A novel sweet potato potyvirus open reading frame (ORF) is expressed via polymerase slippage and suppresses RNA silencing

Abstract

The single-stranded, positive-sense RNA genome of viruses in the genus Potyvirus encodes a large polyprotein that is cleaved to yield 10 mature proteins. The first three cleavage products are P1, HCpro and P3. An additional short open reading frame (ORF), called pipo, overlaps the P3 region of the polyprotein ORF. Four related potyviruses infecting sweet potato (Ipomoea batatas) are predicted to contain a third ORF, called pispo, which overlaps the 3' third of the P1 region. Recently, pipo has been shown to be expressed via polymerase slippage at a conserved GA6 sequence. Here, we show that pispo is also expressed via polymerase slippage at a GA6 sequence, with higher slippage efficiency (∼5%) than at the pipo site (∼1%). Transient expression of recombinant P1 or the 'transframe' product, P1N-PISPO, in Nicotiana benthamiana suppressed local RNA silencing (RNAi), but only P1N-PISPO inhibited short-distance movement of the silencing signal. These results reveal that polymerase slippage in potyviruses is not limited to pipo expression, but can be co-opted for the evolution and expression of further novel gene products.

Keywords: RNA silencing suppression; RNA virus; frameshift; gene expression; overlapping gene; transcriptional slippage.

Figure 1

Genome organization of Sweet potato feathery mottle virus (SPFMV)‐group potyviruses. (a) Map of the ∼10.8‐kb genome showing the polyprotein open reading frame (ORF) (light blue) and overlapping pipo and pispo ORFs (pink). The P1‐N and P1‐pro domains and hypervariable region (HVR) are marked. For consistency with previous naming (SPFMV P1‐N and potyvirus P3N‐PIPO), we adopt the convention that ‘P1‐N’ refers to the N‐terminal domain, whereas ‘P1N’ refers to the entire region of P1 encoded upstream of the G₂A₆ slippage site. (b) Positions of stop codons (blue) in the three forward reading frames, and alignment gaps (grey) in an alignment of 31 SPFMV‐group sequences. (c) Conservation at synonymous sites in the alignment, using a 231‐codon sliding window (green) or a 15‐codon sliding window (red and brown). The upper panels (green, red) depict the probability that the degree of conservation within a given window could be obtained under a null model of neutral evolution at synonymous sites, whereas the bottom panel (brown) depicts the absolute amount of conservation as represented by the ratio of the observed number of substitutions within a given window to the number expected under the null model. The broken grey lines indicate a P = 0.05 threshold after applying a rough correction for multiple testing (namely 3519 codons/15‐ or 231‐codon window size).

Figure 2

Representative nucleotide and peptide sequences. (a) Nucleotide sequences flanking the proposed slippage sites in representative Sweet potato feathery mottle virus (SPFMV)‐group sequences (GenBank accession numbers shown on left, species on right). Spaces separate polyprotein‐frame codons; pispo is in the +2 frame. The G₂A₆ sequence, where slippage is proposed to occur, is highlighted in orange. Numbers on the left indicate the genomic coordinates of the first nucleotide in each line. (b) Amino acid sequences of P1 and P1N‐PISPO in the Ruk73 isolate of SPFMV. The amino acids encoded at the slippage site are highlighted in orange. Sequences corresponding to the epitopes of the two polyclonal antibodies (pAbs) are highlighted in pale blue. GW/WG motifs are underlined. (c) Sequences of the epitopes against which the two pAbs were raised. Sequence differences from the Ruk73 isolate are highlighted in red. SPV2, Sweet potato virus 2; SPVC, Sweet potato virus C; SPVG, Sweet potato virus G.

Figure 3

Single nucleotide insertions in short‐RNA sequencing of sweet potato virus‐infected plants. Short‐RNA reads from Sweet potato virus 2 (SPV2), Sweet potato virus G (SPVG) and two Sweet potato feathery mottle virus (SPFMV) [10, sweet potato only infected with SPFMV; 12, sweet potato infected with SPFMV + Sweet potato chlorotic stunt virus (SPCSV)] samples were mapped to the corresponding reference genomes. For each sample, the sequencing depth of fully matched reads is plotted as a 100‐nucleotide moving average in grey (log scale on left axis). The mean coverage is indicated at the top right. Positions of single nucleotide insertions are indicated in red (linear scale on right axis). For each virus, the positions of the G₂A₆ sequences at the beginning of the pispo and pipo open reading frames (ORFs) are indicated with arrows. An asterisk (*) denotes the off‐scale value of 43 insertions at the pispo G₂A₆ site for sample SPFMV‐12.

Figure 4

Transcriptional slippage at the pispo and pipo G₂A₆ sequences in Sweet potato feathery mottle virus (SPFMV)‐infected plants. Results from targeted high‐throughput sequencing of systemically infected Ipomoea batatas and I. nil. The frequencies of transcripts with a single ‘A’ insertion at the G₂A₆ sequence are shown in blue; the frequencies of transcripts with two or more inserted ‘A’ nucleotides are shown in orange; and the frequencies of transcripts with one or more ‘A’ nucleotides deleted are shown in yellow. Controls from plasmid template were included to assess the variability introduced during amplification and sequencing.

