Opium Poppy Mosaic Virus Has an Xrn-Resistant, Translated Subgenomic RNA and a BTE 3' CITE

Abstract

Opium poppy mosaic virus (OPMV) is a recently discovered umbravirus in the family Tombusviridae OPMV has a plus-sense genomic RNA (gRNA) of 4,241 nucleotides (nt) from which replication protein p35 and p35 extension product p98, the RNA-dependent RNA polymerase (RdRp), are expressed. Movement proteins p27 (long distance) and p28 (cell to cell) are expressed from a 1,440-nt subgenomic RNA (sgRNA2). A highly conserved structure was identified just upstream from the sgRNA2 transcription start site in all umbraviruses, which includes a carmovirus consensus sequence, denoting generation by an RdRp-mediated mechanism. OPMV also has a second sgRNA of 1,554 nt (sgRNA1) that starts just downstream of a canonical exoribonuclease-resistant sequence (xrRNA_D). sgRNA1 codes for a 30-kDa protein in vitro that is in frame with p28 and cannot be synthesized in other umbraviruses. Eliminating sgRNA1 or truncating the p30 open reading frame (ORF) without affecting p28 substantially reduced accumulation of OPMV gRNA, suggesting a functional role for the protein. The 652-nt 3' untranslated region of OPMV contains two 3' cap-independent translation enhancers (3' CITEs), a T-shaped structure (TSS) near its 3' end, and a Barley yellow dwarf virus-like translation element (BTE) in the central region. Only the BTE is functional in luciferase reporter constructs containing gRNA or sgRNA2 5' sequences in vivo, which differs from how umbravirus 3' CITEs were used in a previous study. Similarly to most 3' CITEs, the OPMV BTE links to the 5' end via a long-distance RNA-RNA interaction. Analysis of 14 BTEs revealed additional conserved sequences and structural features beyond the previously identified 17-nt conserved sequence.IMPORTANCEOpium poppy mosaic virus (OPMV) is an umbravirus in the family Tombusviridae We determined that OPMV accumulates two similarly sized subgenomic RNAs (sgRNAs), with the smaller known to code for proteins expressed from overlapping open reading frames. The slightly larger sgRNA1 has a 5' end just upstream from a previously predicted xrRNA_D site, identifying this sgRNA as an unusually long product produced by exoribonuclease trimming. Although four umbraviruses have similar predicted xrRNA_D sites, only sgRNA1 of OPMV can code for a protein that is an extension product of umbravirus ORF4. Inability to generate the sgRNA or translate this protein was associated with reduced gRNA accumulation in vivo We also characterized the OPMV BTE structure, a 3' cap-independent translation enhancer (3' CITE). Comparisons of 13 BTEs with the OPMV BTE revealed additional stretches of sequence similarity beyond the 17-nt signature sequence, as well as conserved structural features not previously recognized in these 3' CITEs.

Keywords: 3′ CITE; BTE; Xrn1; cap-independent translation enhancer; exoribonuclease-resistant sites; subgenomic RNA; umbravirus.

FIG 1

Maximum-likelihood analysis of the umbravirus genus based on 37 full-length umbravirus gRNA sequences available in GenBank. Viruses are highlighted in different colors. GenBank accession numbers are indicated. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to each branch. Vertical branches are arbitrary, and horizontal branches are proportional to calculated mutation distances. The tree is rooted at the midpoint. CMoMV, Carrot mottle mosaic virus; CMoV, Carrot mottle virus; ETBTV, Ethiopian tobacco bushy top virus; GRV, Groundnut rosette virus; IxYMoV2, Ixeridium yellow mottle-associated virus 2; OPMV, Opium poppy mosaic virus; PEMV2, Pea enation mosaic virus 2; PatMMoV, Patrinia mild mottle virus; RCUV, Red clover umbravirus; TBTV, Tobacco bushy top virus.

