Crucial role of the 5' conserved structure of bamboo mosaic virus satellite RNA in downregulation of helper viral RNA replication

Abstract

Satellite RNA of Bamboo mosaic virus (satBaMV), a single-stranded mRNA type satellite encoding a protein of 20 kDa (P20), depends on the helper BaMV for replication and encapsidation. Two satBaMV isolates, BSF4 and BSL6, exhibit distinctly differential phenotypes in Nicotiana benthamiana plants when coinoculated with BaMV RNA. BSL6 significantly reduces BaMV RNA replication and suppresses the BaMV-induced symptoms, whereas BSF4 does not. By studies with chimeric satBaMVs generated by exchanging the components between BSF4 and BSL6, the genetic determinants responsible for the downregulation of BaMV replication and symptom expression were mapped at the 5' untranslated region (UTR) of BSL6. The 5' UTR of BSL6 alone is sufficient to diminish BaMV RNA replication when the 5' UTR is inserted in cis into the BaMV expression vector or when coinoculation with mutants that block the synthesis of P20 protein takes place. Further, the 5' UTR of natural satBaMV isolates contains one hypervariable (HV) region which folds into a conserved apical hairpin stem-loop (AHSL) structure (W. B. Yeh, Y. H. Hsu, H. C. Chen, and N. S. Lin, Virology 330:105-115, 2004). Interchanges of AHSL segment of HV regions between BSF4 and BSL6 led to the ability of chimeric satBaMV to interfere with BaMV replication and symptom expression. The conserved secondary structure within the HV region is a potent determinant of the downregulation of helper virus replication.

FIG. 1.

Schematic diagram of chimeric cDNAs of pBSF4 and pBSL6 and their effects on BaMV replication and BaMV-induced symptoms. (A) Chimeric cDNAs were made up of parts from either pBSF4 (red) or pBSL6 (green) cDNA using the restriction sites BstXI and EcoNI. Open boxes represent the UTRs of satBaMVs. (B) Protoplasts of N. benthamiana were inoculated with BaMV-S RNA alone (lane 1) or coinoculated with the following satBaMV variants: BSF4 (lane 2), BSL6 (lane 3), BS446 (lane 4), BS464 (lane 5), BS466 (lane 6), BS644 (lane 7), BS646 (lane 8), or BS664 (lane 9). At 24 hpi, total RNAs extracted from 3 × 10⁴ protoplasts were glyoxylated, electrophoresed in a 1% agarose gel, and transferred to a nylon membrane. Blots were hybridized with ³²P-labeled BaMV-specific L (top panel) (21) and satBaMV-specific S (middle panel) (25) probes. Additionally, a slot blot of total RNA from inoculated protoplasts was hybridized with a BaMV-specific probe for minus-strand RNA (18). Positions of BaMV genomic RNA (6.4 kb), subgenomic RNAs (2.0 and 1.0 kb), and satellite (7) are indicated on the left. (C) Each bar represents average accumulation levels of genomic RNA and two subgenomic RNAs obtained from six independent experiments with standard deviation. Numbers along the x axis represent lane numbers shown in panel B. (D and E) Effects of chimeric satBaMVs on symptom formation. Relative numbers of N. benthamiana plants showing symptoms after 12 and 24 dpi (D) and appearance of symptoms at 24 dpi (E).

FIG. 2.

Schematic diagram of satBaMV mutants derived from pBSL6 (A) and their effects on BaMV replication (B and C). (A) Mutants of BSL6 satBaMV in which the P20 protein gene was blocked. The first ATG of the P20 protein gene was changed to TAG in mutant BS23, whereas the additional C was inserted after the first ATGG, causing the frameshift in mutant BS24. Open box, noncoding sequence; filled box, coding region of satBaMV; hatched box, altered reading frame of the coding region. (B) Northern blot analysis of BaMV and satBaMV RNA accumulation in inoculated protoplasts, as described in the legend to Fig. 1. (C) Each bar represents relative average accumulation levels of genomic RNA and two subgenomic RNAs from six independent experiments with standard deviation.

FIG. 3.

Replication and symptoms of BaMV expression vector carrying 5′ UTR of satBaMV variants in C. quinoa plants. (A) Schematic representation of the pBV, pBVF4-5′, and pBVL6-5′ genomes. ORFs are denoted by open boxes, and the solid green circle denotes insertion of the duplicated subgenomic promoter. A small solid rectangular box represents the 5′ UTR derived from BSF4 or BSL6 satBaMV, respectively. (B and C) Northern blot of total RNA extracted from plants inoculated with these three constructs and hybridized with a probe specific to detect plus- (B) and minus- (C) strand BaMV RNA. The arrow represents the accumulation of additional synthesized subgenomic RNA under the control of a duplicated subgenomic promoter. (D) Development of local lesions on C. quinoa leaves inoculated with pBV, pBVF4-5′, or pBVL6-5′ at 10 dpi. (E) Each bar represents relative average accumulation of genomic RNA obtained from three independent experiments with standard deviation.

FIG. 4.

Effect of natural satBaMV isolates on the accumulation of BaMV in N. benthamiana protoplasts. (A) AHSL structures of HV regions from different natural satBaMV isolates, predicted by the mfold program (59). Nucleotides in the HV regions different from those of BSL6 satBaMV are shown in red. (B and C) Northern analysis of BaMV and satBaMV RNA accumulation in N. benthamiana protoplasts, as described in the legend to Fig. 1. (D) Total RNA in ethidium bromide-stained agarose gel showing equal loading on each lane.

FIG. 5.

Secondary structure of 5′ UTR of satBaMV variants and their effect on BaMV replication and symptom expression. (A) Schematic representation of the secondary structure of 5′ UTR of pBSF4 (green), pBSL6 (red), and two satBaMV mutants, BSF20 and BSF21, in which the entire HV regions were interchanged between BSF4 and BSL6. (B and C) Accumulation of BaMV and satBaMV RNAs in the inoculated protoplasts of N. benthamiana at 24 hpi, as shown at the top of each lane. (D) Total RNA as loading controls. (E) Symptom formation in the N. benthamiana plants inoculated with BaMV or coinoculated with satBaMV. Photos were taken 24 days after inoculation.