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. 2019 Sep 5;47(15):8255-8271.
doi: 10.1093/nar/gkz564.

A conserved RNA structure is essential for a satellite RNA-mediated inhibition of helper virus accumulation

Lu He  1 , Qian Wang  1 , Zhouhang Gu  1 , Qiansheng Liao  1 , Peter Palukaitis  2 , Zhiyou Du  1
Affiliations

A conserved RNA structure is essential for a satellite RNA-mediated inhibition of helper virus accumulation

Lu He et al. Nucleic Acids Res. .

Abstract

As a class of parasitic, non-coding RNAs, satellite RNAs (satRNAs) have to compete with their helper virus for limited amounts of viral and/or host resources for efficient replication, by which they usually reduce viral accumulation and symptom expression. Here, we report a cucumber mosaic virus (CMV)-associated satRNA (sat-T1) that ameliorated CMV-induced symptoms, accompanied with a significant reduction in the accumulation of viral genomic RNAs 1 and 2, which encode components of the viral replicase. Intrans replication assays suggest that the reduced accumulation is the outcome of replication competition. The structural basis of sat-T1 responsible for the inhibition of viral RNA accumulation was determined to be a three-way branched secondary structure that contains two biologically important hairpins. One is indispensable for the helper virus inhibition, and the other engages in formation of a tertiary pseudoknot structure that is essential for sat-T1 survival. The secondary structure containing the pseudoknot is the first RNA element with a biological phenotype experimentally identified in CMV satRNAs, and it is structurally conserved in most CMV satRNAs. Thus, this may be a generic method for CMV satRNAs to inhibit the accumulation of the helper virus via the newly-identified RNA structure.

