Cucumber mosaic virus-induced RNA polymerase: Partial purification and properties of the template-free enzyme

Abstract

The RNA-dependent RNA polymerase induced in cucumber cotyledons by infection with cucumber mosaic virus (CMV) has been purified one-hundred-fold and its properties investigated. RNA polymerase activity was followed during the purification steps using CMV RNA and poly(C) as templates. The initial enzyme extract (Step 1) was partitioned in a dextran-polyethylene glycol liquid polymer phase system and the enzyme in the polyethylene glycol phase (Step 2) was further purified by adsorption onto and step-wise elution from DEAE-Sephadex; the enzyme (Step 3) was now free of nucleic acid templates. Stepwise elution from columns of phosphocellulose (Step 4) and then singlestranded DNA-agarose (Step 5) gave enzyme which was one-hundred-fold purified for CMV RNA and poly(C) as templates. Enzyme activity for these two templates coupurified through all steps; however, salt gradient elution from DNA-agarose gave separation of the two activities but the poly(C) copying activity was very labile under the conditions used. The Step 5 enzyme was completely dependent on added template for activity. It copied poly(C) and was not specific for CMV RNA as it copied three other RNA species tested. It showed properties expected of an RNA-dependent RNA polymerase, e.g., sensitivity to added pyrophosphate and ribonuclease, low activity with either single- or double-stranded DNA as template, and insensitivity to actinomycin D, rifampicin, α-amanitin, deoxyribonuclease, and orthophosphate. The enzyme was unstable at 0° and −15°, but has been stored in liquid nitrogen for 5 mo without loss of activity. When the same purification procedure was carried out using healthy plant material, negligible RNA replicase activity was found at all stages of the purification. Further purification steps will need to overcome the instability of the enzyme which could be due to the protease activity in purified fractions and possibly also to a labile subunit structure of the enzyme. The possible structure of the enzyme has been discussed in relation to the molecular properties of the phage Qβ and f2 RNA replicases.