title: Purification of a host-encoded RNA-dependent RNA polymerase from cowpea mosaic virus-infected cowpea leaves.
abstract: A membrane-bound RNA-dependent RNA polymerase from Cowpea mosaic virus (CPMV)-infected cowpea leaves (Vigna unguiculata) has been purified 15,000-fold by DEAE-Sepharose CL-6B chromatography, affinity chromatography on poly(U)-Sepharose 4B, and glycerol gradient centrifugation. Particularly, poly(U)-Sepharose 4B chromatography was a very efficient purification step and, in addition, achieved the separation of a host-encoded terminal uridylyl transferase activity from the RNA polymerase activity. On glycerol gradient centrifugation, the polymerase activity sedimented as a homogeneous peak with a rate corresponding to a molecular weight of 120,000. Analysis of the protein composition of the gradient fractions revealed that only one polypeptide with a molecular weight of 130,000 cosedimented with the polymerase activity, suggesting a monomeric enzyme. The most purified enzyme preparations from CPMV infected leaves did not contain polypeptides encoded by RNA from CPMV B-component which presumably carries functions essential for CPMV replication. Using the same purification procedure, an RNA-dependent RNA polymerase has also been purified from mock-inoculated leaves, which appeared to be identical to the RNA polymerase from infected leaves. This host enzyme was strongly stimulated in cowpea leaves infected with CPMV. The role, if any, of the RNA-dependent RNA polymerase from cowpea leaves in CPMV-RNA replication is discussed in view of the recent evidence for virus encoded functions involved in CPMV multiplication.
authors.affiliation: Department of Molecular Biology, Agricultural University, De Dreijen 11, 6703 BC Wageningen, The Netherlands.
authors.forename: L
authors.initials: L
authors.lastname: Dorssers
authors.forename: P
authors.initials: P
authors.lastname: Zabel
authors.forename: J
authors.initials: J
authors.lastname: van der Meer
authors.forename: A
authors.initials: A
authors.lastname: van Kammen