Intrinsically-disordered N-termini in human parechovirus 1 capsid proteins bind encapsidated RNA (original) (raw)

Human parechoviruses (HPeV) are picornaviruses with a highly-ordered RNA genome contained within icosahedrally-symmetric capsids. Ordered RNA structures have recently been shown to interact with capsid proteins VP1 and VP3 and facilitate virus assembly in HPeV1. Using an assay that combines reversible cross-linking, RNA affinity purification and peptide mass fingerprinting (RCAP), we mapped the RNA-interacting regions of the capsid proteins from the whole HPeV1 virion in solution. The intrinsically-disordered N-termini of capsid proteins VP1 and VP3, and unexpectedly, VP0, were identified to interact with RNA. Comparing these results to those obtained using recombinantlyexpressed VP0 and VP1 confirmed the virion binding regions, and revealed unique RNA binding regions in the isolated VP0 not previously observed in the crystal structure of HPeV1. We used RNA fluorescence anisotropy to confirm the RNA-binding competency of each of the capsid proteins' N-termini. These findings suggests that dynamic interactions between the viral RNA and the capsid proteins modulate virus assembly, and suggest a novel role for VP0. Human parechoviruses (HPeV) are important human pathogens for which we lack antivirals or vaccines. They have a positive-sense, single-stranded RNA genome and belong to the Picornaviridae family. The mature virion is icosahedrally-symmetric with a triangulation number of T = 1 (pseudo T = 3) and is composed of capsid proteins VP0, VP1 and VP3 1-4. Unlike in other picornaviruses, the parechovirus VP0 is not proteolytically cleaved in the final maturation of the virions 5. There is also an extensive network of VP0 N-termini on the inner capsid surface that enhance inter-pentamer stability, along with an annulus of VP3 termini under the vertex 3. Regions of structured RNA were recently identified as packaging signals (PSs) that interact with VP1 and VP3 in the HPeV virion 4. Upon interaction with viral pentameric assembly intermediates, these PSs drive capsid assembly. Multiple VP1 and VP3 residues were found to contact the viral RNA in the atomic models of HPeV1 (PDB: 4Z92 & 5MJV) 3,4. When these residues were mutated to alanine, virus assembly was prevented 4. The atomic models do not cover the complete sequences of the capsid proteins or the full genome. The N-terminal regions of all three capsid proteins were apparently disordered 3,4. The virion population may contain multiple states of both the RNA and the capsid, as was recently observed for bacteriophage MS2 6-8. Hence, we expect that there are more RNA-protein interactions to be discovered in the virion. More direct methods could be utilized to identify the regions of the HPeV1 capsid that interact with the encapsidated RNA. One such method is reversible cross-linking, affinity purification, and peptide-mass fingerprinting (RCAP) which has previously been used to map protein-nucleic acid interaction sites. RCAP has been successfully used to map regions of the capsid protein that interact with the virion RNA in brome mosaic virus, adenovirus, and bacteriophage MS2 9-12. The MS2 protein-RNA interactions identified by the RCAP assay have since been confirmed in asymmetric cryoEM reconstructions of MS2 6,13. Here we mapped regions within the HPeV1 capsid proteins that interact with the encapsidated RNA using RCAP. Several regions within VP1 and VP3 were found to interact with the RNA. Surprisingly, VP0 was also identified to contact the genomic RNA within the HPeV virion. The N-terminal regions of all capsid proteins not visualized in the HPeV1 atomic model apparently contact viral RNA. Recombinantly-expressed VP0 and