Identification and characterization of RNA-binding activity in the ORF1-encoded replicase protein of Pelargonium flower break virus

• Published in: Journal of general virology 2010-12, Vol.91 (Pt 12), p.3075-3084
• Main Authors: MARTINEZ-TURINO, Sandra, HERNANDEZ, Carmen
• Format: Article
• Language: English
• Subjects: Binding Sites; Biological and medical sciences; Carmovirus; Carmovirus - enzymology; Carmovirus - physiology; Cloning, Molecular; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Microbiology; Miscellaneous; Pelargonium - virology; Pelargonium flower break virus; Phytopathology. Animal pests. Plant and forest protection; Plant viruses and viroids; Protein Binding; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; RNA Replicase - chemistry; RNA Replicase - genetics; RNA Replicase - metabolism; RNA, Viral - metabolism; Sequence Deletion; Tombusviridae; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism; Virology; Virus Replication
• ISSN: 0022-1317; 1465-2099; 1465-2099
• DOI: 10.1099/vir.0.023093-0

Pelargonium flower break virus (PFBV) belongs to the genus Carmovirus (family Tombusviridae) 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 Escherichia coli and tested its ability to bind RNA in vitro. 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.