Figure 1.
In vitro translation of the CIRV genome assessing the role of the 3'UTR in RT.
(A) Schematic linear representation of the CIRV RNA genome with boxes representing encoded proteins and a thick horizontal line representing non-coding regions. p36 and its RT product, p95 (depicted by the hashed lines), are translated directly from the viral genome. Initiation sites for sg mRNA1 (sg1) and sg2 are indicated below the genome. Relevant RNA structures in the 5' and 3'UTRs, the T-shaped domain and 3'CITE, respectively, are shown schematically above the genome with complementary adapter sequences shown in white. Double-headed arrows connect RNA sequences that base pair over long distances, as shown for the 5'UTR-3'CITE interaction. An additional long-range RNA-RNA interaction, the UL-DL interaction, unites two important RNA replication elements, RII and RIV, so that replicase complex assembly can occur. (B) SDS-10%PAGE analysis of proteins translated from the CIRV genome. The mock lane consists of a translation reaction in the absence of RNA, while the BMV lane contains Brome mosaic virus RNAs 1 and 3, that template translation of proteins of 109 and 32 kDa, respectively, which served as molecular mass markers. Wt and mutant CIRV genomes are indicated above the center lanes and the positions of the CIRV p36 and p95 are indicated to the left. Protein products in this and all subsequent in vitro translation experiments were generated by translating 0.5 pmol of viral genome in wheat germ extract (wge) for 1 hr at 25°C, and, unless specified, the messages were uncapped. (C) SDS-10%PAGE analysis of RT for CIRV genomes containing various modifications to the 3'UTR. Note, the KOAc concentration in this and subsequent in vitro translation assays was optimized for efficient readthrough, not for 3'CITE-dependent translation. The presence or absence of the 3'CITE, RIV or a cap structure in the genome is indicated above each lane as a + or -, respectively. In this and subsequent experiments, the p95:p36 ratio was determined for each lane, and the relative RT percentages (Rel. RT) below each lane correspond to means (± standard error) from three independent experiments that were normalized to the p95:p36 ratio for the wt genome, set at 100.
Figure 2.
Role of the PRTE-DRTE interaction in mediating RT and genome replication.
(A) Predicted RNA secondary structures of SL-PRTE and RIV, with the complementary PRTE and DRTE sequences shown in green. Nucleotides involved in a functional intra-RIV interaction between an internal loop in the replication silencer element SL-3 and the 3'-terminal sequence are shown in white within circles. Wt and mutant PRTE-DRTE interactions are shown with substituted nucleotides depicted in red. (B) In vitro translation in wge of CIRV genomes containing various mutations disrupting and restoring the PRTE-DRTE interaction as shown in panel A. (C) Northern blot analysis and quantification of genomic plus-strand accumulation 22 hr post-transfection of plant protoplasts. The viral genomes analyzed are indicated above each lane and correspond to those depicted in panel A. The positions of the genomic (g) and subgenomic RNAs (sg1 and sg2) are indicated to the left of the blot. The relative values for viral genome accumulation (Rel. g), in this and subsequent experiments, correspond to means (± standard error) from three independent experiments and were normalized to the accumulation of wt genomic RNA levels, set at 100.
Figure 3.
Structural analysis of the PRTE-DRTE interaction.
(A) SHAPE analysis of the PRTE and its flanking sequence. Sequence corresponding to the PRTE is shown in green. Relative reactivity of each nucleotide is plotted graphically, with larger values corresponding to increased flexibility. The genomic mutants assayed are indicated in the key at the top right and correspond to those shown in Figure 2A. (B) Predicted secondary structure of the PRTE and its flanking regions showing residues in the mutants with notably increased reactivity (circled), as determined from the results in panel A. (C) RNA-RNA EMSA assessing SL-PRTE binding to RIV using wt and B-series compensatory mutants shown in Figure 2A. Equimolar amounts of unlabeled RNA fragments were incubated at 25°C for 30 min and the mixtures were then separated in a nondenaturing 15% acrylamide gel and stained with ethidium bromide to allow for visualization of the RNAs (the negative image is shown). The left-most lanes show the positions of free SL-PRTE and RIV, and the asterisk depicts the position of the upward shift observed when binding occurs.
Figure 4.
Analysis of RT using replication-defective genomes and a DI RNA replicon reporter.
