Structure of RNA Stem Loop B from the Picornavirus Replication Platform

Abstract

The presumptive RNA cloverleaf at the start of the 5'-untranslated region of the picornavirus genome is an essential element in replication. Stem loop B (SLB) of the cloverleaf is a recognition site for the host polyC-binding protein, which initiates a switch from translation to replication. Here we present the solution structure of human rhinovirus isotype 14 SLB using nuclear magnetic resonance spectroscopy. SLB adopts a predominantly A-form helical structure. The stem contains five Watson-Crick base pairs and one wobble base pair and is capped by an eight-nucleotide loop. The wobble base pair introduces perturbations into the helical parameters but does not appear to introduce flexibility. However, the helix major groove appears to be accessible. Flexibility is seen throughout the loop and in the terminal nucleotides. The pyrimidine-rich region of the loop, the apparent recognition site for the polyC-binding protein, is the most disordered region of the structure.

Figure 1

Secondary structure prediction of the HRV-14 5′CL. SLB, the subject of this investigation, is circled. SLB is predicted to contain six Watson–Crick base pairs with one wobble base pair shown in color and an eight-nucleotide hairpin loop. Nucleotides implicated in PCBP binding are located in the loop and are shown in color.

Figure 2

Assigned imino proton portion of the 250 ms ¹H–¹H NOESY spectrum at 278 K of HRV-14 SLB in H₂O with the 1D ¹H NMR spectrum shown above. A diagram of the helix is shown at the right.

Figure 3

Base to H1′/H5 region of a 250 ms ¹H–¹H NOESY spectrum of SLB in D₂O. Peaks representing intranucleotide H1′–H6/H8 NOEs are labeled with the nucleotide number. The lines connect the labeled peaks through sequential H1′–H6/H8 NOE cross peaks. The H2 resonances not involved in the walk are underlined.

Figure 4

Superposition of the final 10 structures of HRV-14 SLB determined in this study. The loop and terminal regions are shown as gray ribbons. The bases of hydrogen-bonded base pairs are shown in the following colors: orange for cytosine, yellow for uracil, green for guanine, and blue for adenine.

Figure 5

(A) Comparison between SLB ¹³C chemical shifts and chemical shifts of nucleotide monophosphates (NMPs). The chemical shift difference [δ(NMP) – δ(SLB)] is shown in parts per million. (B) Normalized intensity of C6, C8, and C2 resonances in the ¹H–¹³C HSQC spectrum. (C) SLB ¹H–¹³C RDC values extracted from ¹H–¹³C ARSTY spectra. In each section, the regions comprising the helix are shaded gray, and the color/shape coding of the bars is defined to the right of the figure.

Figure 6

Space-filling view of the average calculated structure of HRV-14 SLB. The helix region is colored gray, and the loop (top) and termini (bottom) are colored blue. The red dashed line shows the shortest distance between phosphorus atoms P_i and P_i–5 across the major groove (G3–C17).