Activation of viral transcription by stepwise largescale folding of an RNA virus genome

Figures

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  Figure 1.. Carnation Italian ringspot virus (CIRV) genome and subgenomic mRNAs. (A) Schematic representation of the CIRV genome (top) and sg mRNAs (below in shaded boxes) with encoded proteins indicated. The small black arrows below the genome indicate the initiation sites for sg mRNA1 (sg1, red asterisk) and 2 (sg2, turquoise asterisks) in the genome. Proteins translated from the genome and sg mRNAs are depicted as dark gray bars immediately below encoded proteins. The positions of the sequences that form long-distance RNA-RNA interactions (LDRIs) involved in translation (5′UTR/3′CITE), readthrough (PRTE/DRTE), sg mRNA2 transcription (DE/CE, brown and AS2/RS2, turquoise) and sg mRNA1 transcription (AS1/RS1, red) are indicated by double-headed arrows. The minus-strand intermediates in transcription are shown above each sg mRNA as dashed grey arrows, with promoter sequences (Pr) indicated at their 3′-ends. (B) Simplified model of the RNA secondary structure for the TBSV genome (48) with relevant large RNA domains (LD2 and LD3), RNA elements (RTSL, green shading, PRTE and DRTE), and LDRIs indicated. The LDRI color-coding scheme corresponds to that depicted in panel (A).

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  Figure 2.. RTSL regulates sg mRNA1 transcription. (A) RNA secondary structure of RTSL (39) with the p36 stop codon highlighted in red, the PRTE depicted in yellow, and a sequence of interest (RTSL-TL) in the terminal loop colored in green. Nucleotide substitutions introduced into RTSL in genomic mutants are shown in red. All substitutions were made at wobble positions and did not alter the p95 amino acid sequence. (B) In vitro translation analysis in wheat germ extract of wt and mutant CIRV RNA genomes shown in panel (A). Samples, labeled at the top, were separated in a 12% SDS polyacrylamide gel and the positions of CIRV proteins are indicated on the left. Average p95 readthrough levels (Rel. RT) for each mutant relative to that of the wt (set as 100%) are shown below the gel, along with standard errors from three independent experiments. (C) Northern blot analysis of total nucleic acids isolated from plant protoplasts transfected with wt and mutant CIRV RNA genomes shown in panel (A). Respective viral infections, labeled at the top, were probed for plus-strand viral RNAs. The positions of the viral genome (g), sg mRNA1 (sg1) and sg mRNA2 (sg2), are shown on the left. Average sg mRNA1…

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  Figure 3.. Functional analysis of candidate LDRIs in regulating sg mRNA1 transcription. (A, D) Compensatory nucleotide substitutions introduced in RTSL-TL/SL59–5′ (green) and AS1-SL3′/SL59–3′ (pink) LDRIs in mutant genomes are shown as red and white nucleotides, respectively. Any corresponding changes in the p95 amino acid sequence are shown under the mutant sequences. (B, E) Northern blot analysis of plus-strand CIRV RNAs isolated from protoplasts transfected with wt and mutant CIRV RNA genomes depicted in panels (A) and (D), respectively. Identities of the viral genomes tested are indicated above the blots and positions of positive-sense genome and sg mRNAs are shown on the left. Average sg mRNA1 accumulation levels relative to that of the wt are provided below the blots with standard errors obtained from three independent experiments. (C, F) Northern blot analysis of minus-strand CIRV RNAs isolated from protoplast infections. Identities of the viral genomes tested are indicated above the blots and positions of the minus-sense genome and sg mRNAs are shown on the left. Average minus-strand sg mRNA1 accumulation levels relative to that of the wt are provided below the blots with standard…

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  Figure 4.. RTSL-TL and SL59–5′ sequences interact physically and via an intragenomic interaction. (A) Simplified depiction of CIRV RTSL (106 nt) and LD2 (1040 nt) RNA fragments tested in RNA–RNA EMSAs. (B) Ethidium bromide-stained native 4% polyacrylamide gel of EMSA with the RNA fragments containing mutations shown in Figure 3A. The contents of each lane are indicated above the gel, with the fragment type shown to the far left. Lane 1 represents a mock lane containing only RNA binding buffer and glycerol. The black arrow on the right side of the gel points to the position where the RTSL/LD2 complexes migrate. The percentages with standard errors of shifted LD2 RNAs are provided below and were obtained from three independent EMSA experiments. (C) Northern blot analysis of protoplast co-transfection and single transfections. Identities of the viral genomic and DI RNAs tested are indicated above the gel. Modifications in the RNAs are shown in Figure 3A. Positions of positive-sense genome, sg mRNAs, and DI8 are indicated on the left and right of the blot. Average sg mRNA1 accumulation levels relative to that of the wt are provided below the blot with standard errors obtained from three…

