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. 2013 Jan 18;5(1):279-94.
doi: 10.3390/v5010279.

Altering SARS coronavirus frameshift efficiency affects genomic and subgenomic RNA production

Ewan P Plant  1 , Amy C Sims, Ralph S Baric, Jonathan D Dinman, Deborah R Taylor
Affiliations

Altering SARS coronavirus frameshift efficiency affects genomic and subgenomic RNA production

Ewan P Plant et al. Viruses. .

Abstract

In previous studies, differences in the amount of genomic and subgenomic RNA produced by coronaviruses with mutations in the programmed ribosomal frameshift signal of ORF1a/b were observed. It was not clear if these differences were due to changes in genomic sequence, the protein sequence or the frequency of frameshifting. Here, viruses with synonymous codon changes are shown to produce different ratios of genomic and subgenomic RNA. These findings demonstrate that the protein sequence is not the primary cause of altered genomic and subgenomic RNA production. The synonymous codon changes affect both the structure of the frameshift signal and frameshifting efficiency. Small differences in frameshifting efficiency result in dramatic differences in genomic RNA production and TCID50 suggesting that the frameshifting frequency must stay above a certain threshold for optimal virus production. The data suggest that either the RNA sequence or the ratio of viral proteins resulting from different levels of frameshifting affects viral replication.

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Figures

Figure 1
Figure 1
Removing stem 3 does not reduce programmed -1 ribosomal frameshifting (-1 PRF) efficiency. The predicted secondary structure of the SARS coronavirus pseudoknot is shown, along with a series of mutants with stem 3 truncated. The stems are labeled S1 to S3 and the loops labeled L1 to L3. Site directed mutagenesis was used to replace the wild-type loop 2 with a CUUG tetraloop (L2Tetra). Additional truncations to stem3 were made (L2Tetra/S3-4bp and L2Tetra/S3-2bp) and frameshifting efficiency analyzed by dual luciferase assay. Frameshifting efficiency is expressed as a percentage with standard error as described in the experimental section.
Figure 2
Figure 2
Stabilizing stem 3 with a tetraloop preserves -1 PRF efficiency. Site directed mutagenesis was used to change loop2 of the wild-type SARS pseudoknot into tetraloop (L2Tetra from Figure 1). The bulged adenosines in stems 2 and 3 were removed by site directed mutagenesis singly or together in conjunction with the tetraloop (L2TetraS2Δ, L2TetraS3Δ, and L2TetraS2ΔS3Δ). Both bulged adenosines were removed from the wild-type pseudoknot (S2ΔS3Δ). Frameshifting efficiency is expressed as a percentage with standard error as described in the experimental section.
Figure 3
Figure 3
Mutations to stem 3 alter pseudoknot structure. (A) The sequence and structure of the wild-type SARS coronavirus pseudoknot is shown on the left (WT). The stems are labeled S1 to S3 with the 5’ portion denoted ‘a’ and the 3’ portion ‘b’. The loops connecting the stems are labeled L1 and L3, and the loop capping the third stem is labeled L2. Changes made to the pseudoknot are shaded and underlined in mutants S2D and L2-UCC. The efficiency of frameshifting as determined by dual luciferase assay is expressed as a percentage below each mutant with standard error shown. (B) Comparison of L2-UCC and WT pseudoknots by SHAPE analysis (see Experimental Section). An autoradiograph of the primer extension for the wild-type and L2-UCC pseudoknots is shown. The sequencing ladder is shown on the left side of the gel and SHAPE reactions are shown on the right. Reactions performed with (+) or without (-) NMIA are indicated. The closed carrots show increased reactivity and the open carrots show reduced reactivity. (C) Diagrams of pseudoknots with mutations (shaded and underlined) in Stem 3. Asterisks mark the position of deleted adenosines. Frameshifting efficiency is shown below each mutant. Positions of increased or decreased NMIA reactivity are indicated. (D) SHAPE analysis of stem 3 mutants. The position of the stems is indicated on the gel, note that because S2ΔS3Δ contains deletions the position of the stems differ slightly from the S3D stems. The changes for S2ΔS3Δ are indicated on the left and the changes to S3D are indicated on the right of the gel.
Figure 4
Figure 4
Mutations to stem 3 can affect virus infectivity and replication. (A) Schematic of the wild-type SARS pseudoknot and viral mutants. The specific bases that were altered are shaded and underlined. (B) Tissue culture infectious dose was determined as described in the materials and methods. The values shown are the average of six measurements with error bars showing the standard deviation. Asterisks indicate values that were at the lower limit of the assay. (C) Relative abundance of genomic and subgenomic RNA in viral stocks. Plaque purified virus was used to infect Vero cells. Four days post infection CPE was observed. Media and detached cells were removed. RNA was extracted from a 100μL aliquot using Trizol. Taqman analysis was used to determine the total number of genomic and subgenomic RNA molecules compared to a reference RNA transcribed from a SARS replicon. The number of copies per mL of viral stock is shown with standard deviation.
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