Specific mutations in a viral RNA pseudoknot drastically change ribosomal frameshifting efficiency

Abstract

Many viruses regulate protein synthesis by -1 ribosomal frameshifting using an RNA pseudoknot. Frameshifting is vital for viral reproduction. Using the information gained from the recent high-resolution crystal structure of the beet western yellow virus pseudoknot, a systematic mutational analysis has been carried out in vitro and in vivo. We find that specific nucleotide tertiary interactions at the junction between the two stems of the pseudoknot are crucial. A triplex is found between stem 1 and loop 2, and triplex interactions are required for frameshifting function. For some mutations, loss of one hydrogen bond is sufficient to abolish frameshifting. Furthermore, mutations near the 5' end of the pseudoknot can increase frameshifting by nearly 300%, possibly by modifying ribosomal contacts. It is likely that the selection of suitable mutations can thus allow viruses to adjust frameshifting efficiencies and thereby regulate protein synthesis in response to environmental change.

Figure 1

Tertiary structure of BWYV RNA pseudoknot (A) Ribbon diagram of the overall fold of the pseudoknot. (B) Triplex hydrogen bonding interactions in the minor groove involving A20, A21, and C22. loop 2 is green, stem 1 is gold. The adenine base of A20 contacts G4 at the 2′-OH and N2 position. The 2′-OH of A20 forms a bifurcating hydrogen bonding network with two layers of base pairs. An additional bond (not shown) goes from the 2′-OH of C5 to N7 of A20 (15). A21 and C22 both bond to G16 at the 2′-OH position, and the amino group of G16 makes a phosphate contact to A21. In the crystal structure, a sodium ion (not shown) was found making base–base contacts between G16, A21, and C22. (C) Triplex interactions of A23 and A24 in the junction region. A23 and A24 use their Watson–Crick faces to interact with the minor groove side of the bases and 2′-OH groups of G7 and C15, which are in different strands of stem 1. (D) Junctional core interaction involving the four bases. The protonated C8 (indicated by +) simultaneously interacts with three other bases, G12, C26, and A25. C8 is on the same level as the G12⋅C26 base pair. Thick dashed lines represent the hydrogen bonding on the top layer, and gray dashed lines represent hydrogen bonds in the lower layer. Junctional base A25 tilts between C14 and C8. C26 propeller twists in its base pair to stack on A25.

Figure 2

(A) Template construct for ribosomal frameshifting. A stop codon is found immediately after the slippery sequence. Because of the requirement for restriction sites, the spacer sequence differs from the wild type. The in vitro template is transcribed by using T7 RNA polymerase at the T7 promoter, and the resulting transcripts are translated by a rabbit reticulocyte lysate in the same reaction tube. For the in vivo ribosomal frameshifting assay, the −1 ribosomal frameshifting elements, including the slippery sequence and the pseudoknot, are inserted between the 5′ β-galactosidase gene and the luciferase gene. A CMV promoter is used in human embryonic kidney cells. The nonframeshifted product is β-galactosidase, whereas frameshifting yields a fusion protein with luciferase. (B) SDS/PAGE analysis of [³⁵S]methionine-labeled translation products from ribosomal frameshifting assay of wild type and selected mutants in the reticulocyte lysate. Translation products are labeled with incoporation of [³⁵S]methionine. The nonframeshifting product (NFS) is the GST protein, and the −1 frameshift product (FS) is a GST–GFP fusion protein.

Figure 3

Mutations in the BWYV pseudoknot and their effects on −1 ribosomal frameshifting activity. The unmutated frameshifting efficiency in this experiment is 10.8%. The numbers near the mutations correspond to the frameshifting efficiency. (A) Mutations in the junction and loops, 1 and 2. (B) Mutations and inversions in stem 1 and stem 2 base pairs. (C) Mutations at the stem 1–loop 2 linker.

Figure 4

Comparison of in vivo and in vitro ribosomal frameshifting efficiencies of selected BWYV pseudoknot mutants. The G19(UC) mutation has a UC insertion instead of G19.