The Potyvirus helper component-proteinase (HC-Pro) binds nonspecifically to single-stranded nucleic acids with a preference for RNA To delineate the regions of the protein responsible for RNA binding, deletions were introduced into the full-length Potato potyvirus Y HC-Pro gene carried by an Escherichia coil expression vector. The corresponding proteins were expressed as fusions with the maltose-binding protein, purified, and assayed for their RNA-binding capacity. The results obtained by UV cross-linking and Northwestern blot assays demonstrated that the N- and C-terminal regions of HC-Pro are dispensable for RNA binding. They also revealed the presence of two independent RNA-binding domains (designated A and B) located in the central part of HC-Pro. Domain B appears to contain a ribonucleoprotein (RNP) motif typical of a large family of RNA-binding proteins involved in several cellular processes. The possibility that domain a consists of an RNP domain is discussed and suggests that HC-Pro could constitute the first example of a plant viral protein belonging to the RNP-containing family of proteins. (C) 2000 Academic Press.2681104111