(A) Schematic diagrams of non-replicating CIRV genomes used to assess p95 production. The functional properties of the different mutants are shown above the genomes and the names of the mutants are shown to the left. The C-to-G mutation in RII that renders all the genomes replication defective is shown above each genome. At the top, the mutants Rd and B3Rd are able to produce both p36 and p95, as indicated by the two hashed lines corresponding to these proteins. In the middle, mutants B1Rd and B2Rd are RT-defective and can produce only p36. Additional modifications in the PRTE and DRTE in mutants B1Rd, B2Rd, and B3Rd correspond to those in mutants B1, B2, and B3, respectively, as shown in Figure 2A. At the bottom, RTRd contains a G-to-U mutation in its stop codon, which changes it to a Tyr codon so that only p95 is produced. (B) Analysis and quantification of DI-7 accumulation when cotransfected with non-replicating CIRV genomes in plant protoplasts. The CIRV genomes cotransfected with DI-7 are indicated below each bar in the graph. DI-7 RNAs were analyzed by Northern blotting (top) 22 hr post-transfection. The relative values for DI-7 accumulation shown in the bar graph, in this and other experiments with DI RNA replicons, correspond to means (± standard error) from three independent experiments and were normalized to the accumulation of DI-7 cotransfected with Rd, set at 100.
Figure 5.
Restoring RT and genome replication with a heterologous RT sequence.
(A) At the top, a portion of the SL-PRTE structure is shown with the insertion site (arrow) of wt and mutant forms of the 6 nt long TMV RTE sequence. Wt and mutant TMV RTE sequences in corresponding genomic mutants are shown below. Substitutions in the mutant TMV RTE are underlined. (B) In vitro translation in wge of CIRV genomes containing mutations disrupting the PRTE-DRTE interaction (B2 and BS2) as well as the wt TMV RTE (B2T and BS2T) or the mutated TMV RTE (B2TN and BS2TN). (C) Northern blot analysis of wt and mutant CIRV genomes in protoplasts and quantification of plus-strand viral genome accumulation.
Figure 6.
Assessing the role of the PRTE and DRTE in modulating viral RNA replication.
(A) Schematic linear representation of the CIRV RNA genome and CIRV-derived DI-8 and DI-7 RNA replicons. The relative locations of the PRTE, RII, and DRTE are indicated above the genome. DI-8 and DI-7 are represented as a series of black boxes and thin intervening lines. The black boxes represent corresponding regions of the CIRV genome that are retained in the DI RNAs and thin lines represent regions that are absent. (B) Northern blot analysis and quantification of DI RNA accumulation when cotransfected with wt CIRV into plant protoplasts. DI-7 and DI-8 RNAs were analyzed 22 hr post-transfection and relative values below the lanes correspond to means (± standard error) from three independent experiments. (C and D) Northern blot analysis of DI-8 RNA accumulation when cotransfected with wt CIRV into plant protoplasts. Mutants DI-8-B1,-B2, -B3, -BS1, -BS2 and -BS3 contain the same substitutions as in genomic mutants B1, B2, B3, BS1, BS2, and BS3, respectively, as shown in Figure 2A. DI-8 RNAs were analyzed by Northern blot as described above. (E) Northern blot analysis of DI-7 RNA accumulation when cotransfected with wt CIRV in plant protoplasts. Mutants DI-7-B1, and -B2 contain the same substitutions as in genomic mutants B1 and B2, respectively, as shown in Figure 2A. (F) SHAPE analysis of the PRTE and its flanking sequence in wt DI-8 and mutant DI-8-B2 and DI-8-BS2. Relative reactivity of each nucleotide is plotted graphically, with larger values corresponding to increased flexibility. The PRTE is shown in green.
Figure 7.
Effect of alternative conformations of RIV on RT and replication.
(A) Schematic diagram of two possible RNA secondary structure conformations that RIV could adopt and influence the context of the DRTE. In the structure on the left, the replication-essential SL-2 is formed, whereas in the structure on the right the proposed SL-T is shown. Dotted lines denote the potential alternative base pairing in the two different structures and the double-headed arrow denotes the dynamic and mutually-exclusive relationship between the two conformations. (B) Substitutions in RIV (in red) predicted to influence formation of SL-2 and SL-T. “SL-2-preferential” mutants contain substitutions predicted to retain SL-2 formation and suppress SL-T formation. Conversely, “SL-T-preferential” mutants are predicted to do the opposite. The mutants in the two columns to the right of “SL-2-preferential” or “SL-T-preferential” are predicted to restore the balance of formation of “SL-2 & SL-T”, respectively, through additional substitutions. (C) Relative RT levels in wge for wt and mutant CIRV genomes containing substitutions shown in B. (D) Relative DI-7 accumulation levels in protoplasts for cotransfections of wt CIRV with wt DI-7 or DI-7 mutants containing the substitutions depicted in panel B. (E) Relative viral genome accumulation levels in protoplasts for wt and mutant CIRV genomes containing the substitutions shown in panel B.
