Generation of subgenomic RNA directed by a satellite RNA associated with bamboo mosaic potexvirus: analyses of potexvirus subgenomic RNA promoter

Abstract

Satellite RNA of bamboo mosaic potexvirus (satBaMV), a single-stranded positive-sense RNA encoding a nonstructural protein of 20 kDa (P20), depends on bamboo mosaic potexvirus (BaMV) for replication and encapsidation. A full-length cDNA clone of satBaMV was used to examine the sequences required for the synthesis of potexvirus subgenomic RNAs (sgRNAs). Subgenomic promoter-like sequences (SGPs), 107 nucleotides (nt) upstream from the capsid protein (CP) gene of BaMV-V, were inserted upstream of the start codon of the P20 gene of satBaMV. Insertion of SGPs gave rise to the synthesis of sgRNA of satBaMV in protoplasts of Nicotiana benthamiana and leaves of Chenopodium quinoa when coinoculated with BaMV-V genomic RNA. Moreover, both the satBaMV cassette and its sgRNA were encapsidated. From analysis of the SGPs by deletion mutation, we concluded that an SGP contains one core promoterlike sequence (nt -30 through +16), two upstream enhancers (nt -59 through -31 and -91 through -60), and one downstream enhancer (nt +17 through +52), when the transcription initiation site is taken as +1. Site-directed mutagenesis and compensatory mutation to disrupt and restore potential base pairing in the core promoter-like sequence suggest that the stem-loop structure is important for the function of SGP in vivo. Likewise, the insertion of a putative SGP of the BaMV open reading frame 2 gene or a heterologous SGP of potato virus X resulted in generation of an sgRNA. The satBaMV cassette should be a useful tool to gain insight into sequences required for the synthesis of potexvirus sgRNAs.

FIG. 1

Genome organization of BaMV and satBaMV mutants (A) and the time course accumulation of BaMV-V genomic RNA (B) and satBaMV mutants (C) in inoculated protoplasts. (A) In these schematic diagrams of BaMV and satBaMV cassettes, the hatched regions represent the ORF regions of BaMV (designated ORFs 1 through 5) or satBaMV (designated P20); the black bar represents the 107 nt upstream of the CP gene of BaMV (designated SGP); restriction enzyme sites ApaI, BstXI, and BseRI in pBSF4 are also indicated as are the positions of primers B56 and BS9. (B and C) Protoplasts of barley were inoculated with BaMV-L RNA only (lanes 1 through 3) or coinoculated with pBSF4 (lanes 4 through 6) or pICF4 (lanes 7 through 9) transcripts. Total RNAs extracted from 5 × 10⁴ protoplasts at 8 h.p.i. (lanes 1, 4, and 7), 16 h.p.i. (lanes 2, 5, and 8), and 24 h.p.i. (lanes 3, 6, and 9) were glyoxylated and fractionated in a 1% agarose gel, blotted to a nylon membrane, and hybridized to a BaMV-specific (20) (B) or satBaMV-specific (23) (C) riboprobe. The star indicates the position of the 0.7-kb sgRNA transcribed from pICF4.

FIG. 2

Schematic diagrams of pICF4 mutants (A) and their transcription activities (B and C). (A) pICF4 and its mutants carrying various lengths of SGP sequences extended downstream to the coding region of the CP gene. The black region represents the 107 nt upstream of the CP gene of BaMV (designated SGP). The start codon of the CP gene is indicated. (B) Northern blot analysis was performed on total RNAs extracted from 5 × 10⁴ protoplasts of N. benthamiana coinoculated with BaMV-L RNA and pICF4 (lane 1), pICN9 (lane 2), pICN18 (lane 3), pICN36 (lane 4), and pICN54 (lane 5) transcripts at 24 h.p.i. with a satBaMV-specific riboprobe. The asterisk indicates the position of the 0.7-kb sgRNA transcribed from satBaMV cassette. (C) Quantitative analyses of the transcription ratios of pICF4 and its mutants in coinoculated protoplasts of N. benthamiana were performed. The transcription ratio was determined by quantifying the 0.7-kb sgRNA relative to that of satBaMV RNA (a), and the transcription ratio was the transcription ratio of ICF4 mutants over that of ICF4 at 24 h.p.i. (b). These data were collected in three independent experiments by PhosphorImager and using the ImageQuant version 3.3 program (Molecular Dynamics); standard deviations are indicated.

FIG. 3

The secondary structure of the minus-strand SGP sequence of the 1.0-kb sgRNA of BaMV-V (A), schematic diagrams of satBaMV mutants (B), and their transcriptional-activity assays (C through E). (A) The secondary structure was predicted by the STAR computer program. The stem-loop structures, designated SL1 (ΔG = −1.3 kcal/mol), SL2 (ΔG = −2.5 kcal/mol), SL3 (ΔG = −8.3 kcal/mol), and SL4 (ΔG = −4.8 kcal/mol) and 18 nt of the N-terminal CP gene of BaMV (designated N18) are shown. The asterisk denotes the transcription initiation site, and the conserved octanucleotide motif is in bold. (B) For SatBaMV mutants, the numbers of nucleotides for each structure are given in parentheses. (C through E) For Northern (C and D) and quantitative (E) analyses of the sgRNAs transcribed by the mutants, total RNAs were extracted from 5 × 10⁴ protoplasts of N. benthamiana at 24 h.p.i. (C) or 5-mg leaves of C. quinoa at 6 d.p.i. (D). These RNAs were coinoculated with BaMV-L RNA and the pICN18 (lane 1), pIC5 (lane 2), pIC4 (lane 3), pIC3 (lane 4), or pIC2 (lane 5) transcript and were subjected to Northern analyses with a satBaMV-specific riboprobe. The asterisk indicates the position of the 0.7-kb sgRNA transcribed from satBaMV cassettes. (E) For quantitative analyses of the transcription ratio of satBaMV mutants in protoplasts of N. benthamiana or in leaves of C. quinoa in three independent experiments, the transcription ratio (a) and the relative transcription ratio over that of pICN18 (b) were determined as described in the legend to Fig. 2.

