Conformational flexibility in the enterovirus RNA replication platform

Abstract

A presumed RNA cloverleaf (5'CL), located at the 5'-most end of the noncoding region of the enterovirus genome, is the primary established site for initiation of genomic replication. Stem-loop B (SLB) and stem-loop D (SLD), the two largest stem-loops within the 5'CL, serve as recognition sites for protein interactions that are essential for replication. Here we present the solution structure of rhinovirus serotype 14 5'CL using a combination of nuclear magnetic resonance spectroscopy and small-angle X-ray scattering. In the absence of magnesium, the structure adopts an open, somewhat extended conformation. In the presence of magnesium, the structure compacts, bringing SLB and SLD into close contact, a geometry that creates an extensive accessible major groove surface, and permits interaction between the proteins that target each stem-loop.

Keywords: NMR; RNA; SAXS; cloverleaf; enterovirus; replication.

FIGURE 1.

Secondary structure prediction of RV-B14 5′CL. The RV-B14 5′CL is predicted to form a four helix junction containing stem A (SA; blue), stem–loop B (SLB; magenta), stem–loop C (SLC; red), and stem–loop D (SLD; green). Protein binding sites in SLB (magenta stars) and SLD (green stars) are shown, along with colored numbering for key nucleotides discussed in the text.

FIGURE 2.

2D ¹H,¹H-NOESY of RV-B14 5′CL. Imino NOESY spectra in the absence (blue) and presence (red) of magnesium. The multiple resonances for nucleotide U29 in the absence of magnesium (solid box) collapse to a single resonance in the presence of magnesium. The imino resonance of nucleotide U67 is only observed upon the addition of magnesium, with sequential imino–imino cross-peaks to U67 shown in the dashed box.

FIGURE 3.

Chemical shift differences for RV-B14 5′CL. ¹H chemical shift changes within SLB (magenta) and within SLD (green) induced by incorporation into the 5′CL (A) in the absence of magnesium and (B) in the presence of magnesium.

FIGURE 4.

SAXS data plots for RV-B14 5′CL. Plots are shown for data in the absence (blue) and presence (red) of magnesium. (A) Kratky plot. (B) Pairwise distance distribution function. (C) SAXS-only ab initio models (gray surface) of 5′CL in the absence (left) and presence (right) of magnesium, overlaid with SAXS/NMR-based structures with the helices colored as shown in Figure 1. (D) Overlay of experimental and calculated SAXS profiles.

FIGURE 5.

Structure of RV-B14 5′CL. Superimposition of the eight lowest energy structures of RV-B14 5′CL in (A) the absence of magnesium and (B) the presence of magnesium. The loop and terminal regions are colored in gray, the stem regions are colored as in Figure 1. Atoms from the bases of U29, U66, and U67, each of which show significant changes in NMR spectra upon addition of magnesium, are shown as white spheres.

FIGURE 6.

Changes in orientation induced by magnesium. (A) Structure in the absence of magnesium. (B) Structure in the presence of magnesium. The black arrow in part A illustrates the change in position of SLD due to the addition of magnesium. Stem regions are colored as in Figure 1.

FIGURE 7.

Major groove accessibility within RV-B14 5′CL. SLB and SLD from RV-B14 each contain accessible major groove regions, which align in the presence of magnesium. The gray arrow illustrates the position of the combined/aligned major groove in two different views of the structure in the presence of magnesium. Stem regions are colored as in Figure 1.

FIGURE 8.

RNA–protein interaction modeling. Modeled binding of PCBP KH1 domain (yellow) with SLB and 3C protease (orange) with SLD (A) in the absence and (B) in the presence of magnesium. Stem regions are colored as in Figure 1.

FIGURE 9.

RNA–protein interaction modeling. Modeled binding of PCBP KH1 domain (yellow) with SLB and poliovirus 3CD protease (3C: orange, 3D: rose) with SLD, using the 5′CL structure in the presence of magnesium. Stem regions are colored as in Figure 1.