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Figures

Figure 1.
Figure 1.
Sat-T1 attenuated viral symptoms, accompanied with a decreased accumulation of viral RNAs in plants. Nicotiana benthamiana plants were inoculated with CMV alone or plus sat-T1 via agroinfiltration. Mock plants were treated with infiltration solution. (A) Viral symptoms in N. benthamiana plants. The plants were photographed at 6 days post-infiltration. Bar scale, 1 cm. (B) Northern blot hybridization analysis of viral RNAs and sat-T1 in the inoculated or upper systemic leaves at different time points as indicated. The DNA oligonucleotide probes targeting CMV 3′ UTR, 1a and 2b were used to detect all viral RNAs, RNA1, and RNA2 and its subgenomic RNA4A, respectively. The ethidium bromide-stained rRNAs were used as a loading control. The signal intensities of viral RNAs were arbitrarily quantified using the program Gel Pro Analyzer 4.0. The relative level of each viral RNA is shown in the chart below. All of viral RNAs in the CMV-infected plants at each time point were assigned a value of 1. The columns represent the mean value and standard error from three independent biological experiments.
Figure 2.
Figure 2.
Sat-T1 inhibited the replication of a modified RNA1 (RNA1Δ1a) and RNA2 (RNA2Δ2a2b), but not RNA3. (A) Replication of sat-T1 promoted by the transiently expressed CMV replicase. Sat-T1 was transiently co-expressed with the CMV 1a and 2a proteins or a vector (pCB301) by agroinfiltration into the 6th true leaves of Nicotiana benthamiana plants. RNA silencing suppressor, tomato bushy stunt virus-encoded p19 was co-expressed in the experiment. The accumulation of sat-T1 in the infiltrated leaves was analyzed by northern blot hybridization at 5 days post-infiltration (dpi). (B-D) Replication of RNA1Δ1a (lacking the 1a protein), RNA2Δ2a2b (lacking the 2a and 2b proteins) or RNA3 in the presence or absence of sat-T1 promoted by the transiently expressed CMV replicase. The CMV 1a and 2a proteins were co-expressed with RNA1Δ1 (B), RNA2Δ2a2b (C) or RNA3 (D), combined with or without the expression of sat-T1 via agroinfiltration as described above. The p19 silencing suppressor was co-expressed in this experiment. Viral and satellite RNAs in the infiltrated leaves were examined by northern blot hybridization at 5 dpi. Their relative levels were arbitrarily quantified and shown below. Equal loading was confirmed by staining of rRNAs with ethidium bromide.
Figure 3.
Figure 3.
The 5′ UTR played no role in the reduced accumulation of CMV RNAs 1 and 2 caused by sat-T1. (A) Schematic diagrams of CMV genomic RNAs and their derivatives. An identical secondary structure shown at the 5′ end of RNAs 1 and 2 was proposed previously (69). RNA2u and RNA3u are chimeric RNA2 and RNA3, respectively, generated by exchanging their 5′ UTR between RNA2 and RNA3. (B) Base paring between the proposed structure in the 5′ UTR of CMV RNA2 (RNA1) and the predicted structure at the 5′ end of sat-T1. The complementary sequences in both structures are underlined. The residue in parentheses is present in RNA1. The names of the mutations are bracketed. (C–F) Northern blot hybridization analysis of the accumulation of CMV, sat-T1 and their mutants in the infiltrated leaves. All the mutants are shown in the panel (B). Nicotiana benthamiana plants were inoculated via agroinfiltration with CMV wild-type (wt) or mutant in the presence or absence of sat-T1 or its mutants. Inoculation of CMV alone is indicated as ‘-’. Total RNAs were extracted from the infiltrated leaves at 6 days post-infiltration. In panels E and F, the DNA oligonucleotide probes targeting CMV 3′ UTR, 1a and 2b were used to detect all viral RNAs, RNA1, and RNA2 and its subgenomic RNA4A, respectively. The relative accumulation levels of CMV RNAs 1 and 2 are shown below (C, D) or in the chart (F). The relative levels of RNAs 1 and 2 in the CMV-inoculated plants are assigned a value of 1. The columns shown in the chart represent the mean value and standard error from three independent biological experiments. The ethidium bromide-stained rRNAs were used as a loading control.
Figure 4.
Figure 4.
Different satellite RNAs had differential ability to attenuate symptom expression and inhibit viral accumulation. (A) Viral symptoms in Nicotiana benthamiana plants infected with CMV alone or plus satRNA T1, D4 or SD. Plant inoculation was performed via agroinfiltration, and the plants were photographed at 6 days post-inoculation (dpi). Mock plants were treated with infiltration solution. Bar: 1cm. (B) Northern blot hybridization analysis of the accumulation of viral RNAs and satRNAs in the upper systemic leaves at 6 and 10 dpi. The DNA oligonucleotide probes targeting CMV 3′ UTR, 1a and 2b were used to detect all viral RNAs, RNA1, and RNA2 and its subgenomic RNA4A, respectively. The signal intensities of viral RNAs were arbitrarily quantified using the program Gel Pro Analyzer 4.0. The relative level of each viral RNA was shown in the chart on the right. All of viral RNAs in the CMV-infected plants at each time point were assigned a value of 1. The columns represent the averaged value and standard error from three independent biological experiments. (C) Northern blot hybridization analysis of the accumulation of satRNA-derived siRNAs (sat-siRNAs) in the upper systemic leaves. The signal intensities of sat-siRNAs were arbitrarily quantified using the program Gel Pro Analyzer 4.0. The numbers shown below represent the relative accumulation levels of sat-siRNAs.
Figure 5.
Figure 5.
Comparison of the primary sequences and predicted secondary structures of CMV satRNAs. (A) Alignment of the nucleotide sequences of sat-T1, SD and D4 ranging from nucleotide positions 185–246, or 186–248. The nucleotides of sat-SD or sat-D4 that are different from sat-T1 are colored red. In total, there are four nucleotides varying between sat-SD and sat-D4 underlined. (B) The predicted secondary structures for these primary sequences shown in the panel (A). Sat-T1 and sat-SD can be folded into a three-way branched structure containing two hairpins (γH1 and γH2) branched from the basal stem γBs. Compared with sat-T1 or sat-SD, sat-D4 lacks the γH2 hairpin. The nucleotides colored red or underlined are the same as shown in the panel (A). (C) Conservation analysis of the predicted structures. All 182 sequences of CMV satRNAs deposited in GenBank were analyzed. The sequences of these three stems with a frequency of ≥5% are shown here.
