The 23-kDa protein coded by the 3 '-terminal gene of Citrus tristeza virusis an RNA-binding protein

The 23-kDa protein (p23), encoded by the 3'-proximal gene of the RNA of Cit rus tristeza virus (CTV), was overexpressed in Escherichia coil fused to th e maltose-binding protein and purified by affinity chromatography. Gel reta rdation and UV crosslinking assays demonstrated that. p23 has the ability t o cooperatively bind single-stranded RNA in a non-sequence-specific manner. Formation of the p23-RNA complex was dependent on the conformational state of p23 and on the presence of a basic region, but the complex was stable a t high salt concentrations, suggesting that interactions other than those b etween the negatively charged RNA and the basic region of p23 are involved. Competition assays showed that the affinity of p23 for single-stranded and double-stranded RNA was similar but considerably higher than for single-st randed and double-stranded DNA. By use of a series of artificially generate d mutants, the RNA-binding domain of p23 was mapped between positions 50-86 , a region containing several basic amino acids and a putative zinc-finger domain. Additional pea-derivatives lacking the conserved residues presumabl y involved in coordinating the tine ion showed RNA-binding activity, but wi th an apparent dissociation constant higher than the wild-type protein. The se conserved residues might confer binding specificity or increase binding stability in vivo. Within the Closteroviridae family. p23 is the only prote in characterized so far showing RNA-binding activity. (C) 2000 Academic Pre ss.