3'-End stem-loops of the subviral RNAs associated with turnip crinkle virus are involved in symptom modulation and coat protein binding

Abstract

Many plant RNA viruses are associated with one or more subviral RNAs. Two subviral RNAs, satellite RNA C (satC) and defective interfering RNA G (diG) intensify the symptoms of their helper, turnip crinkle virus (TCV). However, when the coat protein (CP) of TCV was replaced with that of the related Cardamine chlorotic fleck virus (CCFV), both subviral RNAs attenuated symptoms of the hybrid virus TCV-CP(CCFV). In contrast, when the translation initiation codon of the TCV CP was altered to ACG and reduced levels of CP were synthesized, satC attenuated symptoms while diG neither intensified nor attenuated symptoms. The determinants for this differential symptom modulation were previously localized to the 3'-terminal 100 bases of the subviral RNAs, which contain six positional differences (Q. Kong, J.-W. Oh, C. D. Carpenter, and A. E. Simon, Virology 238:478-485, 1997). In the current study, we have determined that certain sequences within the 3'-terminal stem-loop structures of satC and diG, which also serve as promoters for complementary strand synthesis, are critical for symptom modulation. Furthermore, the ability to attenuate symptoms was correlated with weakened binding of TCV CP to the hairpin structure.

FIG. 1

Genomic and subviral RNAs used in this study. (A) Genomic RNAs. ORFs and untranslated regions are represented by thick and thin boxes, respectively. The percent sequence similarity between TCV and CCFV genomes is shown. TCV-CPm has a point mutation in the CP initiation codon as indicated, which causes translation initiation at an upstream CUG codon resulting in two additional N-terminal amino acids and a reduction in CP levels to 20% of that of the wt (54). (B) Subviral RNAs associated with TCV. Similar sequences among TCV genomic and subviral RNAs are shaded alike. The sizes of the RNAs are given. (C) Alignment of the 3′-end sequences of satC, diG, and the TCV genomic RNA, which is identical to satC* (TCV/C∗) (16). Only differences among the RNAs are indicated. Lines indicate absence of the bases in satC and diG, compared with TCV or satC∗. The six positional differences between satC and diG are shaded. The last two positions (5 and 6) each have two consecutive nucleotide differences between the two subviral RNAs.

FIG. 2

Symptom modulation by the TCV subviral RNAs coinoculated with the helper TCV (A), TCV-CP_CCFV (B), and TCV-CPm (C). Seedlings of A. thaliana ecotypes Col-0 and Di-0 at the six- to eight-leaf stage were inoculated with buffer alone (Mock), helper virus genomic RNA without any subviral RNA (None), or with satC (C), satC56G (C56G), satC∗ (C∗), diG (G), and diG56C (G56C), as indicated below the plants. Representative plants were photographed at 17 dpi. The helper virus used to inoculate the plant is shown on the right. Di-0 plants are resistant to infection by TCV. T/C, TCV-CP_CCFV; CPm, TCV-CPm.

FIG. 3

3′-terminal stem-loop structures and replication of satC, diG, and mutant subviral RNAs. (A) 3′-end stem-loop structures of wt and mutant subviral RNAs as predicted by the computer structure program MFOLD (Genetics Computer Group, University of Wisconsin, Madison). Similar 3′-terminal stem-loop structures are assigned to the same class (class I through IV). Subviral RNAs that have been previously studied for symptom modulation (16) are shaded. Mutations in each mutant subviral RNA are shown in italics and underlined. V, location of deleted nucleotides in diG5C and diG56C that are present in wt diG. (B) Accumulation of wt and mutant subviral RNAs in protoplasts. A. thaliana protoplasts (5 × 10⁶) were inoculated with 20 μg of wt TCV with (lanes 1 to 9) or without (lane 10) the addition of 2 μg of wt or mutant subviral RNAs, as shown above each lane. Total RNA extracted at 40 h postinoculation was subjected to RNA gel blot analysis with a probe specific for TCV and the subviral RNAs (Table 1) or rRNA (39). Species corresponding to TCV genomic RNA (gRNA), the two subgenomic RNAs (1.72 and 1.45 kb), and the subviral RNAs and their dimer forms are indicated.

FIG. 4

Gel retardation analysis of TCV CP binding to RNA fragments. (A) TCV CP binding to the C3′ (64 nt), C56G3′ (66 nt) and SK70 (70 nt) RNAs. ³²P-labeled RNAs (100 pM) were incubated in the presence of a series of increasing concentrations of CP, indicated above each gel. Incubation mixtures were subjected to electrophoresis on a 5% polyacrylamide gel that was fixed and dried prior to autoradiography. (B) Quantification of the results presented in panel A and two additional independent experiments (data not shown). Autoradiograms were scanned by densitometry, and the fractions of unbound RNA remaining in the presence of different CP concentrations were determined and plotted against CP concentrations. Each point represents the average of three experiments. Standard deviation bars that are not within the limits of the symbols are shown.

FIG. 5

Competition for CP binding between 3′-terminal RNA fragments of satC and satC56G. (A) Autoradiogram of a representative competition binding assay between the ³²P-labeled C3′ RNA and increasing concentrations (indicated above each lane) of unlabeled C3′ (lanes 1 to 8) and C56G3′ (lanes 9 to 16) RNAs. (B) Quantification of the results from two independent experiments. Autoradiograms were scanned by densitometry, and the fractions of unbound ³²P-labeled C3′ RNA in the presence of different competitor concentrations were determined and plotted against the competitor concentrations. Each point represents the average of two experiments. Standard deviation bars that are not within the limits of the symbols are shown.

FIG. 6

The TCV CP and CCFV CP differentially bind to C3′ (A) and C56G3′ (B) RNAs. ³²P-labeled C3′ or C56G3′ RNA (100 pM) in lanes 1 was incubated with increasing concentrations (indicated above each lane) of TCV CP (lanes 2 and 3), CCFV CP (lanes 4 to 9), and bovine serum albumin (lanes 10 to 15). Incubation mixtures were subjected to electrophoresis on a 5% polyacrylamide gel that was fixed and dried prior to autoradiography.

FIG. 7

Putative model for symptom modulation by TCV subviral RNAs in Arabidopsis. In this model, the TCV CP (indicated by small gray circles) binds directly to the 3′ ends of satC and diG and competes for binding with a putative host factor, X (indicated by large black circles), that is involved in virus long-distance movement. I, symptom intensification; R, resistance (i.e., symptom attenuation); U, unaffected symptoms. (A) When high levels of CP are present, CP outcompetes X for binding to the 3′-terminal stem-loops of both satC and diG, leaving X available for virus long-distance movement (i.e., systemic infection). (B) When low levels of CP are present, X outcompetes the CP for binding to the 3′-terminal stem-loop of satC but not that of diG, and sequestration of X by satC restricts virus movement and results in symptom attenuation. In contrast, CP outcompetes X for binding to the 3′-terminal stem-loop of diG, due to the higher affinity of the diG-like hairpin for binding to CP. Why symptoms are not intensified under these conditions is not known. (C) X outcompetes non-TCV CP such as CCFV CP (indicated by small brick-patterned circles) for binding to the 3′ ends of both satC and diG, and sequestration of X by the subviral RNAs restricts virus movement and results in symptom attenuation. Note that satC and diG are more abundant in the absence of TCV CP than in the presence of TCV CP as previously found (16).