Rose spring dwarf-associated virus has RNA structural and gene-expression features like those of Barley yellow dwarf virus

Abstract

We determined the complete nucleotide sequence of the Rose spring dwarf-associated virus (RSDaV) genomic RNA (GenBank accession no. EU024678) and compared its predicted RNA structural characteristics affecting gene expression. A cDNA library was derived from RSDaV double-stranded RNAs (dsRNAs) purified from infected tissue. Nucleotide sequence analysis of the cloned cDNAs, plus for clones generated by 5'- and 3'-RACE showed the RSDaV genomic RNA to be 5808 nucleotides. The genomic RNA contains five major open reading frames (ORFs), and three small ORFs in the 3'-terminal 800 nucleotides, typical for viruses of genus Luteovirus in the family Luteoviridae. Northern blot hybridization analysis revealed the genomic RNA and two prominent subgenomic RNAs of approximately 3 kb and 1 kb. Putative 5' ends of the sgRNAs were predicted by identification of conserved sequences and secondary structures which resembled the Barley yellow dwarf virus (BYDV) genomic RNA 5' end and subgenomic RNA promoter sequences. Secondary structures of the BYDV-like ribosomal frameshift elements and cap-independent translation elements, including long-distance base pairing spanning four kb were identified. These contain similarities but also informative differences with the BYDV structures, including a strikingly different structure predicted for the 3' cap-independent translation element. These analyses of the RSDaV genomic RNA show more complexity for the RNA structural elements for members of the Luteoviridae.

Figure 1

Schematic representation of the RSDaV genome organization. Numbered boxes represent open reading frames, with functions of RNA-dependent RNA polymerase (pol) and coat protein (CP) indicated. Gray bars above the genome map represent the positions of the indicated RNA probes used in the northern blot hybridizations (Fig. 2). Bold black lines indicate genomic (gRNA) and subgenomic RNAs (sgRNA), with 5′ ends of sgRNAs predicted as discussed in text. Black boxes on genome indicate predicted cis-acting structures (left to right): BTE-complementary loop of genomic RNA (gBCL, Fig. 3), shifty heptanucleotide and bulged stem-loop at frameshift site (FS, Fig. 4), BTE-complementary loop at predicted 5′ end of sgRNA1 (sg1BCL, Fig. 3), BYDV-like cap-independent translation element (BTE, Fig. 5) and long-distance frameshift element that interacts with the frameshift site (LDFE, Fig. 4).

Figure 2

Northern hybridization analysis of double-stranded RSDaV RNAs from infected plant tissue. Three probes specific for various regions of the genome were derived from pRSDaV5′ (complementary to bases 1938–2574), lane 1; pRSDaVCP (bases 3201–3694), lane 2; and pRSDaV3′ (complementary to bases 5075–5799), lane 3. Mobilities of genomic RNA (gRNA) (~6kb), subgenomic RNA1 (sgRNA1) (~3kb) and subgenomic RNA2 (sgRNA2) (~1kb) are indicated. Also see Fig. 1 for probe positions. Sizes of the RNAs were estimated from stained RNA standards in the same gel (not shown).

Figure 3

Predicted 5′ ends of RSDaV sgRNA1 and sgRNA2. A. Alignment of 5′ end of the RSDaV genomic RNA with sequences located at sites consistent with the 5′ ends of sgRNAs. The conserved sequence at the 5′ ends of BYDV genomic and sgRNAs 1 and 2 is shown below the RSDaV sequence. Bases in gray do not fit the consensus. There are two sites, nts 2815, 2817 that fit consensus start sites for sgRNA1. B. Predicted secondary structures around the known (genomic RNA) and predicted (sgRNAs) 5′ ends of RSDaV RNAs. Bent arrows, bases in bold indicate the predicted 5′ ends of sgRNAs. Bases in loops, in bold italic are predicted to base pair with the 3′ BTE (see Fig. 5).

Figure 4

Predicted secondary structures of frameshift sites of RSDaV and BYDV-PAV RNAs. Predicted long-distance base pairing between a stem-loop 4 kb downstream and a bulged loop adjacent to the frameshift site is indicated at left. Similar long-distance stem-loop-bulge-loop interaction is known to be required for BYDV-PAV frameshifting, (right). The shifty heptanucleotide is in italics. Amino acid sequences of the end of ORF 1 and the overlapping portion of ORF 2 are shown below the shifty site.

Figure 5

Predicted secondary structures of the RSDaV BTE compared with the known structure of the BYDV BTE (boxed in B). The three most stable suboptimal structures of the RSDaV sequence are shown. The 17 nt consensus sequence in all BTEs is in green, and the bases that form a kissing interaction with a stem-loop in the 5′ UTR of genomic RNA and sgRNA1 are in blue bold italics. These two features allow identification of SL-I and SL-III respectively, as in the BYDV BTE. The stem-loop adjacent to SL-III (SL-II) and the helical region upstream of SL-I (S-IV) are present in all 31 MFOLD-predicted structures that have a stability (ΔG) within 20% of the most stable.

Figure 6

Phylogenetic relationship between the RSDaV complete genome nucleotide sequence compared with other members of the family Luteoviridae. The tree was constructed by using the Minimum evolution algorithm provided in the MEGA 2 software package (Kumar et al., 2001). The numbers at each node indicate the bootstrap values resulting from 1,000 replicates. The scale bar represents the number of residue substitution per site. Barley yellow dwarf virus-GAV (BYDV-GAV), Barley yellow dwarf virus-MAV (BYDV-MAV), Barley yellow dwarf virus-PAS (BYDV-PAS), Barley yellow dwarf virus-PAV (BYDV-PAV), Bean leafroll virus (BLRV), Beet chlorosis virus (BChV), Beet mild yellowing virus (BMYV), Beet western yellows virus (BWYV), Cereal yellow dwarf virus-RPS (CYDV-RPS), Cereal yellow dwarf virus-RPV (CYDV-RPV), Cucurbit aphid-borne yellows virus (CABYV), Pea enation mosaic virus-1 (PEMV-1), Potato leafroll virus (PLRV), Soybean dwarf virus (SbDV), Sugarcane yellow leaf virus (ScYLV) and Turnip yellows virus (TuYV).