Figure 1.
General structure of the TBSV genome and the DI-73 replicon.
(A) Schematic linear representation of the TBSV RNA genome with boxes representing encoded proteins. p33 and p92 share a start codon, and both are translated directly from the viral genome; the latter by read-through of the p33 stop codon (UAG). Proteins encoded further downstream are translated from two sg mRNAs that are transcribed during infections (represented as horizontal arrows, top). Regions in the TBSV genome that are present in DI-73 are delineated by thick horizontal lines under the genome. (B) Linear structure of the non-coding DI-73 RNA replicon. The three contiguous regions of DI-73 that are derived from the TBSV genome are delineated by the dotted arrows with the corresponding genomic coordinates. The contiguous 3′-proximal segment is defined by three regions: RIII, R3.5, and RIV. Replicase complex assembly requires both RII and RIV, as indicated.
Figure 2.
RNA segments of the TBSV genome containing a putative long-range RNA–RNA interaction.
(A) Cartoon depicting secondary structures of RII and contiguous segment RIII-R3.5-RIV. RIII and RIV are separated by R3.5 (grey), which forms an extended Y-shaped domain containing the 3′CITE. The relative positions of two complementary 11 nt long sequences in RII and RIII (open rectangles), termed UL and DL, respectively, are shown. The proposed interaction between these two segments in the folded and linear forms of the sequences is indicated by the dashed double-headed arrows. TBSV genome coordinates for the termini of the segments shown are indicated. (B) The putative UL–DL base-pairing interaction between RII and RIII is presented in detail for TBSV (left) and mono- and co-variations that occur in the corresponding sequences in different tombusviruses are shown to the right (TBSV-N, TBSV nipplefruit isolate; TBSV-S, TBSV statice isolate; MNeSV, Maize necrotic streak virus; CBV, Cucumber Bulgarian virus).
Figure 3.
Analysis of the UL–DL interaction in two different tombusvirus genomes.
(A) The set of compensatory mutations in the UL and DL sequences that were introduced into the TBSV and CIRV viral genomes is shown. Substituted nucleotides are in bold and underlined. Note that the mutations in dU are predicted to preferentially inhibit the base pairing interaction in the plus-strand (i.e. the AC and CC mismatches in the plus-strand would be less disruptive GU wobbles and GG mismatches in the minus strand). Conversely, the dD mutations would favor plus-strand formation (for reasons similar to those described above). (B, C) Northern blot analysis and quantification of plus- and minus-strand accumulation of viral RNAs (top and bottom panels, respectively) from TBSV (B) or CIRV (C) infections of plant protoplasts. The viral genomes analyzed are labeled above the lanes. The positions of the genomes (g) and corresponding subgenomic mRNAs (sg1 and sg2) are indicated. Viral RNAs were analyzed by northern blot analysis 22 hr post-transfection of plant protoplasts. The relative values below the lanes correspond to means (±standard deviations, SD) from three independent experiments and were normalized to the accumulation level of the wt genome, set at 100.
Figure 4.
Translational analysis of the CIRV genome in wheat germ extract.
(A) SDS-PAGE analysis of proteins synthesized from the CIRV genome and its mutant counterparts. The viral genomes analyzed are labeled above the lanes. The mock lane consists of a translation reaction with no RNA added, while the CIRVΔTE lane contains a genomic control that does not contain a 3′CITE. The position of the expected p36 product is indicated to the left. Products were generated by translating 0.5 pmol of uncapped full-length viral genomes in wheat germ extract for a period of 1 hr at 25°C. The relative accumulation levels of p36 were quantified and the values correspond to means from three independent experiments that were normalized to the accumulation level for the wt genome, set at 100. (B) Stability assay of CIRV genomes in wheat germ extract. Aliquots were removed from translation reactions at various time intervals and the viral RNAs were analyzed by northern blotting. Means of RNA levels (±SD) from three independent experiments were plotted versus time.
Figure 5.
Analysis of the requirement of the UL–DL helix for viral RNA accumulation.
