Studies on reovirus RNA. I. Characterization of reovirus genome RNA

Chemical treatment does not release the genome of reovirus in intact form. Irrespective of the procedure used, the same mixture of fragments is released with sedimentation coefficients of about 14, 12 and 10.5 s. On electrophoresis in polyacrylamide gels these size classes can be further resolved, the two former into two components each, the last into three. These fragments are double-stranded as judged by the following criteria: they exhibit a sharp melting profile with a T_m dependent on the ionic concentration; they are resistant to ribonuclease, provided that the concentrations of both monovalent and divalent ions, as well as that of the enzyme, are suitably adjusted; their sedimentation behavior is independent of the ionic concentration over a wide range; and their base composition displays equality of A and U, as well as of G and C.In addition to this mixture of double-stranded fragments there is released from virions an RNA of small size (2 to 3 s) which amounts to about 20% of the mass of the viral genome. The base composition of this RNA is 82% A, 13% U, 3% G and 3% C. This A-rich RNA is synthesized in step with the viral genome RNA.RNA molecules corresponding to intact viral genomes were not detected in infected cells at times when the viral genome was replicating. Irrespective of the method of breaking open the cells, or of the cell strain used, the same mixture of RNA fragments was obtained as was released from virions.These results suggest that the reovirus genome consists of a number of segments of double-stranded RNA; further, it would appear that the reovirus genome does not replicate as one intact molecule, but rather in the form of these same segments. The molecular weights of the L, M and S segments have been estimated by reference to three sets of data in the literature; the most likely values are 2.3 ± 0.2 × 10⁶, 1.3 ± 0.2 × 10⁶ and 8 ± 2 × 10⁵. L, M and S segments are released from virions in the ratio of n : n : 1.5n. Owing to uncertainty concerning the size of the reovirus genome, the exact number of segments of which it is composed cannot be specified. The most likely value of n is 2, although a value of 3 or even 4 is not ruled out.The function of the A-rich material is not known. It is conceivable that it serves to link the double-stranded segments, giving rise to a structure which is so labile that it does not withstand any but the most gentle manipulation. However, the precise manner in which linking is accomplished, and the role, if any, of the Arich material in this process must await further work.