NMR structure of stem-loop D from human rhinovirus-14

Abstract

The 5'-cloverleaf of the picornavirus RNA genome is essential for the assembly of a ribonucleoprotein replication complex. Stem-loop D (SLD) of the cloverleaf is the recognition site for the multifunctional viral protein 3Cpro. This protein is the principal viral protease, and its interaction with SLD also helps to position the viral RNA-dependent RNA polymerase (3Dpol) for replication. Human rhinovirus-14 (HRV-14) is distinct from the majority of picornaviruses in that its SLD forms a cUAUg triloop instead of the more common uYACGg tetraloop. This difference appears to be functionally significant, as 3Cpro from tetraloop-containing viruses cannot bind the HRV-14 SLD. We have determined the solution structure of the HRV-14 SLD using NMR spectroscopy. The structure is predominantly an A-form helix, but with a central pyrimidine-pyrimidine base-paired region and a significantly widened major groove. The stabilizing hydrogen bonding present in the uYACGg tetraloop was not found in the cUAUg triloop. However, the triloop uses different structural elements to present a largely similar surface: sequence and underlying architecture are not conserved, but key aspects of the surface structure are. Important structural differences do exist, though, and may account for the observed cross-isotype binding specificities between 3Cpro and SLD.

FIGURE 1.

SLD sequences for which 3D structures have been determined. (A) Sequence of the HRV-14 stem–loop D (SLD) region investigated in this study. (B) Consensus sequence derived from rhinoviral and enteroviral SLDs, as defined by Du et al. (2004). (C) Coxsackievirus-B3 (CVB3) SLD sequence (Ohlenschlager et al. 2004). Figures 3–8 follow the coloring scheme introduced in this figure, to allow easy identification of mismatch and loop nucleotides. To facilitate comparison with the HRV-14 sequence, the third and fourth nucleotides in the tetraloops of parts B and C are numbered 15.1 and 15.2, respectively. Nucleotides in bold italic indicate points of divergence between the three sequences.

FIGURE 2.

Portion of 700 MHz 2D NOESY spectrum of HRV-14 SLD in H₂O containing imino–imino connectivities. A 1D ¹H NMR spectrum of the full imino region is plotted across the top. Key cross-peaks are labeled by nucleotide number.

FIGURE 3.

Stereo views of the superposition of the 10 lowest-energy structures of HRV-14 SLD determined (A) with RDCs, and (B) without RDCs. Bases are colored as in Figure 1A.

FIGURE 4.

A zoom view of the pyrimidine–pyrimidine-mismatch region. Base-pairing hydrogen bonds are indicated by dotted lines, while base hydrogens that do not participate in hydrogen bonds are omitted. Each of the two U–U base pairs form an H3–O2 and an O4–H3 hydrogen bond. However, in the lower pairing (U5–U23), the O2 is from U5 while the O4 is from U23. In the top pairing, the O2 is from U21 while the O4 is from U7. The central C6–U22 base pair forms hydrogen bonds from C6(amino group) to U22(O4) and from U22(H3) to C6(N3). The phoshpodiester backbone is shown as a worm, and the color scheme is that of Figure 1A, with the addition of red, white, and blue for base oxygen, hydrogen, and nitrogen atoms, respectively.

FIGURE 5.

The HRV-14 SLD triloop. (A) Cartoon representation. U13 (green) lies on the major groove side, while A14 (blue) lies along the minor groove. U15 is shown in pink. (B) Stereo view showing key NOE connectivities (red lines) and CH bonds specifically oriented by RDC data (red bonds). Nucleotides are otherwise colored as in Figure 1A, with the addition of violet phosphorous atoms to assist in viewing the path of the backbone.

FIGURE 6.

Space-filling views of NMR-determined structures of SLD from (A) HRV-14, (B) consensus sequence (Du et al. 2004), and (C) Coxsackievirus B3 (CVB3) (Ohlenschlager et al. 2004). Widened major grooves are evident at the center of each part of the figure. Sequences and colors are as in Figure 1A–C, respectively.

FIGURE 7.

(A) Plot of HRV-14 SLD major groove width, calculated as distance of closest phosphorous–phosphorous approach across the groove, minus 5.6 Å. Distances have been measured from the nucleotides listed directly below the x axis to the nucleotides listed below the error bars. Groove widths for the standard A and B forms are shown as horizontal lines within the figure. Base-paired nucleotides and coloring of the mismatch region have been added below the x axis for clarity. (B) A CPK rendering of HRV-14 SLD. Bases are again colored as in Figure 1A. Backbone phosphorous nuclei are shown in purple, while backbone oxygens not covalently bound to sugar carbons are shown in red. The widening of the major groove upon approach to the triloop is clearly evident.

FIGURE 8.

(A) Top view of HRV-14 SLD showing sugars and bases only of the nucleotides from the triloop, the central mismatch (orange and yellow), and the closing base pair (C12–G16). In this view, the triloop is closest to the observer. U13 (green), which lies in the major groove side, stacks onto C12 (gray). A14 (blue) lies on the minor groove side. (B) Similar view of the consensus SLD structure (see Fig. 1B). The closing base pair (U12–G16) is omitted for clarity. NMR structure of HRV-14 stem–loop D. However, the tetraloop base pair formed by C13–G15.2 (gray) plays a somewhat analogous role. A14 (blue) again lies along the minor groove, while C15.1 (green) lies on the major groove side. There is no base analogous to U15 from HRV-14 (pink base in A). (C) The CVB3 SLD structure (see Fig. 1C) shows similarity to the consensus structure in B.