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. 2006 Apr;80(8):3781-91.
doi: 10.1128/JVI.80.8.3781-3791.2006.

Cap-independent translation mechanism of red clover necrotic mosaic virus RNA2 differs from that of RNA1 and is linked to RNA replication

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Cap-independent translation mechanism of red clover necrotic mosaic virus RNA2 differs from that of RNA1 and is linked to RNA replication

Hiroyuki Mizumoto et al. J Virol. 2006 Apr.

Abstract

The genome of Red clover necrotic mosaic virus (RCNMV) in the genus Dianthovirus is divided into two RNA molecules of RNA1 and RNA2, which have no cap structure at the 5' end and no poly(A) tail at the 3' end. The 3' untranslated region (3' UTR) of RCNMV RNA1 contains an essential RNA element (3'TE-DR1), which is required for cap-independent translation. In this study, we investigated a cap-independent translational mechanism of RNA2 using a firefly luciferase (Luc) gene expression assay system in cowpea protoplasts and a cell-free lysate (BYL) prepared from evacuolated tobacco BY2 protoplasts. We were unable to detect cis-acting RNA sequences in RNA2 that can replace the function of a cap structure, such as the 3'TE-DR1 of RNA1. However, the uncapped reporter RNA2, RNA2-Luc, in which the Luc open reading frame (ORF) was inserted between the 5' UTR and the movement protein ORF, was effectively translated in the presence of p27 and p88 in protoplasts in which RNA2-Luc was replicated. Time course experiments in protoplasts showed that the translational activity of RNA2-Luc did not reflect the amount of RNA2. Mutations in cis-acting RNA replication elements of RNA2 abolished the cap-independent translational activity of RNA2-Luc, suggesting that the translational activity of RNA2-Luc is coupled to RNA replication. Our results show that the translational mechanism differs between two segmented genomic RNAs of RCNMV. We present a model in which only RNA2 that is generated de novo through the viral RNA replication machinery functions as mRNA for translation.

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Figures

FIG. 1.
FIG. 1.
(A) Schematic diagram of RCNMV RNA1 and RNA2, reporter Luc mRNAs of RNA1 and RNA2 (R1-5′-XbS, RNA2-Luc, and R2-UTR-Luc), and Luc mRNA that contains vector-derived sequence (thin line) and poly(A) tail of 60 nt (pA60). The RCNMV genome is shown as a thick line with the protein-coding regions depicted as boxes. The dotted box indicates an untranslatable MP ORF. The bent line indicates the deleted region. Numbers indicate the positions of nucleotides. (B) Translational activities of capped and uncapped Luc RNA transcripts in cowpea protoplasts. Luc RNA was cotransfected with R-Luc mRNA that was used as an internal control. Transfected protoplasts were incubated at 17°C for 6 h. Luc activities are expressed as percentages of uncapped R1-5′-XbS activity. Error bars show the standard errors, and the mean values are given. Assays were performed at least three times.
FIG. 2.
FIG. 2.
Translational activity of uncapped Luc RNA transcripts in the presence or absence of RNA1. For other conditions, refer to the legend to Fig. 1B.
FIG. 3.
FIG. 3.
(A) Schematic diagrams of DNA-mediated expression plasmids. The pentagon labeled 35S represents the 35S promoter of CaMV. The boxes labeled Ribo and Ter represent the ribozyme of a sTRSV and the terminator of CaMV, respectively. The RCNMV genome is shown as a thick line with the protein-coding regions indicated by boxes. The four nucleotides used to generate the p27 and CP frameshift mutants are indicated by the ▿ symbol at the insertion sites. (B) Translational activity of RNA2-Luc cotransfected with DNA-mediated expression plasmids in cowpea protoplasts. Luc activities for RNA2-Luc are presented as Luc/R-Luc ratios. Transfected protoplasts were incubated at 17°C for 24 h. For other conditions, refer to the legend to Fig. 1B. (C) Accumulation of RNA1, sgRNA, and RNA2-Luc in cowpea protoplasts. Total RNA was extracted from protoplasts 24 h after transfection, separated by gel electrophoresis, and blotted onto membranes. The membranes were then probed with DIG-labeled RNA specific for RCNMV RNA1 (upper) and Luc ORF (lower).
FIG. 4.
FIG. 4.
(A) Schematic diagrams of deletion or nucleotide substitution mutants of RNA2-Luc. The genome organization of RCNMV RNA2 is shown at the top. The bent line indicates the deleted region. The position of nucleotide substitution is indicated by the ▿ symbol. The dotted box indicates untranslatable MP ORF. (B) Translational activity of RNA2-Luc mutants in cowpea protoplasts. Transfected protoplasts were incubated at 17°C for 24 h. Luc activities for RNA2-Luc mutants are presented as Luc/R-Luc ratios. For other conditions, refer to the legend to Fig. 1B. (C) Accumulation of positive-strand RNA1 and sgRNA (top), positive-strand RNA2-Luc (middle), and negative-strand RNA2-Luc (bottom) in cowpea protoplasts. Total RNA was extracted from protoplasts 24 h after transfection. The membranes were then probed with DIG-labeled RNA specific for RCNMV RNA1 (top), Luc ORF (middle), and negative-strand RCNMV RNA2 (bottom). For other conditions, refer to the legend to Fig. 3C.
FIG. 5.
FIG. 5.
(A) Temporal changes in the accumulation pattern of Luc RNA. Total RNA was extracted from protoplasts immediately after transfection (0 h) and 2, 4, 6, 8, and 24 h after transfection, separated by gel electrophoresis, and blotted onto membranes. The membranes were then probed with DIG-labeled RNA specific for the Luc ORF. Relative values for the accumulation of Luc RNA (0 h in inoculation of R1-5′-XbS was defined as 100%) were calculated from four independent experiments, and a representative pattern on a Northern blot is shown below. Error bars show the standard errors, and mean values are given. (B) Temporal changes in the accumulation pattern of negative-strand RNA2-Luc. (C) Temporal changes in the translational activity of Luc RNA. Relative values for Luc activity (24 h in inoculation of R1-5′-XbS alone was defined as 1) were calculated from four independent experiments. Error bars show the standard errors.
FIG. 6.
FIG. 6.
(A) Translational activity of uncapped Luc mRNAs in cell-free lysate (BYL) prepared from evacuolated tobacco BY2 protoplasts (26). Luc mRNAs (5 fmol) were translated at 17°C for 2.5 h. Luc activities were expressed as percentages of uncapped R1-5′-XbS activity; error bars show the standard errors from four independent experiments. (B) Western blot analysis of viral proteins in BYL using RCNMV p27 antibody. RNA2-Luc (125 fmol) was incubated together with RNA1 mutants (190 fmol) or a mixture of RNA1-p27 and RNA1-p88 (95 fmol each) at 17°C for 2.5 h.

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