molecular determinants of RNA binding to 3C using a wide range of biophysical and computational methods. These studies showed that 3C binds to a broad spectrum of RNA oligonucleotides, displaying minimal sequence and structure dependence, at least for these shorter RNAs. However, they also uncovered a novel aspect of these interactions, that is, 3C-RNA binding can induce liquid-liquid phase separation (LLPS), with 3CD-RNA interactions likewise leading to LLPS. This may be a general phenomenon for other 3C and 3C-like proteases and polyproteins incorporating 3C domains. These findings have potential implications in understanding virally induced apoptosis and the control of stress granules, which involve LLPS and include other proteins with known interactions with 3C/3CD.