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RNA-binding activities of cocksfoot mottle sobemovirus proteins
1Gene Technology Center, Institute of Chemical Physics and Biophysics, Tallinn Technical University, Akadeemia tee 23, EE12618, Tallinn, Estonia.
Abstract:
Cocksfoot mottle virus (CfMV) has a positive-sense ssRNA genome containing four open reading frames (ORFs). ORF1 encoded protein (P1) is the putative movement protein; the product of ORF2a (P2a) contains VPg and the motifs characteristic of serine proteases. P2b, encoded by ORF2b, is the putative RNA-dependent RNA polymerase. P3, the coat protein, is encoded by ORF3. CfMV P1, P2a, P2b, and P3, containing a six histidine tag at the amino terminus, were expressed in Escherichia coli, purified and their RNA-binding activities were analysed. The northwestern blot assay showed that His-tagged P1, P2a, P2b, and P3 were able to interact with ssRNA transcripts in a sequence-nonspecific manner. The filter-binding assay confirmed the ssRNA-binding capacity of recombinant P1, P2a, and P3. The RNA-binding activities of His-tagged P3 and native coat protein were similar. P1 and P2a binding to ssRNA decreased markedly by increasing NaCl concentrations. In contrast, P3 had the RNA-binding optimum at 100-200 mM NaCl. We discuss the possible amino acid motifs involved in the RNA-binding of CfMV proteins.
Insights
Cocksfoot mottle virus (CfMV) proteins P1, P2a, P2b, and P3 bind to single-stranded RNA (ssRNA) non-specifically. Their binding affinities vary with salt concentration, suggesting distinct roles in viral replication.
Area of Science:
- Virology
- Molecular Biology
- Plant Pathology
Background:
- Cocksfoot mottle virus (CfMV) possesses a positive-sense single-stranded RNA (ssRNA) genome.
- Four open reading frames (ORFs) encode key viral proteins: movement protein (P1), VPg/serine protease (P2a), RNA-dependent RNA polymerase (P2b), and coat protein (P3).
Purpose of the Study:
- To investigate the RNA-binding activities of CfMV proteins P1, P2a, P2b, and P3.
- To characterize the sequence-nonspecific RNA-binding properties and salt concentration optima for these proteins.
Main Methods:
- Expression and purification of His-tagged CfMV proteins (P1, P2a, P2b, P3) in Escherichia coli.
- Northwestern blot assays to detect ssRNA-protein interactions.
- Filter-binding assays to quantify ssRNA-binding capacity.
- Analysis of RNA-binding activity under varying NaCl concentrations.
Main Results:
- All tested CfMV proteins (P1, P2a, P2b, P3) exhibited sequence-nonspecific binding to ssRNA.
- Recombinant P1, P2a, and P3 demonstrated confirmed ssRNA-binding capacity.
- RNA-binding activity of P1 and P2a decreased significantly with increasing NaCl concentrations.
- P3 displayed optimal RNA-binding at 100-200 mM NaCl, similar to the native coat protein.
Conclusions:
- CfMV proteins P1, P2a, P2b, and P3 possess inherent ssRNA-binding capabilities.
- Differential salt concentration optima suggest distinct functional roles for these proteins in the viral life cycle.
- Further investigation into specific amino acid motifs involved in RNA binding is warranted.