(A) Depiction of TBSV-based replicons with deleted intervening sequences that either did (CorP1) or did not (CorP2) include the UL and DL segments. The regions of the TBSV genome (middle) present in CorP1 and CorP2 are shown by the dashed arrows along with corresponding genomic coordinates. The UL–DL sequences, present only in CorP1, are predicted to form the RNA hairpin shown. (B) Northern blot analysis and quantification of CorP replicon accumulation in co-transfections of plant protoplasts.
Figure 6.
Analysis of the UL–DL interaction in the TBSV DI-73 replicon.
(A) Northern blot analysis of the accumulation of DI-73 plus-strands in co-transfections with TBSV genome in plant protoplasts. DI-73 and its variants contained the same set of UL–DL modifications that are shown in Figure 3A. (B) Northern blot analysis of DI-73 minus-strand accumulation. (C) Stability analysis of DI-73 and its mutants in plant protoplasts. Protoplasts were transfected with DI-73 or its variants in the absence of helper genome and the relative levels of these RNAs were determined over time by northern blot analysis. (D) Representative analysis of wt and mutant DI-73 template activities determined in vitro using a plant-derived replicase extract. Terminally-initiated (ti) and internally-initiated (ii) minus-strand products are indicated and levels of the former were quantified.
Figure 7.
Replication and replicase assembly assays of DI-73 and its mutants in yeast.
(A) Representative replication assay showing the accumulation levels of DI-73 (Y73, being the wt yeast plasmid counterpart) and its variants in yeast cells, as assessed by northern blot analysis. These replicons contained the same set of modifications that are shown in Figure 3A. (B) Representative replicase assembly assay showing the efficiency with which affinity-purified replicase (prepared from cells expressing the different DI-73 variants described above) copies an added DI-72(−) template in vitro. Terminally-initiated (ti) and internally-initiated (ii) products are indicated and the former was quantified. (C) Western blot showing levels of p33 present in the cells used for replicase preparation. Similar results were obtained when p92 levels were assessed (not shown). (D, E, F) are as described for (A, B, C), respectively, except that replication-defective forms of DI-73 (designated by the prefix “m”) were used in these assays.
Figure 8.
Secondary structures of portions of the TBSV genome depicting the UL–DL interaction and linear representations of similar interactions in other viruses.
(A) Secondary structure cartoon showing RII and RIII-R3.5-RIV and the UL–DL interaction (open rectangles). The RNA elements that are essential for replicase assembly are enclosed by dashed ovals, and the double-headed arrow depicts the communication required between these structures. p33 and p92 are shown as shaded ovals and the arrow indicates binding to RII(+)-SL. (B) Linear representation of positive-strand RNA viral genomes that infect hosts from three different kingdoms. The long-range RNA–RNA interactions in these viral genomes that are required for RNA replication are depicted by dotted lines, with the approximate lengths of the intervening sequences indicated. The approximate positions of replicase binding sites are indicated by asterisks.
Figure 9.
Linear and higher-order structural models for the functional long-distance RNA–RNA interactions that occur in the TBSV genome.
(A) Linear representation of the TBSV genome showing RNA–based interactions involved in translation, replication, and sg mRNA transcription. (B) Higher-order structural model for long-range RNA–based interactions in the TBSV genome. The sequences that are directly involved in forming long-range base pairing interactions are shown in red, while associated sequences and/or structures that are involved in translation, replication, and sg mRNA transcription are color coded as orange, blue, and green, respectively. Relevant structures are labeled (TSD, T-shaped domain; DSD, downstream domain; AS, activator sequence; RS, receptor sequence; CE, core element; DE, distal element). The SL3–SLB interaction is required for translation of p33/92 from the genome. The AS1–RS1 interaction is required for sg mRNA1 transcription, while both the AS2–RS2 and DE–CE interactions are required for sg mRNA2 transcription. The TSD, SL5, and DSD are located 5′-proximally in the 5′UTR of the genome and are involved in mediating genome plus-strand RNA synthesis [65]. Large intervening sections of sequence, which are predicted by mfold to form domains (see Figure S1), are shown as ovals and roughly correspond to the ORFs for p33, p92, p41, and p19/22 (not to scale). The start codon for p33/92, as well as the termination codons for these two proteins, are labeled and denoted by asterisks. Initiation sites of the two sg mRNAs are indicated by small arrows. See text for additional details.