Figure 6.
Figure 6.
The predicted secondary structure was supported by the SHAPE data. (A) The SHAPE phosphorimage showing flexibility of the nucleotides in the secondary structure. G, U, C, A, nucleotide ladder lanes; D, DMSO-treated control; N, NMIA-treated. (B) Nucleotide flexibilities were determined using SHAPE structural probing and quantified using semiautomated footprinting analysis software. The nucleotides with high flexibility (reactivity ≥ 0.6) are denoted by a red circle or colored red, those with medium flexibility (reactivity from 0.3 to 0.6) are denoted by a green circle or colored green, and those with low to no flexibility (reactivity < 0.3) are colored black. γBs is the basal stem of the γ-shaped structure. γH1 and γH2 are the 5′ side and 3′ side hairpins, respectively.
Figure 7.
Figure 7.
Conservation and mutational analysis of a tertiary pseudoknot in the γSS element. (A) A potential pseudoknot (Ψ1) interaction formed between the loop and flanking sequences of γH1. The residues assumingly engaged in the Ψ1 interaction are underlined, and their base paring is denoted by connected arrowheads. Names of the compensatory mutations generated in the interacting partners are bracketed. (B) Conservation of the putative Ψ1 interaction was analyzed in all 182 sequences of CMV satRNA deposited in GenBank. The residues of the pseudoknot distinct from that in sat-T1 are colored red. (C) Accumulation of sat-T1 with various compensatory mutations in Nicotiana benthamiana plants. Total RNAs were extracted from the infiltrated leaves at 3 days post-infiltration, and sat-T1 and its mutants were detected by northern blot hybridization. ‘–’ shown on the top indicates infection of CMV alone. The numbers represent the mean value and standard error from three independent experiments. The ethidium bromide-stained rRNAs were used as a loading control.
Figure 8.
Figure 8.
Mutational analysis of the γSS element. Residues in the structure that were targeted for mutation are shaded and the residues mutated in corresponding mutants are colored red. Northern blot hybridization analysis was used to determine the accumulation of sat-T1 and its mutants in the inoculated leaves at 3 days post-infiltration. ‘–’ shown on the top indicates infection of CMV alone. The values represent the mean percentages of relative accumulation levels from three independent experiments with standard errors. The ethidium bromide-stained rRNAs were used as a loading control.
Figure 9.
Figure 9.
The γH2 hairpin engaged in the inhibition of the accumulation of CMV RNAs by sat-T1. (A) Disease symptoms in Nicotiana benthamiana plants infected with CMV alone (–) or plus sat-T1 wild-type (wt) or γH2 mutants as indicated on the top. These mutants have been depicted in Figure 8. The plants were photographed at 6 days post-infiltration (dpi). Mock plants were infiltrated with infiltration solution. Bar: 1 cm. (B) Accumulation of CMV, sat-T1 and its mutants in the infected plants analyzed by northern blot hybridization. Total RNAs were extracted from the upper systemic leaves at 6 dpi. The DNA oligonucleotide probes targeting CMV 3′ UTR, 1a and 2b were used to detect all viral RNAs, RNA1, and RNA2 and its subgenomic RNA4A, respectively. The signal intensities of CMV and satellite RNAs were arbitrarily quantified using the program Gel Pro Analyzer 4.0. The relative level of each RNA species is shown in the chart on the right. All of viral RNAs in the CMV-infected plants were assigned a value of 1, as well as wt sat-T1. The columns represent the averaged value and standard error from three independent biological experiments. The ethidium bromide-stained rRNAs were used as a loading control. (C) Immunoblot analysis of the accumulation of the CMV CP and 2b proteins in the infected plants. Total proteins were prepared from the upper systemic leaves at 6 dpi, and separated in a SDS-contained polyacrylamide gel for immunoblotting using antiserum against CMV CP or 2b. The values represent relative accumulation levels. (D) Northern blot hybridization analysis of the accumulation of satRNA-derived siRNAs (sat-siRNAs) in the upper systemic leaves at 6 dpi. The signal intensities of sat-siRNAs were arbitrarily quantified using the program Gel Pro Analyzer 4.0. The numbers shown below represent the relative accumulation level of sat-siRNAs.
Figure 10.
Figure 10.
Biological relevance of the mutations that disrupted or generated the γH2 hairpin in sat-SD or sat-D4, respectively. (A) The γSS structure of sat-SD and its disruptive mutation to γH2 (top), and the equivalent structure of sat-D4 and its mutation (bottom). The mutation introduced to sat-D4 presumably generates a γH2 hairpin in sat-D4. (B) Disease symptoms in Nicotiana benthamiana plants infected with CMV alone (–) or plus a satRNA or its mutant as indicated on the top. The plants were photographed at 6 days post-infiltration (dpi). Mock plants were infiltrated with infiltration solution. Bar: 1 cm. (C) Northern blot hybridization analysis of the accumulation of CMV, satRNA or satRNA mutants in the infected plants. Total RNAs were extracted from the inoculated leaves or upper systemic leaves at 6 dpi. The DNA oligonucleotide probes targeting CMV 3′ UTR, 1a and 2b were used to detect all viral RNAs, RNA1 and RNA2 and its subgenomic RNA4A, respectively. The ethidium bromide-stained rRNAs were used as a loading control.
Figure 11.
Figure 11.
A proposed model for satRNA inhibiting the accumulation of CMV RNAs. RNAs 1 and 2 released from infected CMV are used as translation templates to produce viral replicase components 1a and 2a, respectively. Differential host factors are subsequently recruited by viral replicase and RNAs to form replication machinery for replication of RNAs 1 and 2 or RNA3. CMV satRNAs specifically compete the replication machinery for replication of RNAs 1 and 2, by which it reduces the accumulation of both viral RNAs. Once the accumulation of RNAs 1 and 2 is reduced to a level at which the 1a and 2a proteins produced by both viral RNAs become limited for RNA3 replication, the accumulation of RNA3 is reduced as observed in the case of sat-SD shown in this study.
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