Reconstituted and Cellular Viroid-Protein Complexes

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Abstract
Summary Complexes of potato spindle tuber viroid (PSTV) with plant nuclear proteins were investigated by in vitro reconstitution of purified viroids and proteins from nuclear extracts, on nitrocellulose filters and in solution. Well defined complexes were formed under both conditions. Competition for complex formation was achieved by greater than a 1000-fold excess of 5S RNA. Linear PSTV transcripts form complexes with protein that are different from those of circular PSTV. Natural viroid-protein complexes were crosslinked covalently by u.v. radiation in nuclei isolated from infected plant tissue. They have a sedimentation coefficient of approximately 10S, and exhibit defined bands in SDS-PAGE gels which are similar to those of viroid-protein complexes reconstituted in solution. A 43K protein was isolated from the cellular complexes by RNase digestion. Complexes crosslinked by u.v. radiation eluted together with non-crosslinked PSTV RNA in anion-exchange HPLC on Nucleogen columns. Using HPLC of non-irradiated samples a 43K protein eluted at a KCl concentration of 0.61 m, but only in samples from infected material. The relevance of the viroid-protein complexes for viroid function is discussed.