Boehr, D. D. | bioRxiv Science

bioRxiv2025

Interactions between the picornavirus 3C(D) main protease and RNA 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 dependence on RNA sequence and structure. 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 polyprotein incorporating 3C domains. These findings have potential implications in understanding virally induced apoptosis and controlling stress granules, which involve LLPS and include other proteins with known interactions with 3C/3CD. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/648818v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@16bbd2corg.highwire.dtl.DTLVardef@50a2cborg.highwire.dtl.DTLVardef@1efc3beorg.highwire.dtl.DTLVardef@1beff0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2017

The RNA-binding site of poliovirus 3C protein doubles as a phosphoinositide-binding domain

Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using NMR spectroscopy. The PIP-binding site was located on a highly dynamic -helix that also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses.

biochemistry↗