The 5'-proximal hairpin of turnip yellow mosaic virus RNA: its role in translation and encapsidation

Abstract

The RNA genome of turnip yellow mosaic virus (TYMV) consists of more than 6,000 nucleotides. During a study of the roles of the two hairpins located in its 90-nucleotide 5' untranslated region, it was observed that stabilization of the 5'-proximal hairpin leads to a delay in the development of symptoms on plants. This delay in symptom development for both locally and systemically infected leaves was found to be dependent on a change in the free energy of the hairpin caused by introduced mutations. A protoplast transfection assay revealed that the accumulation of plus-strand full-length RNA and subgenomic RNA, as well as protein expression levels, was affected by hairpin stability. Stabilization of this hairpin inhibited translation. A model is proposed in which a destabilized 5'-proximal hairpin allows maximal translation of the viral proteins. It is suggested that this hairpin may exist in close proximity to the 5' cap as long as its stability is low enough to enable translation. However, at an acidic pH, the hairpin structure becomes more stable and is functionally transformed into the initiation signal for viral packaging. Slightly acidic conditions can be found in chloroplasts, where TYMV assembly is driven by a low pH generated by active photosynthesis.

FIG. 1.

Overview of the substitution mutations made in the internal loop of the 5′-proximal hairpin of TYMV RNA. (A) 5′ UTR of TYMV with the two hairpins containing protonatable internal loops (HP1 and HP2). The mutagenesis site is boxed, and the start codons of the MP ORF and the RdRp ORF are indicated by arrows. (B) Overview of substitution mutants S1 to S10 used in this study. Base substitutions in the internal loop, relative to the wild type, are boxed. The predicted stability of each mutant hairpin (as calculated at pH 7.0) is indicated in kilocalories per mole.

FIG. 2.

Appearance of symptoms in Chinese cabbage plants inoculated with T7 RNA transcripts. The first appearance of local (black bars) and systemic (white bars) symptoms is indicated. The standard deviation calculations were based on the inoculation of 15 plants per mutant, except for mutant S6, where 44 plants were tested. Delay in symptom development is indicated as days postinoculation (D.P.I.). Probability factors were calculated by using Student's t test and reflect the significance of delay in symptom development compared to the results obtained for the wild-type (WT) TYMV construct (*, P < 0.001; **, P < 0.0001; ***, P < 0.00001).

FIG. 3.

Analysis of delay in symptom development. (A) Relationship between delay in the development of local symptoms versus systemic symptoms on Chinese cabbage plants during infectivity assays with TYMV mutants. (B) Relationships between the observed delay of development of local (○) and systemic (•) symptoms and the calculated stability of the 5′-proximal hairpin. Delay in symptom development is indicated as days postinoculation (D.P.I.).

FIG. 4.

Analysis of viral product formation for several TYMV (mutant) constructs during protoplast infection. (A) Western blot analysis of TYMV MP. The marker positions are indicated on the left. WT, wild type. (B) Western blot analysis of TYMV CP. (C) Northern blot analysis of the production of genomic full-length TYMV RNA (FL-RNA) and subgenomic TYMV RNA (sgRNA). The positions of 28S and 18S rRNAs are indicated on the left. The negative control (−) consisted of protoplasts transfected with water only.

FIG. 5.

TYMV product accumulation after protoplast infection (Fig. 4) as a function of the predicted stability of the 5′-proximal hairpin (Fig. 1B). (A) Genomic full-length TYMV RNA (FL-RNA). (B) sgRNA. (C) MP. (D) CP. All values in panels A to D were normalized to the level of expression found for the wild-type TYMV construct, which was set to 1. (E) Summary of the results shown in panels A to D. The shaded region reflects the area in the graph which accommodates at least 90% of the data points retrieved from panels A to D. (F) Levels of [³⁵S]methionine incorporation determined with a wheat germ in vitro translation system for the wild-type and mutant TYMV RNAs. The values obtained were normalized to that of the wild-type TYMV construct. The shaded area was taken from panel E.

FIG. 6.

Reversion requirement observed for mutant forms of the 5′-proximal hairpin of TYMV RNA. (A) Dependence of the reversion requirement on the stability of HP1. Three types of reversions were distinguished: required (+), preferred (+/−), and not required (−). Arrows indicate the change in stability resulting from an observed reversion in the progeny over multiple rounds of infection (2). When the original mutation could not be recovered in the virus progeny, a reversion was required. Mutants in the preferred reversion layer did not necessarily revert in the first or subsequent rounds of infection, whereas mutants in the layer that was not required (−) never introduced reversions during the four rounds of infection that were monitored. The areas of gray shading mark the apparent thresholds between the three types of reversion requirements. WT, wild type. (B) The reversion pathway of mutant S6 exhibited a turnover from S6 to S6.1, S6.2, and the WT over several rounds of infection (2). Initially, the infection assay started with the inoculation of mutant S6 (ΔG, −16.3 kcal · mol⁻¹). In the following rounds of infection, the predominant (pseudo) revertants that were observed belonged to one of three genotypes: S6.1 (ΔG, −10.1 kcal · mol⁻¹), S6.2 (ΔG, −8.1 kcal · mol⁻¹), or WT (ΔG, −3.6 kcal · mol⁻¹).

FIG. 7.

Model for dichotomy on the basis of pH conditions. In the presence of a nearly neutral pH in the cytoplasm, the 5′-proximal hairpin is proposed to be relatively unfolded, allowing maximal translation initiation. At a low pH (e.g., 4.5), at the necks of invaginations of the chloroplasts, the stability of HP1 is increased due to the protonation of C · C and C · A base pairs, and the 5′-proximal hairpin functions as an encapsidation initiation signal.