Polyhedral cytoplasmic deoxyribovirus virions contain a DNA-dependent RNA polymerase which catalyzes the incorporation of ribonucleotides into an acid-precipitable product. Treatment of virions with sodium deoxycholate and dithiothreitol resulted in the formation of subviral particles which could be separated from virions by rate zonal centrifugation in sucrose gradients. Subviral particles were RNA polymerase-positive and more active per unit mass of protein than virions. In vitro enzyme activity associated with subviral particles required addition of ribonucleotides, Mg2+, and exogenous denatured DNA template. Optimal enzyme activity occurred over a broad pH (7.2 to 8.8) and Mg2+ concentration (2 to 10 limol) range. The specific activity of the RNA polymerase was maximal at 37 C. Addition of DNase or actinomycin D to the reaction mixture reduced the incorporation of [3H ]UMP into an acid-precipitable product. The product of the reaction was sensitive to degradation by RNase but not to DNase or Pronase. These data suggest that the enzyme copies DNA into RNA. serum albumin, dithiothreitol, ATP, GTP, CTP, UTP, and DNase I (pancreatic, RNase free, electrophoretically purified) from Sigma Chemical Co.;RNase (pancreatic, 2x crystallized) from Worthington Biochemicals; actinomycin D, rifampin, aamanitin, and pronase from Calbiochem; sodium deoxycholate from Fisher Scientific; poly(dA-dT) from P-L Biochemicals, Inc.; Liquifluor, [3H JTdR (20 Ci/mmol), and ["CC]TdR (50 mCi/mmol) from New England Nuclear; and [3H]UTP (12.6 Ci/mmol) and sucrose (RNase free) from Schwarz/Mann. Preparation of purified PCDV virions. A 20x cell-associated virus (CAV) stock was prepared from PCDV-infected fathead minnow cells as previously described (14). The resulting CAV stock was treated with DNase (100 Ag/ml of CAV suspension, 37 C, 30 min) and centrifuged (SW27 rotor, 52,000 x g, 2 h, 10 C) through 10% (wt/wt) potassium tartrate in 0.01 M Tris buffer (TB) (pH 7.8) (8). The virus pellets were resuspended in 0.05 M sodium phosphate-buffered saline, pH 7.2, supplemented with 0.5% bovine serum albumin to 1/20 the original volume of the 20x CAV 231 on July 6, 2020 by guest