Figure 5

Locations of GW/WG sequences in P1 and P1N‐PISPO proteins of Sweet potato feathery mottle virus (SPFMV)‐group viruses. Schematic diagram of the P1 protein of Sweet potato mild mottle virus (SPMMV) and the P1 and P1N‐PISPO proteins of SPFMV‐group viruses, showing the positions of GW/WG sequences. The P1 sequence is indicated in light blue; the PISPO sequence is indicated in pink. GW/WG sequences are indicated by vertical coloured lines, with homologous instances (by blastp alignment) indicated with the same colour. SPV2, Sweet potato virus 2; SPVC, Sweet potato virus C; SPVG, Sweet potato virus G.

Figure 6

Binary vectors for the expression of viral proteins in leaves by agroinfiltration. Cauliflower mosaic virus 35S promoter‐driven binary vectors were designed to express the following proteins: P1, P1 of Sweet potato feathery mottle virus (SPFMV) [the shaded bar indicates the position of the +2 frameshift pispo open reading frame (ORF) encoding 230 residues]; P1N‐PISPO, engineered transframe sequence for the expression of the N‐proximal part of P1 together with PISPO; P1ΔPISPO, P1 including a stop codon inserted in the +2 frame pispo ORF to ensure that P1N‐PISPO is not produced; P1N‐PISPO(ΔWG2,3,4), P1N‐PISPO mutated at three positions (W₅₁₄G₅₁₅, W₆₂₄G₆₂₅ and W₆₆₅G₆₆₆; designated as WG motifs 2, 3 and 4, respectively) to substitute the tryptophan residues for alanines; P1(ΔWG₁), P1 mutated to substitute residue W₂₅ for alanine (WG motif 1); P1N‐PISPO(ΔWG₁), P1N‐PISPO with W₂₅ mutated to alanine; P1N‐PISPO(ΔWG1,2,3,4), P1N‐PISPO with all four WG motif tryptophans mutated to alanine; HCpro, HCpro of SPFMV; YN‐HCpro, the N‐proximal part of the yellow fluorescent protein (YN) fused to the N‐terminus of SPFMV HCpro; P1(SPLV), P1 of Sweet potato latent virus; YN‐P1(SPLV), the N‐proximal part of the yellow fluorescent protein (YN) fused to the N‐terminus of SPLV P1.

Figure 7

Suppression of sense‐mediated RNAi of green fluorescent protein (GFP) expression by potyviral proteins in leaves of GFP‐transgenic Nicotiana benthamiana line 16c. (a) Silencing of gfp was induced by overexpression of GFP mRNA ‘on the spot’ from a binary vector pBIN:GFP introduced by agroinfiltration, and interference with silencing was tested by co‐introduction of binary vectors for the expression of: (1) Sweet potato feathery mottle virus (SPFMV) P1; (2) SPFMV P1N‐PISPO; (3) SPFMV HCpro; (4) Sweet potato latent virus (SPLV) P1; (5) Potato virus A (PVA) HCpro; or (6) β‐glucuronidase (GUS) (control). The leaves were photographed at 4 days post‐infiltration (dpi) (a), 6 dpi (d) and 8 dpi (e). Scale bars indicate 2 cm. (b) Expressed proteins were detected at 4 dpi using antibodies to GFP, peptide antibodies generated against an epitope common to P1 and P1N‐PISPO, and peptide antibodies specific to the PISPO domain of P1N‐PISPO. In the P1 panel, lane 2 was loaded in a different order in the gel, and spliced electronically afterwards (indicated by black vertical lines), for consistency with other panels. Staining of total proteins by Coomassie blue was used as a loading control (shown for GFP gel). (c) Northern blot analysis for the detection of GFP mRNA and gfp‐derived small interfering (si)RNA in leaf tissues co‐infiltrated with pBIN:GFP and the viral constructs in (A) at 4 dpi; gfp was used as a probe and 28S and 5S ribosomal RNAs (rRNA) were used as loading controls. Note the different order of samples 5 and 6 in panels (b) and (d). (d) The infiltrated leaf tissue expressing P1 was surrounded by a red halo (left), in contrast with the tissue expressing P1N‐PISPO (right), which was most apparent at 6 dpi. (e) Suppression of silencing by P1, P1N‐PISPO and PVA HCpro was indicated by GFP fluorescence in the co‐infiltrated leaf tissue at 8 dpi, whereas no suppression of silencing was observed with SPFMV HCpro and SPLV P1.

Figure 8

Effects of mutation of the WG motifs in P1 and P1N‐PISPO on the suppression of sense‐mediated RNAi of green fluorescent protein (GFP) expression in leaves of GFP‐transgenic Nicotiana benthamiana line 16c. Silencing of gfp was induced by overexpression of GFP mRNA from a binary vector pBIN:GFP introduced by agroinfiltration, and interference with silencing was tested by co‐introduction of binary vectors for expression of Sweet potato feathery mottle virus (SPFMV) P1, P1N‐PISPO or their mutated forms (see Fig. 3). (a) Suppression of silencing by: (1) P1, (2) P1N‐PISPO, (3) P1ΔPISPO, (4) P1N‐PISPO(ΔWG_2,3,4), (5) Potato virus A (PVA) HCpro or (6) β‐glucuronidase (GUS) control at 4 days post‐infiltration (dpi). (b) Half‐agroinfiltrated leaf arrays were used to test the suppression of silencing by: (1) P1, (2) P1(ΔWG₁), (3) P1N‐PISPO, (4) P1N‐PISPO(ΔWG₁), (5) P1N‐PISPO(ΔWG_2,3,4) or (6) P1N‐PISPO(ΔWG_1,2,3,4). Scale bars indicate 2 cm.