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.

Conformational flexibility in the enterovirus RNA replication platform

“…The imino chemical shift difference between SLB and SLD in previously described isolated stem-loops (Headey et al 2007;Warden et al 2017) versus when incorporated into the 5 ′ CL (without magnesium) are shown in Figure 3A. The differences are small (RMSD 0.028 ppm), FIGURE 1.…”

“…In this overlay, part of the SLB loop region extends outside of the ab initio model. However, this SLB loop has been shown to be highly dynamic (Warden et al 2017), and so its position in this figure is somewhat misleading. As a dynamic loop, it can explore many conformations and positions, and so may not give rise to intense density in the ab initio model.…”

“…In general, imino proton resonances were assigned based on sequential imino-imino NOESY cross-peaks ( Fig. 2), with the assistance of imino proton resonance assignments from previously published works with isolated RV-B14 SLB (Warden et al 2017) and SLD (Headey et al 2007). The observed pattern of sequential imino-imino NOE cross-peaks confirmed the formation of SA, SLB, SLC, and SLD helices, and the absence of additional imino resonances indicated nucleotides that are not involved in stable hydrogen bonding (for a full list of the hydrogen bond constraints, see Materials and Methods).…”

“…Furthermore, when high-resolution structures of the isolated stem-loops are available (via conventional NMR or crystallographic analyses), and when the conservation of these separate structures is verified upon incorporation into the complex, the SAXS/NMR approach is capable of producing high-resolution information. In the present case, prior high-resolution structures are available for the largest two stem-loops (SLB and SLD [Headey et al 2007;Warden et al 2017]); which together comprise 61% of the total cloverleaf. As discussed in the Results section, chemical shift analysis, together with RDC and SAXS fitting, confirm that SLB and SLD structures are largely conserved upon incorporation into the cloverleaf, and pertinent measures were taken to adjust the individual structures to the few data that indicate localized rearrangement.…”

“…The narrow major groove restricts protein access for basespecific interactions to the minor groove side. However, the isolated solution structures of RV-B14 SLB and SLD each contain atypically wide and accessible major grooves (9 Å and 13 Å width, respectively) (Headey et al 2007;Warden et al 2017), a feature that is retained upon incorporation into the cloverleaf (see below). In this context, it is interesting to note that magnesium has been shown to help stabilize non-Watson-Crick base pairs (Correll et al 1997).…”