Structural lability in stem-loop 1 drives a 5' UTR-3' UTR interaction in coronavirus replication

Abstract

The leader RNA of the 5' untranslated region (UTR) of coronaviral genomes contains two stem-loop structures denoted SL1 and SL2. Herein, we show that SL1 is functionally and structurally bipartite. While the upper region of SL1 is required to be paired, we observe strong genetic selection against viruses that contain a deletion of A35, an extrahelical nucleotide that destabilizes SL1, in favor of genomes that contain a diverse panel of destabilizing second-site mutations, due to introduction of a noncanonical base pair near A35. Viruses containing destabilizing SL1-DeltaA35 mutations also contain one of two specific mutations in the 3' UTR. Thermal denaturation and imino proton solvent exchange experiments reveal that the lower half of SL1 is unstable and that second-site SL1-DeltaA35 substitutions are characterized by one or more features of the wild-type SL1. We propose a "dynamic SL1" model, in which the base of SL1 has an optimized lability required to mediate a physical interaction between the 5' UTR and the 3' UTR that stimulates subgenomic RNA synthesis. Although not conserved at the nucleotide sequence level, these general structural characteristics of SL1 appear to be conserved in other coronaviral genomes.

Fig. 1

Predicted secondary structure model of MHV-A59 SL1 constructs discussed here. (a) WT; (b) WT*, which corresponds to SL1-Δ(C16/C19/C20) with a 5′-GA overhang required for initiation of in vitro transcription by SP6 polymerase; and (c) second-site revertant SL1s derived from infection with the SL1-ΔA35 virus (ΔA35 RNA sequence shown; recovered single-nucleotide substitutions highlighted in red). The constructs labeled WT*, ΔA35, ΔA35/U33C, ΔA35/C34U, and ΔA35/A36U RNAs (b and c) were used for the NMR and thermal unfolding studies. The 5′ g is a non-native nucleotide, shown in lower case.

Fig. 2

One-step growth curves of viable mutant and WT (MHV-A59 1000) viruses. (a) Viruses containing mutations in the upper (SL1-B and SL1-AB) and lower (SL1-C and SL1-D) portions of SL1 (see Table 1 for sequences). (b) Viruses recovered from infection with SL1-ΔA35 viruses.

Fig. 3

Phenotypes of recombinant SL1-ΔA35 second-site suppressor viruses. (r) indicates recombinant virus versus those recovered from infection with SL1-ΔA35 virion genomes. (a) Plaque size (mm). (b) One-step growth curves.

Fig. 4

Sequence scans of selected mutants. (a) Selection of the mutation at position 3′A29G in the SL1-ΔA35/C34U virus. (b) True reversion of the introduced mutant G7-C38 base pair to the WT A7-U38 base pair. The scans shown correspond to negative-sense sequences.

Fig. 5

Analysis of MHV-specific RNA synthesis. In all cases, BHK-R cells were electroporated with in vitro transcripts corresponding to the genomes of MHV-A59 1000 (WT), SL1-A, or a A59/nsp12-FS frameshift mutant incapable of directing the synthesis of viral RNAs. Total RNAs were extracted at the times indicated (4 hpe, 8 hpe, and 12 hpe) and analyzed by RT-PCR (see Materials and Methods) for (a) negative-sense anti-gRNA; (b) negative- and positive-sense sgRNA 7; or (c) gRNA. Note that total RNA is used as the template for RT in (a) and (b); poly(A) ⁺ RNA was used as the template in (c), and duplicate samples were analyzed. The arrows indicate the position of the amplicons expected for each RNA species. GAPDH, RNA recovery control.

Fig. 6

Comparison of the thermal unfolding of the WT*, ΔA35, ΔA35/U33C, ΔA35/C34U, and ΔA35/A36U SL1 RNAs. The experimental optical melting profiles show every fifth data point collected at 260 nm (•) and 280 nm (○), with the calculated fits (dashed lines) shown. For the ΔA35 RNA, the nonlinear least-squares simultaneous composite fit to a single transition unfolding model, and the transition is shown in solid line. For the WT* model and three recovered SL1 mutant ΔA35/U33C, ΔA35/C34U, and ΔA35/A36U RNAs, a nonlinear least-squares simultaneous composite fit to a two-transition unfolding model, and component transitions 1 and 2 (solid lines) are shown. The thermodynamic parameters derived from these fits are compiled in Table 2.

Fig. 7

Imino proton regions of 1D jump–return echo spectra acquired at 10 °C and 10 mM KP_i (pH 6.0) for WT* (a), ΔA35 (b), ΔA35/U33C (c), ΔA35/C34U (d), and ΔA35/A36U (e). Imino protons corresponding to noncanonical base pairs are shown in bold. Note that some spectra (WT*, ΔA35/U33C, and ΔA35/C34U) are characterized by slow conformational heterogeneity at the base portion of SL1 (A5-U40, G6-C39, and A7-U38 base pairs).

Fig. 8

Graphical representation of the imino proton solvent exchange rates (k_ex) for the SL1 WT* and mutants. (a) Secondary structure of the WT* RNA. (b) k_ex is plotted versus base-pair position (from the bottom to the top of the SL1 helix, from left to right). The mutations are shaded in red. k_ex could not be unambiguously measured for the G8 for the middle three RNAs due to spectral overlap with G15; the same is true of G14 and G17 imino protons in all spectra. In these cases, the average k_ex is plotted.

Fig. 9

Model of a dynamic SL1 that is consistent with the functional and structural data presented here. The fully based-paired SL1 (A; modeled by the ΔA35 RNA) exists in equilibrium with one or more higher-energy conformers (A′; WT* and ΔA35 second-site revertants) that are partially unfolded or that experience dynamic destabilization as a result of noncanonical pairing. A hypothetical protein (B) binds to both A and A′ to form the same partially unwound AB complex, but the affinity of B for A′ will be higher, since the full energetic cost of unfolding the lower stem will not have to be paid; this interaction then mediates a long-distance RNA–RNA, RNA–protein, or protein–protein interaction, which is crucial for the viral replication.