1887

Abstract

(PFBV) belongs to the genus (family ) and, as with the remaining members of the group, possesses a monopartite genome of single-stranded, positive-sense RNA that contains five ORFs. The two 5′-proximal ORFs (ORFs 1 and 2) encode two polypeptides of 27 and 86 kDa (p27 and p86), respectively, that show homology with replication proteins. The p27 does not present any motif to explain its presumed involvement in replication, while p86 has the motifs conserved in RNA-dependent RNA polymerases. In this work, we have confirmed the necessity of p27 and p86 for PFBV replication. To gain insights into the function(s) of p27, we have expressed and purified the protein from and tested its ability to bind RNA . The results have shown that p27 is able to bind ssRNA with high affinity and in a cooperative fashion and that it is also capable of binding other types of nucleic acids, though to a lesser extent. Additionally, competition experiments suggest that p27 has a preference for PFBV-derived ssRNAs. Using truncated forms of p27, it can be concluded that several regions of the protein contribute to its RNA-binding properties and that this contribution is additive. This study is the first to show nucleic acid-binding ability of the ORF1 product of a carmovirus and the data obtained suggest that this product plays an essential role in selection and recruitment of viral RNA replication templates.

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2010-12-01
2019-10-15
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Control EMSA with distinct amounts of BSA. The P-labelled 3′-PFBV ssRNA probe (at 33 pM) was incubated with no protein (lane 1) or with increasing concentrations (50, 75 and 100 nM) of BSA (lanes 2–4, respectively). The unbound, free RNA probe is marked on the right; no shifted (bound) RNA complexes were detected.

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Illustrative EMSA with distinct amounts of recombinant proteins. The P-labelled 3′-PFBV ssRNA probe (at 33 pM) was incubated with no protein (lane 1) or with two concentrations (100 nM in lanes 2, 4, 6, 8, 10, 12 and 14 and 0.1 µM in lanes 3, 5, 7, 9, 11, 13 and 15) of His-tagged p27 and derivatives. The unbound, free RNA probe and the shifted (bound) RNA complexes are marked on the right.

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RNA-binding predictions for PFBV p27 and related carmoviral proteins using the program RNABindR. Residues predicted to bind RNA (in 'optimal prediction mode') are indicated by grey boxes. Numbers denote positions of the amino acid residues in the corresponding full-length proteins. Sequences used for comparison were retrieved from the sequence databases and correspond to the following viruses: PFBV ( flower break virus; GenBank accession no. AJ514833), SCV (saguaro cactus virus; NC_001780), CbMV ( mottle virus; GQ244431), NLVCV ( vein-clearing virus; EF207438), CarMV (carnation mottle virus; X02986), AnFBV ( flower break virus; DQ219415), TCV (turnip crinkle virus; M22445), CCFV (cardamine chlorotic fleck virus; L16015), HCRSV (hibiscus chlorotic ringspot virus; X86448), MNSV (melon necrotic spot virus; M29671), PSNV (pea stem necrosis virus; NC_004995), JINRV (Japanese iris necrotic ring virus; NC_002187), CPMoV (cowpea mottle virus; U20976) and GaMV ( mosaic virus; Y13463).

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