Figure 8.
SHAPE analysis of the PRTE in viral genomes containing substitutions in RIV.
Relative reactivity of each nucleotide is plotted graphically, with larger values corresponding to increased flexibility. The genomic mutants assayed are indicated in the key at the top right and correspond to those described in Figure 7B.
Figure 9.
Analysis of the role of the UL-DL interaction in RT.
(A) Schematic linear representation of the CIRV RNA genome showing the UL-DL and PRTE-DRTE interactions. The relative locations of RII and RIV are shown above and below the genome, respectively. (B) CIRV genomic mutants with compensatory mutations in the UL-DL interaction. Substitutions are shown in red. Previous results of viral genome accumulation levels from protoplast transfections are shown in the box below [24]. (C) In vitro translation in wge of CIRV genomes containing mutations shown in panel B. (D) Analysis and quantification of DI-7 accumulation by Northern blot analysis (top) 22 hr post-cotransfection with non-replicating CIRV genomes in plant protoplasts. The CIRV genomes cotransfected with DI-7 are indicated below each bar in the graph.
Figure 10.
Conservation of potential PRTE-DRTE-like interactions in members of the family Tombusviridae.
Predicted RNA secondary structures for the sequences 3'-proximal to the RT stop codons (on left in each panel) and the 3'-terminal sequences (right) in selected species of genera in Tombusviridae. Sequence segments that could potentially form PRTE-DRTE-like interactions are shown in green and the RT stop codon is highlighted in grey. The virus acronyms are: TCV, Turnip crinkle virus; CLSV, Cucumber leaf spot virus; PMV, Panicum mosaic virus; MCMV, Maize chlorotic mottle virus; TNV-D, Tobacco necrosis virus D; OCSV, Oat chlorotic stunt virus.
Figure 11.
Analysis of the proposed PRTE-DRTE in TCV.
(A) TCV genomic mutants with compensatory mutations in the PRTE-DRTE interaction. Mutated residues are shown in red. (B) In vitro translation in wge of TCV genomes containing mutations shown in panel A. The same gel was exposed for different times, as indicated, in order to better visualize bands corresponding to p88 and p28. (C) Northern blot analysis of wt and mutant TCV genomes in protoplasts and quantification of plus-strand viral genome accumulation.
Figure 12.
Proposed RNA-based regulatory network modulating RT and genome replication in CIRV.
(A) Schematic linear representation of the CIRV RNA genome showing all defined functional long-range RNA-RNA interactions in tombusviruses. The 5'UTR-3'CITE interaction facilitates efficient initiation of translation [18]; the AS1-RS1 interaction mediates sg mRNA1 transcription [22]; the AS2-RS2 and DE-CE interactions mediate sg mRNA2 transcription [21], [23]; the UL-DL interaction facilitates replicase complex assembly [24] and RT (this study); and the PRTE-DRTE interaction mediates RT (this study). (B) Events leading to the translation of p36 and p95 and subsequent RNA replicase complex assembly. (i) The 5'UTR-3'CITE interaction mediates efficient initiation of translation that allows for production and accumulation of p36. (ii) Formation of the PRTE-DRTE and UL-DL interactions makes it possible for elongating ribosomes to readthrough the p36 stop codon. Also, since the PRTE-DRTE interaction involves the SL-T-containing conformation of RIV, which inhibits the replication function of RIV, any p95 generated and provided in trans will not be utilized. (iii) Following RT, further 3'-movement of the translating ribosome leads to disruption of both the PRTE-DRTE and UL-DL interactions, which down-regulates RT and p95 production and allows RIV to adopt its SL-2-containing replication-active conformation. (iv) Reformation of the UL-DL interaction with the replication-active RIV generates a functional RII-RIV RNA platform that is used by the p95 translated in cis, along with p36 and host factors, to assemble into an active viral RNA replicase complex (RC) that carries out minus-strand synthesis.