RNA synthesis in reovirus-infected L929 mouse fibroblasts.

Reovirus contains double-helical RNA.' 2 Both singleand double-stranded reovirus-specific RNA's have been found within phenol extracts of reovirus-infected cells.3-9 In the present study, analyses of extracts of reovirus-infected cells were made under conditions in which the double-stranded RNA within virus or subviral particles is not released. Three new viral-specific single-stranded RNA's have been found. These are associated with polyribosomes and are in part messenger RNA's (mRNA's). No double-stranded RNA has been found free within virus-infected cells other than that in newly synthesized virus and subviral particles. The virus and subviral particles appear to be associated with virus-synthesizing factories. Materials.-Virus: Dearing strain of reovirus 3, cloned,1 was used as stock virus after two passages. Cell culture: L cells, strain 929, were grown in suspension for the preparation of virus stocks and large quantities of RNA labeled with radioactivity. Monolayer cultures were used for titration of virus.10 Other materials: Ribonuclease-free (RNase-free) sucrose was further treated with the coarse Mg-bentonite fraction (20,000 X g), prepared as described by Petermann," before preparing stock solutions of 15 and 30% sucrose in buffer containing 0.01 M Tris HCl, pH 7.4, 0.01 M KCl, 0.0015 M MgCl2 (RSB). Sucrose not treated with bentonite was used in experiments for determination of the base compositions of RNA fractions. Sodium dodecyl sulfate (SDS) was recrystallized according to Mandel.12 Polyoxyethylene (20) cetyl ether (BRIJ 58) was purchased from Atlas Chemical Company. Methods.-Preparation of cellular extracts: L929 cells, in the logarithmic stage of growth, were collected by centrifugation from suspension cultures and inoculated with reovirus 3 at a multiplicity of 7-10 plaque-forming units per cell. After the 2-hr adsorption period, the cells were collected and resuspended at a concentration of 1-3 X 106 cells/ml in reinforced Eagle's spinner medium" containing 2% fetal bovine serum. Controls were carried through the same procedure using growth medium without virus. Where indicated, actinomycin D was added after virus adsorption to a final concentration of 0.15 pg/ml and samples were then incubated at 370C on a roller drum. At various times during the virus growth cycle, 0.33 Mc/ml of H3-labeled uridine, 18,000-30,000 ine/uninole (in the presence of 1000-fold thymidine), or 50 ic/ml of P32-labelcd orthophosphate was added for a chosen time interval at 370C. After the labeling period, the cells were washed one time with RSB, resuspended in iISB at 00C, and ruptured with 25 strokes in a tight-fitting Dounce homogenizer. Cytoplasinic and nuclear extracts were prepared as described by Penman,"4 modified in that nuclear extracts were prepared in buffer containing 0.01 M Tris HCl, pH 7.4, 0.01 Ml sodium chloride, 0.0015 M MgC90. The cytoplasmic extracts contained from 70 to 80% of the total cellular protein, from 70 to 80% of the RNA, and less than 2% of the DNA. Analytical procedures: Gradient centrifugations were done in the Spinco L2 ultracentrifuge. Fractions were collected from below. Absorbancy (at 260 inmM) of fractions was determined, and acid-insoluble radioactivity was obtained by precipitating the fractions with 5% trichloroacetic acid (TCA). The resulting precipitates were collected on Millipore filters, and the amount of radioactivity in the samples was determined in a Tri-Carb scintillation spectrometer. Determination of base composition: Labeled RNA's in sucrose gradient fractions were precipitated with TCA or ethanol and collected by centrifugation. More than 90% of the radioactivity was recovered in the pellets. The pellets were dissolved in 0.3 N KOH and the RNA's