FIG 2

Basic properties of OPMV. (A) Genome organization of umbraviruses. Sizes of OPMV-encoded products are in parentheses. (B) Two N. benthamiana plants infected with OPMV and its uncharacterized helper virus showing mild leaf curling and yellowing. Mock, plant treated with inoculation buffer. (C) 5′ UTRs of OPMV and closely related umbraviruses. RCUV is not included, as a 5′ UTR sequence was not available. Carmovirus consensus sequence is in green and the ORF1 start codon is in red. OPMV*, previously reported OPMV 5′ UTR sequence. TBTV 5′ UTR starts with either G or A, depending on the isolate. (D) Northern blot of total RNA isolated from systemically infected N. benthamiana leaves following agroinfiltration with OPMV. Mock, infiltrated with empty binary vector. Ethidium bromide-stained 28S rRNA was used as a loading control.

FIG 3

sgRNAs of OPMV and their encoded products. (A) Primer extension assay to map the transcription start sites of OPMV sgRNAs. Total RNA isolated from N. benthamiana that was either infected with OPMV (and its uncharacterized helper virus) or treated with transfection buffer (mock) was used. The first four lanes (U, C, G, and A) are sequencing ladders. Positions of the extension products are given. (B) Sequence and structure of a portion of the intergenic region of OPMV showing 5′ ends of the sgRNAs and their encoded products. Mutations used in panels B, C, and D are indicated, with construct names in brackets. Start codons for p27, p28, and p30 are shaded red. Bases are colored according to their N-methylisatoic anhydride (NMIA) reactivity in selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) structure probing, with red being the most reactive and black being the least reactive. Structure was determined using SHAPE probing data and phylogenetic comparisons (see Fig. 4 and 6). Dark blue and light blue asterisks denote conserved residues (>90% or >75%, respectively) in xrRNA_D elements (39). A dashed line connects residues (shaded gray) involved in a critical pseudoknot (noted with dark blue asterisk). The structure just upstream from the sgRNA2 transcription start site is conserved in all umbraviruses (see Fig. 6). Carmovirus consensus sequence (CCS) is shaded green. (C) SDS-PAGE gels of in vitro translated OPMV gRNA (left) or wild-type (WT) and mutant sgRNAs (right). Note that p27 and p28 migrate aberrantly in these gels. (D and E) Northern blots of total RNA isolated from N. benthamiana infiltrated with either wild-type (WT) or mutant OPMV with alterations in either the xrRNA_D site (D) or the p30 initiation codon (E). i2797C is a single-base insert that terminates p30 translation at the adjacent downstream UGA. 28S rRNA served as a loading control. RNA bands intensities were measured using ImageJ. Data are from three experiments, and standard deviations are shown.

FIG 4

xrRNA_D structures in umbraviruses. (A) xrRNA_D structures in the intergenic region (IR) of four umbraviruses. Conserved bases are in red, and less conserved bases are in orange (39). A critical pseudoknot formed between residues shaded gray is shown by a dashed line. Start sites for the sgRNAs and some of their encoded products are shown for the OPMV structure. (B) OPMV and TBTV are the only umbraviruses with a second xrRNA_D structure near the 5′ end of their 3′ UTR. ORF4 termination codon is shaded green.

FIG 5

Umbravirus (assigned and unassigned) intergenic region (IR) alignment. (A) The numbers of isolates included in the alignment are given in parentheses. Degenerate nucleotide codes are as follows: Y (C or U), R (A or G), W (A or U), K (U or G), M (C or A), and H (A or C or U). Start sites of OPMV sgRNA1 and sgRNA2 are indicated. CCS at the beginning of OPMV sgRNA2 and equivalent sgRNAs are shaded green. Start codons of ORF3 and p30 are shaded red and blue, respectively. Stem regions of putative hairpins are underlined in red, and a highly conserved motif found in many apical loops (AAUHGA) is highlighted in yellow. A second conserved loop motif (UGGCU) is highlighted in orange. Other conserved sequences are colored alike. Duplicated stretches within the IR are denoted by arrows. The xrRNA_D structure in four umbraviruses is denoted. The conserved sequence/structure in the vicinity of the sgRNA2 transcription start site is labeled, and structures are shown in Fig. 6. (B). Translation context of p30 initiation codon (blue). Critical purine at −3 and guanylate at +4 are present. (C) N-terminal sequence of p30, which is in frame with downstream ORF4. All other umbraviruses have an in-frame termination codon (in red) just upstream of ORF4 and thus cannot produce an N-terminal extended version of the ORF4 product.

