Helper virus-independent transcription and multimerization of a satellite RNA associated with cucumber mosaic virus

Abstract

Satellite RNAs are the smallest infectious agents whose replication is thought to be completely dependent on their helper virus (HV). Here we report that, when expressed autonomously in the absence of HV, a variant of satellite RNA (satRNA) associated with Cucumber mosaic virus strain Q (Q-satRNA) has a propensity to localize in the nucleus and be transcribed, generating genomic and antigenomic multimeric forms. The involvement of the nuclear phase of Q-satRNA was further confirmed by confocal microscopy employing in vivo RNA-tagging and double-stranded-RNA-labeling assays. Sequence analyses revealed that the Q-satRNA multimers formed in the absence of HV, compared to when HV is present, are distinguished by the addition of a template-independent heptanucleotide motif at the monomer junctions within the multimers. Collectively, the involvement of a nuclear phase in the replication cycle of Q-satRNA not only provides a valid explanation for its persistent survival in the absence of HV but also suggests a possible evolutionary relationship to viroids that replicate in the nucleus.

Fig 1

Multimerization of Q-satRNA in the absence of its HV. (A) Schematic diagram of a T-DNA-based Q-satRNA construct (pQ-satRNA) for in vivo expression via agroinfiltration. The basal binary vector contains, in sequential order, a left border of T-DNA (LB), a double 35S promoter (35S), full-length cDNA of Q-satRNA, a ribozyme sequence (Rz) from HDV, a 35S terminator (Ter), and a right border of T-DNA (RB). The transcription initiation and HDV cleavage sites are shown. (B, C) Specificity of Q-satRNA riboprobes. Duplicate blots containing the indicated amounts of in vitro transcripts of positive- and negative-strand Q-satRNA were hybridized with ³²P-labeled riboprobes designed to detect either Q-satRNA positive strands (B) or Q-satRNA negative strands (C). (D) Northern blot analysis of total RNA recovered at 4 dpi from N. clevelandii leaves agroinfiltrated with the indicated cultures of agroconstructs along with p19 (a suppressor of RNA silencing) and hybridized with riboprobes as shown. The positions of the Q-satRNA monomeric (1×), dimeric (2×), trimeric (3×), and tetrameric (4×) forms are indicated on the left of each panel. In the bottom part of the panel, RNA1 to RNA5 are HV progeny RNAs. rRNA represents a loading control. All samples contained 20 μg of total RNA, except in the top and middle parts, where lanes containing EV plus HV and pQ-satRNA plus HV, respectively, contained 4 and 0.4 μg of total RNA. In the bottom part, these two lanes contained 4 μg of total RNA. In each part, a radiolabeled RNA size marker is shown on the right. (E, top and middle) Northern blot analysis of total RNA recovered from N. clevelandii plants mechanically inoculated with the indicated concentrations of Q-satRNA in vitro transcripts and probed with the indicated riboprobes. (E, bottom) Northern blot hybridization of the Q-satRNA in vitro transcripts used for mechanical inoculation.

Fig 2

Nuclear localization of Q-satRNA in the absence of HV. (A) The predicted in vitro secondary structure of Q-satRNA and SL-C is shown, and a dotted circle indicates the location where the MS2 binding site was engineered. WT, wild type. (B) Schematic representation of pQ-R5-MS2 showing the location where the MS2 binding site (indicated by a dotted circle) was engineered into Q-CMV RNA5. (C) Northern blot assay showing that insertion of the MS2 binding site into Q-satRNA has no significant effect on Q-satRNA replication when it is allowed to coinfiltrate leaves with HV. The blot was hybridized with a riboprobe to detect the positive strand. The positions of the Q-satRNA monomeric (1×) and dimeric (2×) forms are indicated. rRNA represents the loading control. (D) Schematic representation of agroconstructs used for ectopic expression of GFP alone, GFP fused to MS2-CP, or GFP fused to the NLS and MS2-CP. The basal binary PZP vector contains, in sequential order, a left border of T-DNA (LB), a double 35S promoter (35S), multiple cloning sites, a 35S terminator (T35S), and a right border of T-DNA (RB). The bent arrow indicates the translation initiation site. The target genes were subcloned in frame into the StuI and SpeI sites. (E to I) Representative confocal microscopic images of N. benthamiana leaves agroinfiltrated with either single or pairwise combinations of the indicated agrocultures. Fluorescent signals were observed in the epidermal cells at 3 dpi. In each panel, GFP expression (green), light mode, and merged images are shown. In addition, in panel I, CFP expression (blue) is shown. Bars, 20 μm.

Fig 3

In situ evidence for the synthesis of the antigenomic strands of Q-satRNA in the absence of HV. Immunolabeling of dsRNAs in leaves infiltrated with EV (A), HV (B), Q-satRNA (C), NG-satRNA (D), Q-satRNA negative-strand (E), Q-RNA5 (F), and BMV RNA3 (G) agroconstructs. To visualize the nuclei, leaf segments were treated with DAPI (blue). dsRNAs in the leaf segments were probed using J2-Ab and then treated with an Alexa Fluor 633 (emits red fluorescence)-conjugated secondary antibody (see Materials and Methods for details). Leaves infiltrated with the HV (H) or Q-satRNA (I) agrocunstruct alone were treated with RNase III prior to immunolabeling with J2-Ab. Bars, 10 μm.

Fig 4

Analysis of the junction sequences in Q-satRNA multimers formed in the presence or absence of HV. PCR products of the junction regions of Q-satRNA multimers formed in the presence or absence of HV using the primer set Fw1 plus Rv1 (A) or Fw2 plus Rv2 (B). In each panel, agarose gel analysis of the PCR products resulting from each primer set is shown on the left. On the right, a schematic representation of the dimeric form of Q-satRNA and the location of each primer are shown. Primer Fw2 was designed to contain a guanine residue beyond the authentic terminal cytosine residue at the 3′ end of Q-satRNA. To assess the specificity of each primer set, 50 ng of pQSx2-ΔC (a Q-satRNA dimer construct lacking the terminal C residue from the 3′ end of the first monomeric unit) and pQSx2-HNM (a Q-satRNA dimer construct having the HNM [GGGAAAA] between monomers) were used in PCRs. (C) Summary of the sequence analyses of the junction region of the Q-satRNA multimers from the agroinfiltrated or mechanically inoculated leaves. The values on the right are the number of cDNA clones with a specific sequence/total number of cDNAs sequenced. Exp., experiment.

Fig 5

In situ evidence for the nuclear localization of Q-satRNA in the presence of HV. (A to D) Representative confocal microscopic images showing the distribution of GFP in leaves expressing the indicated mixtures of agroconstructs. The agroconstructs, experimental conditions, and confocal microscopy procedures used are as described in the legend to Fig. 2. In all panels, GFP expression (green), light mode, and merged images are shown. In addition, in panel D, CFP expression (blue) is shown. Bars, 20 μm.