Intrinsically-disordered N-termini in human parechovirus 1 capsid proteins bind encapsidated RNA

Abstract

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 recombinantly-expressed 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.

Figure 1

Results of whole-virion HPeV1 RCAP. The top hits arising from each of the capsid proteins from the virion RCAP are shown on the HPeV1 model (PDB: 4Z92). (a) Visualized on the outer surface of the capsid, all but one hit span the capsid and are exposed on the inner surface too. (b) Regions identified by RCAP visualized on the inner capsid surface. The peptides from VP0 are cyan, VP1 are magenta and VP3 are orange. Note that the N-termini of VP0 and VP1, although in the top hits, are disordered and therefore not seen in this representation.

Figure 2

RCAP of capsid protein VP1. RCAP results of recombinantly-expressed VP1 are shown on the HPeV1 model (PDB: 4Z92). (a) Peptides from VP1 identified by RCAP that are localized to the outer capsid surface (b) Peptides from VP1 identified by RCAP that localize to the inner capsid surface. (c) Locations of the peptides that contact RNA on a monomer of VP1 with a view corresponding to the outer capsid surface whereas (d) A view of VP1 that corresponds to the inner capsid surface.

Figure 3

RCAP of capsid protein VP0. RCAP results of recombinantly-expressed VP0 are shown on the HPeV1 model (PDB: 4z92). (a) Peptides derived from VP0 identified to contact RNA that localize to the outer capsid surface whereas (b) shows the peptides on the inner capsid surface. (c) Location of peptides from VP0 that contact RNA on a monomer of VP0 with a view corresponding to the outer capsid surface whereas (d) A view of VP0 that corresponds to the inner capsid surface.

Figure 4

Intrinsically disordered regions in VP0, VP1 and VP3 can interact with HPeV1 RNA. (A) RCAP results of peptides from VP0, VP1, and VP3 that could interact with the HPeV1 RNA. The residues in the peptides crosslinked to RNA are underlined. The VP0-A, VP1-A and VP3-A peptides were all missing from the X-ray structure and predicted to be intrinsically disordered. The VP0-B, VP1-B and VP3-B peptides were all modelled in the X-ray structure. (B) Peptides from intrinsically disordered regions of VP0, VP1, and VP3 can bind to HPeV1 RNA in a fluorescence anisotropy assay. Each error bar shows one standard deviation of three independent measurements. The asterisk denotes the samples whose anisotropy values differ from the anisotropy value of the peptide alone by a p value of <0.02 in the Student’s T-test.