FIG 6

Proposed structure just upstream of the start site of umbravirus sgRNA2. Similar sequences are colored alike. CCS is shaded green and ORF3 start codon is shaded red.

FIG 7

OPMV gRNA and sgRNA 5′ sequences necessary for efficient translation in vivo. (A) Constructs used to examine gRNA 5′ sequences required for efficient translation. PEMV2 control construct containing the PEMV2 5′ 89 nt and 3′ UTR (5′89+3U) was previously described (13). All OPMV constructs contain a full-length OPMV 3′ UTR. Blue box denotes OPMV coding region sequences. 5ΔU+3U contains 18 random nucleotides at the 5′ end. (B). Relative luciferase activity of constructs assayed in Arabidopsis thaliana protoplasts. Error bars denote standard deviation for three replicate assays. (C). Constructs used to assay OPMV sgRNA2 5′ sequences required for efficient translation. All constructs contain a full-length OPMV 3′ UTR. Blue box denotes coding region sequences. (D) Relative luciferase activity of constructs assayed in protoplasts. Error bars denote standard deviation for three replicate assays.

FIG 8

OPMV has a BTE 3′ CITE that connects with the gRNA and sgRNA2 5′ ends through a long-distance RNA-RNA interaction. (A) Proposed secondary structure of the 3′ UTR of OPMV. Residues are colored according to SHAPE NMIA reactivity, with purple being the most reactive and black being the least reactive. BTE and TSS 3′ CITEs are labeled. A red line denotes the BTE 17-nt signature sequence. Potential long-distance RNA-RNA interactions between the BTE and 5′ proximal hairpins in the gRNA (gH1) and sgRNA2 (sgH1) are denoted by dashed lines, with the signature motifs commonly found in carmovirus and umbravirus long-distance interactions shaded light blue. Alterations generated to assay for the interactions are shown with construct names in parentheses. Gray shaded sequences connected by dashed lines denote local tertiary interactions in the TSS and between the TSS and 3′ terminal sequences that are conserved in a subset of carmoviruses and umbraviruses (see Fig. 9). Hairpins and pseudoknots are labeled as previously described (11, 30). Sequence in the apical loop of the 3′ terminal hairpin that engages in the conserved interaction with a hairpin just downstream from the −1 PRF site is shaded tan. End points for ΔBTE and ΔTSS deletions in the parental gRNA luciferase construct 5′38+3U (generating 5′38+ΔBTE and 5′38+ΔTSS) and parental sgRNA construct 5′69+3U (generating 5′69+ΔBTE and 5′69+ΔTSS) are indicated. (B) Relative luciferase activity of constructs assayed in protoplasts. Error bars denote standard deviation for three independent assays.

FIG 9

Conserved structures near the 3′ end of OPMV and some umbraviruses. Structure presented was confirmed by mutagenesis for PEMV2 (11). See legend to Fig. 8.

FIG 10

Comparative analysis of BTE structures. Proposed subclass A (three hairpins) includes BYDV, ETBTV, GRV, OPMV, Rose spring dwarf-associated virus (RSDaV), Soybean dwarf virus (SbDV), and TBTV. Subclass B (two hairpins) includes Beet black scorch virus (BBSV), IxYMaV2, OLV, PatMMoV, TNA-A, and TNV-D. Subclass C (five hairpins) includes Red clover necrotic mosaic virus (RCNMV). All BTEs contain the 17-nt signature sequence (green), and the orange sequence is conserved in subclasses A and B. The red sequence connecting SL2 and SL3 in subclass A and SL2 and SL3 in subclass B is conserved within the subclasses. Bases that differ from consensus sequences within these motifs are in black. Additional base pairings for BYDV BTE are indicated, and these pairings are conserved for multiple BYDV isolates (18, 68). Boxed residues are protected by eIF4G binding from chemical modification (18). Apical loop residues known or predicted to be involved in the long-distance interactions with the 5′ end are shaded light blue (10, 13, 56, 69, 70).