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  Figure 5.. Formation of the RTSL/LD2 complex is dependent on both the AS1/RS1 and AS1-SL3′/SL59–3′ interactions. (A) Simplified depiction of CIRV RTSL (106 nt) and LD2 (1040 nt) RNA fragments tested in RNA-RNA EMSAs. (B, D) Compensatory nucleotide substitutions (in white) that were introduced, respectively, into the AS1/RS1 (red) and AS1-SL3′/SL59–3′ (pink) complementary partner sequences. (C, E) Ethidium bromide-stained native 4% polyacrylamide gels of EMSAs testing the RNA fragments containing mutations shown in panels (B) and (D), respectively. The contents of each lane are indicated above the gels, with the fragment type shown to the far left. Lane 1 represents a mock lane containing just the RNA binding buffer and glycerol. The black arrows on the right side of the gels point to the position where the RTSL/LD2 complexes migrate. The percentages with standard errors of shifted LD2 RNAs are provided below and were obtained from three independent EMSA experiments.

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  Figure 6.. Structural requirements for RTSL/LD2 complex formation. (A, C) Secondary structures of CIRV RTSL (106 nt) and LD2-core (188 nt) RNA fragments tested in RNA-RNA EMSAs. The red nucleotides in the LD2-core secondary structure represent the added UNCG-type tetraloops that replaced LD2 sub1 and sub2 beyond S38 and S56, respectively. The G–C pair shown in red in RTSL mutant TC-184 was added to allow for its transcription from PCR templates using T7 RNA polymerase. (B, D) Ethidium bromide-stained 8% native polyacrylamide gels of EMSAs testing the RNA fragments containing modifications shown in Figure 3A and panel C, respectively. The contents of each lane are indicated above the gels, with the fragment type shown to the far left. Lane 1 represents a mock lane containing only RNA binding buffer and glycerol. The black arrows on the right side of the gels point to the positions where the RTSL/LD2 complexes migrate. The percentages with standard errors of shifted LD2-core RNAs are provided below the gels and were obtained from three independent EMSA experiments.

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  Figure 7.. In-line structural probing analysis of CIRV LD2-core and RTSL RNAs. (A, C) Sequencing gel analysis following in-line probing of radiolabeled LD2-core (LD2-core*) and RTSL (RTSL*) RNA fragments, respectively. Lane 1 contains untreated LD2-core* or RTSL* RNA samples (NR, no reaction). Lane 2 contains the RNase T1-digested LD2-core* or RTSL* RNA samples to generate G ladders. Lane 3 contains LD2-core* or RTSL* RNA samples that were subjected to alkaline hydrolysis reaction (–OH) to generate cleavages at every nucleotide position. Lane 4 contains in-line reactions from free LD2-core* or free RTSL* RNA fragments (free). Lane 5 shows in-line reactions when LD2-core* or RTSL* was incubated with unlabeled RTSL and LD2-core, respectively, to generate a complex. Nucleotide positions of selected G residues are indicated on the left. Different regulatory sequences are color coded and labeled on the right side of the gels. Black bars on the left of lane 4 in panel (A) indicate SL59 and AS1-SL3′ sequences that show high cleavage levels in free LD2-core. The white bar on the left of lane 4 shows moderate cleavage levels for SL59–5′ in free LD2-core. (B) Two alternative RNA secondary structure…

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  Figure 8.. Proposed RNA genome folding pathway leading to activation of sg mRNA1 transcription in CIRV. Note, this is a highly simplified folding pathway based on conjectured temporally-distinct transitions dictated by differences in stability, complexity and the relative spatial positions of the subcomponents. The relative timescales for transitions are not known and the structures shown represent approximations of probable intermediates within ensemble populations. (A) Schematic depiction of partially folded sections of the CIRV genome including RTSL, AS1-SL and SL59. AS1 and RS1 are separated by ∼1000 nucleotides in the linear sequence. Orange and blue double-headed arrows point to the complementary sequences involved in formation of S38 of sub1 and S56 of sub2, respectively. Small secondary structures within sub1 and sub2 are not shown, but are anticipated to form on a similar timescale as RTSL, AS1 and SL59. (B) RNA secondary structure after folding of LD2 sub1 (orange) and sub2 (blue), which are closed by stems S38 (orange) and S56 (blue), respectively. In this conformation AS1 and RS1 are brought within ∼80 nt from one another, which facilitates their base pairing (red double headed…