A series of in-frame deletion mutants was used to identify a domain within the 3a protein of cucumber mosaic virus (CMV) that is required for RNA-binding activity. Deletions in the 3a gene were generated by PCR and restriction digestion, and the resulting mutated 3a sequences were cloned either in pT7-7 or in pGEX-5X3 expression vectors. The mutated 3a proteins or fusions with glutathione S-transferase (GST) were expressed in E. coli, purified, and their nucleic acid-binding activities analysed by photochemical UV cross-linking assays using digoxigenin-UTP-labelled RNA probes. Comparative analyses of seven mutated 3a proteins obtained from inclusion bodies and eight GST fusion proteins revealed that there is an RNA-binding domain located between amino acids 174 and 233. This RNA-binding domain is able to bind single-stranded RNA out of the context of the complete 3a movement protein and is highly conserved within both subgroups of CMV.


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