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. 2011 Aug 15;417(1):79-86.
doi: 10.1016/j.virol.2011.05.008. Epub 2011 Jun 8.

Mutational analysis of three predicted 5'-proximal stem-loop structures in the genome of tick-borne encephalitis virus indicates different roles in RNA replication and translation

Harald Rouha  1 , Verena M Hoenninger, Caroline Thurner, Christian W Mandl
Affiliations

Mutational analysis of three predicted 5'-proximal stem-loop structures in the genome of tick-borne encephalitis virus indicates different roles in RNA replication and translation

Harald Rouha et al. Virology. .

Abstract

Flavivirus gene expression is modulated by RNA secondary structure elements at the terminal ends of the viral RNA molecule. For tick-borne encephalitis virus (TBEV), four stem-loop (SL) elements have been predicted in the first 180 nucleotides of the viral genome: 5'-SL1, 5'-SL2, 5'-SL3 and 5'-SL4. The last three of these appear to be unique to tick-borne flaviviruses. Here, we report their characterization by mutagenesis in a TBEV luciferase reporter system. By manipulating their thermodynamic properties, we found that an optimal stability of the 5'-SL2 is required for efficient RNA replication. 5'-SL3 formation is also important for viral RNA replication, but although it contains the viral start codon, its formation is dispensable for RNA translation. 5'-SL4 appears to facilitate both RNA translation and replication. Our data suggest that maintenance of the balanced thermodynamic stability of these SL elements is important for temporal regulation of its different functions.

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Figures

Fig. 1
Fig. 1
Genome secondary structure and organization of TBEV and derived replicon constructs. (A) The 5′ and 3′ regions of the TBEV genome. The stem-loop structures are shown as predicted for the linear form, without consideration of the long-range interaction of the cyclization sequences (CS) (depicted in green) in the viral5′-SL2 and 3′-SL1. The CS element was characterized in an earlier study (Kofler et al., 2006) and was originally called CS-A. The viral start codon AUG within the 5′-SL3 structure is highlighted in blue. (B) Schematic diagram of the parental replicon C17 (wt), in which the structural protein region of TBEV was replaced by an in-frame insertion of the firefly luciferase gene (fluc) (Rouha et al., 2010). C17, truncated capsid protein gene; NS1–NS5, the coding region for the non-structural proteins 1–5; NCR, non-coding region; TaV2A site, Thosea asigna virus 2A site. (C) Schematic diagram of mutant replicon ΔAUG, which has a deletion starting from nucleotide position 133, causing it to lack the entire region encoding the capsid protein, the 5′-SL3 element, and the viral start codon. Diagrams A–C are not drawn to scale.
Fig. 2
Fig. 2
Characterization of 5′-SL2. (A) Schematic drawing and thermodynamic analysis of the predicted secondary structure of wild-type 5′-SL2 and those of the engineered mutant replicons. Nucleotide changes are depicted in red. ΔG, Gibbs free energy of the secondary structure of the first 194 nucleotides of the viral genome. A decrease in the ΔG value relative to the wild-type indicates stabilization of the secondary structure, whereas an increase indicates destabilization. (B) Translation level of viral input RNA in BHK-21 cells 3 h posttransfection. Normalized luciferase levels are shown as percentages relative to the parental control C17 (wt). RNA of construct ΔAUG, which lacks the viral translational AUG start codon, was used as a negative control. Error bars represent standard deviation of at least two independent experiments. (C) Viral RNA replication efficiencies of the parental C17 replicon (wt) and mutants derived from it, monitored by comparing normalized firefly luciferase activity in BHK-21 cells 15.5–72 h posttransfection. Capped NS5-GAA RNA, which can be translated but cannot replicate due to a GDD-to-GAA mutation in the viral RNA polymerase gene NS5, was used as a control. Error bars represent the standard deviation of two independent experiments, each measured in triplicate.
Fig. 3
Fig. 3
Characterization of 5′-SL3. (A) Schematic drawing and thermodynamic analysis of the predicted secondary structures of wild-type 5′-SL3 (wt, C17, bold letters) and engineered mutant replicons. Nucleotide changes are depicted in red. The viral start codon AUG within the 5′-SL3 structure is highlighted in blue. ΔG, Gibbs free energy of the secondary structure of the first 194 nucleotides of the viral genome. A decrease in the ΔG value relative to the wild-type indicates stabilization of the secondary structure, whereas an increase indicates destabilization. (B) Translation level of viral input RNA in BHK-21 cells 3 h posttransfection. Normalized luciferase levels are shown as percentages relative to the parental control C17 (wt). RNA of construct ΔAUG, lacking the viral translational start codon AUG, was used as a negative control. Error bars represent standard deviation of at least two independent experiments. (C) Viral RNA replication efficiencies of the parental C17 replicon and mutants derived from it, monitored by comparing normalized firefly luciferase activity in BHK-21 cells 15.5–72 h posttransfection. Capped NS5-GAA RNA, which can be translated but cannot replicate due to a GDD-to-GAA mutation in the viral RNA polymerase gene NS5, was used as a control. Error bars represent the standard deviation of two independent experiments, each measured in triplicate.
Fig. 4
Fig. 4
Characterization of 5′-SL4. (A) Schematic drawing of the predicted secondary structure for wild-type 5′-SL4 (C17 wt) and engineered mutant replicons including thermodynamic analysis. Nucleotide changes are depicted in red. In mutant C10 only the codons for the first 10 (instead of 17) amino acid residues were maintained. It therefore lacks the entire 5′-SL4 element. ΔG, Gibbs free energy of the secondary structure of a segment of the first 194 nucleotides of the viral genome. A decrease in the ΔG value relative to the wild-type indicates stabilization of the secondary structure, whereas an increase reflects destabilization. (B) Translation level of viral input RNA in BHK-21 cells 3 h posttransfection. Normalized luciferase levels are shown as percentages relative to the parental control C17. RNA of construct ΔAUG, which lacks the viral translational AUG start codon, was used as a negative control. Error bars represent the standard deviation of at least two independent experiments. (C) Viral RNA replication efficiencies of the parental C17 replicon and mutants derived from it, monitored by comparing normalized firefly luciferase activity in BHK-21 cells 15.5–72 h posttransfection. Capped NS5-GAA RNA, which can be translated but cannot replicate due to a GDD-to-GAA mutation in the viral RNA polymerase gene NS5, was used as a control. Error bars represent the standard deviation of two independent experiments, each measured in triplicate.
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