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Properties of a Viroid-replicating Complex Solubilized from Nuclei

R. F. Rivera-Bustamante

Department of Plant Pathology, University of California, Riverside, California 92521-0122, U.S.A.

An organelle-free fraction with the ability to synthesize citrus exocortis viroid (CEV) RNA was prepared from nuclei-rich samples taken from CEV-infected Gynura aurantiaca D.C. leaf tissue. This extraction was accomplished in the presence of the detergent sarkosyl. Characterization of the viroid synthetic reaction demonstrated that the solubilized complex retained the properties displayed by intact nuclei. These include optima of 1 mM-MnCl₂, 10 mM-MgCl₂ and 25 mM-(NH₄)₂SO₄, as well as inhibition by -amanitin (90% at 4 µg/ml) and by the chelators o-phenanthroline (78% at 5 mM) and hydroxyquinoline (45% at 5 mM). Nucleic acid-binding agents such as ethidium bromide and actinomycin D showed a low, non-specific inhibition at relatively high concentrations. Sucrose gradient sedimentation analysis of the sarkosyl supernatant (SSN) components before and after the RNA synthesis showed that the bulk of the progeny remained associated with the viroid complex which sedimented faster than phenol-treated viroid RNA. A high percentage of the CEV RNA molecules migrated in electrophoresis as the circular form. This suggests that all elements necessary for the synthesis of viroid RNA and processing to circular structures are present in the SSN. This endorses the potential of these subnuclear preparations for the study of the processes involved in viroid replication.

Keywords: citrus exocortis viroid, RNA polymerase, viroid replication

Present address: Centro de Investigaciones y Estudios Avanzados-IPN, Unidad Irapuato, Apdo. Postal 629, Irapuato, Gto. Mexico.

Received 29 March 1989; accepted 20 June 1989.