Inoculation with RNAs 1 and 2 of Cucumber Mosaic Virus Induces Viral RNA Replicase Activity in Tobacco Mesophyll Protoplasts

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Abstract
The activity of membrane-bound RNA-dependent RNA polymerase in tobacco mesophyll protoplasts was measured and in vitro products were characterized after inoculation with cucumber mosaic virus (CMV) or with different combinations of the four virus RNAs and satellite RNA. Activity increased rapidly 4 to 6 h after inoculation with virus particles. When the inoculum contained RNA 1, RNA 2 and RNA 3, most of the in vitro products of the enzyme were full-length positive-sense RNAs (RNAs 1, 2, 3 and 4) that migrated as dsRNA; inoculation of RNA 1 or RNA 2 alone did not increase RNA polymerase activity and no virus RNA was produced. After inoculation with a mixture of RNA 1 and RNA 2, however, the enzyme activity increased and full-length positive-sense RNA 1 and RNA 2 were synthesized, and we speculate that the induced enzyme is CMV RNA replicase as a complex comprising the translation products of RNA 1 and RNA 2, membrane components of the host cell and CMV RNA templates. Protoplasts inoculated with RNA 1, RNA 2 and RNA 4 did not synthesize RNA 4 which suggests that positive-sense RNA 4 does not serve as a template for the replicase complex. Satellite RNA was synthesized in protoplasts inoculated with RNA 1, RNA 2 and satellite RNA which suggests that satellite RNA is replicated by the replicase complex.