The polypeptides of adenovirus. V. Young virions, structural intermediate between top components and aged virions

The turnip yellow mosaic virus (TYMV) RNA-replicase bound to the chloroplast fraction of a participate cell-free preparation isolated from TYMV-infected Chinese cabbage leaves catalyzes the synthesis of RNA of the viral type, “plus” RNA, in the absence of added “minus” RNA template. Addition of TYMV-RNA does not stimulate RNA synthesis. The complex formed by the replicase bound to “minus” RNA template can be removed from the chloroplast fraction by solubilization with a non-ionic detergent, Lubrol W, and separated from insoluble material by centrifugation. The resulting supernatant contains the template-bound replicase as well as a small proportion of template-free replicase molecules. Treatment of this preparation with a polyethylene glycol-dextran two-phase aqueous system at high salt concentration removes the replicase from the template and yields, after centrifugation, a polyethylene glycol upper phase containing the template-free replicase molecules (PEG-enzyme) and a lower dextran phase containing a small proportion of replicase molecules still bound to their template. The soluble PEG-enzyme is markedly stimulated by added RNA. With TYMV-RNA as the added RNA, the product synthesized by the enzyme is RNA complementary to TYMV-RNA, i.e., “minus” RNA, indicating the enzyme uses the viral RNA as template. The “minus”-RNA synthesized remains hydrogen bonded to the viral RNA template and forms a RNase resistant, double-stranded structure. Chromatography on cellulose CF 11 column reveals two different double-stranded structures, one, fully double-stranded, in which the synthesized “minus” RNA strand is hydrogen bonded to template RNA of equal length, and one partly double-stranded and partly single-stranded in which probably the template RNA is very much longer than the synthesized “minus” RNA. The PEG-enzyme seems to be sensitive to high ionic strength since its activity can be completely suppressed by various salts; removal of the salts restores the activity.