FIG. 4

Predicted secondary structure of SL2 and the schematic diagrams of satBaMV mutants (A) and their transcription activities (B and C). (A) Shown are the predicted secondary structure of SL2 and the schematic diagrams of mutants introduced in SL2. The boxed and shaded letters represent the mutated nucleotides. The bold letters indicate the conserved nucleotide motif. (B) Northern blot analysis of total RNAs extracted from 5 × 10⁴ protoplasts of N. benthamiana coinoculated with BaMV-L RNA and pICN18 (lane 1), pIC8-C/C (lane 2), pIC9-G/G (lane 3), pIC10-G/C (lane 4), pIC14-CCC (lane 5), and pIC7 (lane 6) transcripts at 24 h.p.i. with a satBaMV-specific riboprobe. The asterisk indicates the position of the 0.7-kb sgRNA transcribed from the satBaMV cassette. (C) The relative ratio was the transcription ratio of a mutant over that of pICN18 in protoplasts of N. benthamiana (a) or in leaves of C. quinoa (b). These data were collected three times from protoplasts and two times from plants.

FIG. 5

SgRNA transcription assays of pICORF2 and pICPVX. (A) Schematic diagrams of ICORF2 and ICPVX. Hatched regions represent the ORF regions of BaMV, PVX, and satBaMV. The black and dotted regions represent the SGP of the 2.0-kb sgRNA of BaMV and of the 0.9-kb sgRNA of PVX, respectively. (B and C) Northern analyses of total RNAs extracted from 5 × 10⁴ protoplasts of N. benthamiana at 24 h.p.i. (lanes 1 through 3) or 5-mg leaves of C. quinoa at 6 d.p.i. (lanes 4 through 6) coinoculated with BaMV-L RNA and pICORF2 (lanes 1 and 4), pICF4 (lanes 2 and 5), and pICPVX (lanes 3 and 6) transcripts with a BaMV (B) or satBaMV (C) riboprobe. The asterisk indicates the position of the 0.7-kb sgRNA transcribed from satBaMV cassettes.

FIG. 6

Time course accumulation of BaMV-V genomic RNA, satBaMV, and its derived sgRNA in infected leaves of C. quinoa. (A and B) Total RNAs extracted from 5-mg leaves inoculated with BaMV-L RNA only (lanes 1, 4, and 7) or together with pBSF4 (lanes 2, 5, and 8) or pICF4 (lanes 3, 6, and 9) transcripts at 3 d.p.i. (lanes 1 through 3), 6 d.p.i. (lanes 4 through 6), and 9 d.p.i. (lanes 7 through 9) were subjected to Northern blotting with a BaMV-specific (20) (A) or satBaMV-specific (23) (B) riboprobe. (C and D) For staining (C) and Northern blot analysis (D) of encapsidated satBaMV and its sgRNA, virion RNAs were purified from 0.2 g of C. quinoa leaves and inoculated with BaMV-L RNA only (lane 1) or with pBSF4 (lane 2), and pICF4 (lane 3) transcripts were separated by electrophoresis in a 1% agarose gel and stained with EtBr (C) or analyzed by Northern hybridization with the satBaMV probe (D). The asterisks indicate the position of the 0.7-kb sgRNA transcribed from pICF4.

FIG. 7

Identification of the 5′ termini of sgRNAs by primer extension. Total RNAs (lanes 1) extracted from C. quinoa leaves infected with BaMV-L RNA only (A) or together with pICF4 (B) were hybridized with primer B56 (complementary to the CP gene of BaMV) (A) or primer BS9 (complementary to the P20 gene of satBaMV) (B) for primer extension. The products were displayed on a 6% polyacrylamide–7 M urea sequencing gel. Lanes A, C, G, and T contained the products of a dideoxynucleotide-sequencing reaction performed on cDNA clone pBa19 of BaMV-V genomic RNA (40) with primer B56 (A) and on pICF4 with primer BS9 (B). Arrows indicate the position of the transcription initiation site. Letters represent the sequences between the octanucleotide motif (in italics) and the transcription initiation site.

FIG. 8

Primer extension analyses of the sgRNAs transcribed by pICORF2 (A) and pICPVX (B). Messenger RNAs extracted from 2 × 10⁵ protoplasts of N. benthamiana at 24 h.p.i. (lane 1) or total RNAs extracted from 5-mg leaves of C. quinoa at 6 d.p.i. (lane 2) coinoculated with pICORF2 (A) or pICPVX (B) transcripts and BaMV-L RNA were hybridized with primer BS9 for primer extension. The products were displayed on a 6% polyacrylamide–7 M urea sequencing gel. Lanes A, C, G, and T contained the products of a dideoxynucleotide sequencing reaction performed on cDNA clone pICORF2 (A) or pICPVX (B) with primer BS9. Arrows indicate the positions of the transcription initiation sites, and letters represent the sequences between the octanucleotide motif (in italics) and the transcription initiation sites.