Allosteric activation of the RNA and lipid binding capabilities of the picornaviral 3CD polyprotein

In the backdrop of a hostile cellular environment, viral genomes must encode processes to regulate the host cell’s machinery to allow for the efficient replication and packaging of new virus particles. These processes are encoded by relatively small genomes, and so viruses face a formidable information storage problem. Picornaviruses are positive‐strand RNA viruses, and first produce a large polyprotein that must then be cleaved to generate functional components. Proteolytic precursors and fully mature forms of viral proteins often have different functions. The picornavirus 3CD protein exemplifies this concept. The 3C protein is a protease and has RNA‐binding capabilities, and the 3D protein is an RNA‐dependent RNA polymerase (RdRp). The 3CD protein has unique protease and RNA‐binding abilities relative to 3C and is devoid of RdRp activity. How these functional changes in 3CD arise is poorly explained by its X‐ray crystal structure, which suggests that the 3C and 3D domains are merely tethered together by a linker (i.e. ‘beads‐on‐a‐string’ type model), and the domains do not interact. Computer simulations and small angle X‐ray scattering experiments are consistent with a dynamic 3CD conformational ensemble in which transient interactions between the domains may influence function. Our biophysical studies suggest an even simpler mechanism – extension of the C‐terminal tail of 3C by a few amino acid residues dramatically changes function. A network of interactions then transmits these structural/dynamic changes to the protease active site and the RNA‐binding site, which may also affect the ability of 3CD to interact with phosphoinositide‐containing viral replication membranes. Proteolytic processing of the C‐terminus is then the switch between 3CD and 3C/3D function. Such a simple mechanism may not require any additional domain‐domain interactions in the 3CD polyprotein to regulate domain function. In a broader context, we propose that the conformational dynamics of precursor and processed forms of viral proteins regulate the specific function of these molecules during the viral replication cycle to expand the functional proteome beyond the limited storage capacity available within small viral genomes. Support or Funding Information This work was funded through NIH R01 grants from NIAID: AI091985 and AI104878 (DDB) and AI053531 (CEC)( A ) X‐ray crystal structure of 3C (PDB 1L1N) highlighting the protease catalytic triad in black, and residues important for RNA and phosphoinositide lipid binding highlighted in green and orange, respectively. Binding of ( B ) phosphoinositide lipid and ( C ) RNA to the 3C constructs was measured using fluorescence polarization. The Dn designations indicate the number (n) of 3D residues added to the C‐terminus of 3C.