The conserved 5' apical hairpin stem loops of bamboo mosaic virus and its satellite RNA contribute to replication competence

Abstract

Satellite RNAs associated with Bamboo mosaic virus (satBaMVs) depend on BaMV for replication and encapsidation. Certain satBaMVs isolated from natural fields significantly interfere with BaMV replication. The 5' apical hairpin stem loop (AHSL) of satBaMV is the major determinant in interference with BaMV replication. In this study, by in vivo competition assay, we revealed that the sequence and structure of AHSL, along with specific nucleotides (C(60) and C(83)) required for interference with BaMV replication, are also involved in replication competition among satBaMV variants. Moreover, all of the 5' ends of natural BaMV isolates contain the similar AHSLs having conserved nucleotides (C(64) and C(86)) with those of interfering satBaMVs, suggesting their co-evolution. Mutational analyses revealed that C(86) was essential for BaMV replication, and that replacement of C(64) with U reduced replication efficiency. The non-interfering satBaMV interfered with BaMV replication with the BaMV-C64U mutant as helper. These findings suggest that two cytosines at the equivalent positions in the AHSLs of BaMV and satBaMV play a crucial role in replication competence. The downregulation level, which is dependent upon the molar ratio of interfering satBaMV to BaMV, implies that there is competition for limited replication machinery.

Figure 1.

Genetic maps and 5′ and 3′-UTR secondary structures of BaMV and satBaMVs. (A) Genome organization and ORFs of BaMV and satBaMV. ORFs and the molecular weight of encoded proteins are shown in boxes. Two subgenomic RNAs of BaMV are presented by lines. (B) The secondary structures of the 5′-UTRs of BaMV and satBaMVs. The AHSL structures of BaMV and satBaMV are boxed, and contain an apical loop and two internal loops (IL-1 and IL-2). Black circles indicate the start codon of BaMV ORF1. The common GAAA(A) repeats in the 5′-UTRs of BaMV and satBaMV are underlined. (C) The secondary structure of the BaMV 3′-UTR contains a cloverleaf-like ABC domain, a stem loop D (D domain) and a pseudoknot (E domain) (40,41). The secondary structure of the satBaMV 3′-UTR is similar to that of BaMV except for the lack of A and E domains (19).

Figure 2.

Identification of progeny satBaMV in N. benthamiana protoplasts co-infected with various satBaMVs. (A) Schematic representation of the secondary structure of 5′-UTR of BSF4 (green), BSL6 (red), and two satBaMV mutants, BSF4-1 and BSL6-1, in which the entire AHSLs were interchanged between BSF4 and BSL6. Sequences and AHSL structures of BSF4 and BSL6 and their derived mutants, BSF4-5 and BSL6-11 were shown. (B) The BaMV and satBaMV transcripts for inoculation are indicated above each lane. Lane ‘−’: no satBaMV RNA added. Lane ‘+’: satBaMV RNA was added as indicated. At 24-h post-inoculation (hpi), total RNAs isolated 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- (top) or satBaMV-specific probes (below). Positions of the BaMV genomic RNA (6.4 kb), 2.0 and 1.0 kb subgenomic RNA, and satRNA are indicated at the left. EtBr: ethidium bromide staining of the gel prior to the blotting shows approximately equal loading as revealed by ribosomal RNA abundance in each lane. The numbers of cloned cDNA of progeny satBaMV are indicated in the bottom panel.

Figure 3.

The replication efficiency of BSL6 was higher than that of BSF4 under the support of BaMV replicase in N. benthamiana protoplasts. (A) Northern blot analyses of satBaMV replication under the help of BaMV or BaMV replicase in protoplasts of N. benthamiana. The plasmids used for transfection were shown in the upper panel. Plasmids pCB, pCBSF4 and pCBSL6 were the infectious cDNA clones of BaMV-S, BSF4 and BSL6, respectively. Plasmids pBaORF1 and pBaORF1dGDD were the expression clones of wild-type and GDD motif deletion mutant of BaMV ORF1, respectively. (B) The numbers of cloned cDNA of progeny satBaMV in mix-infected population. (C) The accumulation levels of satBaMV RNAs under the support of different dose replicase in N. benthamiana protoplasts. (D) The bar chart shows the relative accumulation fold change of satBaMV RNAs in different doses of BaMV replicase. The input satRNA level in the absence of replicase was set as 1. The data are the average of three independent experiments.

Figure 4.

The predicted AHSL structures of natural BaMV isolates. (A) The 5′ conserved AHSL structures of BaMV-S and satBaMV variants. (B) The secondary structures of BaMV 5′ ends were predicted by Mfold program (56). BaMV variants with or without satellite RNA are shown in black and gray, respectively. Asterisk represents BaMV variants containing interfering satBaMV.

Figure 5.

Specific nucleotides in the AHSL is involved in BaMV replication. (A) Sequences and AHSL structures of BaMV-S and its derived mutants, BaMV-C64U and BaMV-C86U. (B) Accumulation of BaMV RNAs in inoculated protoplasts at 24 hpi by northern blotting as described in Figure 2.

Figure 6.

Non-interfering satBaMV interferes with the replication of the BaMV-C64U mutant. (A) Local lesions on C. quinoa leaves inoculated with pCB or pCB-C64U alone or co-inoculated with pCBSF4 or pCBSL6 at 10 dpi. (B) Northern blot of total RNA extracted from plants inoculated with infectious clones of BaMV and satBaMV at 10 dpi and hybridized with a probe specific to detect BaMV and satBaMV RNA. The different exposure times of films are indicated. To detect satBaMV BSL6 RNAs, the film was processed for 16 h.

Figure 7.

Dose-dependent interference with BaMV replication by satellite RNA. (A) Northern blot analyses of the accumulation of BaMV and satBaMV RNAs in N. benthamiana protoplasts inoculated with 1 µg of BaMV-S RNA alone or co-inoculated with 1 µg of BSF4 or different doses of BSL6 satBaMV transcripts. Northern blot analyses of the accumulation of BaMV and satBaMV RNAs in N. benthamiana protoplasts as described in Figure 2. (B) The bar chart shows the relative accumulation of BaMV RNAs in different molar ratios of satBaMV/BaMV. The data are the average of three independent experiments.