RNA polymerase from tobacco necrosis virus-infected and uninfected tobacco: II. Properties of the bound and soluble polymerases and the nature of their products

Abstract

RNA polymerase activities in extracts of healthy and tobacco necrosis virus (TNV)-infected tobacco plants were investigated. Considerable ”bound“ enzyme activity, associated with endogenous viral RNA template, was found in the 30,000 g pellet obtained from TNV-infected plants. This activity was usually not increased by addition of RNA. In the corresponding fraction from healthy plants a much lower activity was detected which was increased when RNA was added. The conditions favoring the enzyme activity and its general properties were investigated. The RNA synthesized by this enzyme on the endogenous template, be it in the particulate state or solubilized and ammonium sulfate-fractionated, was about 90% double-stranded and of high molecular weight. It corresponds to a viral plus strand. After melting, this RNA comigrated with TNV-RNA. Thus it appears that the membrane-bound enzyme in TNV-infected plants represents a TNV replicase which is able to synthesize or finish full-length TNV-RNA on an endogenous template, the minus strand of TNV-RNA; this enzyme-template complex can be solubilized and fractionated without losing this ability. Nucleotide polymerizing activity was also detected in the 100,000 g supernatant of healthy tobacco plants and this activity was significantly increased upon TNV infection. This activity which resembled in some respects that reported by Duda et al. (1973) was greatly stimulated by added RNA. The products synthesized by this “soluble” enzyme extracted from healthy and infected plants were of similarly low molecular weight; they consisted of both single- and doubles-tranded RNAs and behaved partly as expected for fragments of minus-strand viral RNA.