Transcription of the Genomes of Type 1 and Type 3 Reoviruses

L. Prevec, Y. Watanabe, C. J. Gauntt, A. F. Graham
1968 Journal of Virology  
The double-stranded ribonucleic acid (RNA) genome of type 1 reovirus was fragmented into three size classes on extraction, as has already been shown for reovirus type 3. The relative amounts and molecular weights of the three classes were the same for the two viruses. Cells infected with type 1 virus synthesized three classes of messenger RNA. Each class of messenger RNA hybridized exclusively with a denatured double-stranded RNA fragment of equivalent length, as had also been found for type 3
more » ... n found for type 3 reovirus. The double-stranded RNA segments thus act as specific units for transcription of messenger RNA in the infected cells. In cells infected with type 3 reovirus, the three classes of messenger RNA are made in equal amounts throughout the course of multiplication. In contrast, cells infected with type 1 virus produced only half as much of the largest messenger RNA as they did of the other two classes at all times during the replicative cycle. This finding suggests that transcription of the largest segments of type 1 viral genome is restricted. The genome of type 3 reovirus is a doublestranded ribonucleic acid (dsRNA) considered to have a minimal molecular weight of approximately 107 daltons (6, 10). Apparently, there are weak spots in the molecule since, on extraction from virions or infected cells by a variety of methods, the dsRNA is obtained in lengths which fall into three main size classes (2, 3, 7, 9, 17). These classes have molecular weights of approximately 0.8 X 106, 1.4 X 106, and 2.4 X 106 daltons (17). Hybridization tests on denatured RNA from the three classes, taken in all possible pairs, showed no homology to exist between them; the fragments of dsRNA therefore arise through disruption of the viral genome at specific points rather than by random fragmentation (17) . For each class of dsRNA that can be isolated, a messenger RNA (m.RNA) of similar length is produced in infected cells (1, 6, 17), and each of the three classes of mRNA hybridizes exclusively with its corresponding length of viral RNA (1, 17, 19) . Thus, the various segments of double-stranded viral genome appear to act as individual templates for transcription of mRNA during the cycle of infection. In the present paper, it is shown that type 1 reovirus, although serologically distinct from type 3, is similar to type 3 virus in the above respects. A major problem arising from these observations is associating functions with each class of mRNA. One approach, which has been used here, is to examine the kinetics of synthesis of the three classes of mRNA in an effort to determine whether they may be synthesized at different times in the infected cell and could, therefore, be associated with early or late functions in the replicative process. It will be shown that, insofar as type 3 reovirus is concerned, all three classes of mRNA are synthesized in approximately equal amounts at early as well as late times during infection. With type 1 reovirus, the relative amounts of the three classes of mRNA synthesized also remained constant, although much less of the largest mRNA was made in the type 1 than in the type 3 system. Apparently, transcription of the largest double-stranded segments is markedly restricted in the type 1 system. MATERIALS AND METHODS Cells and virus. Suspension cultures of L cells were used for all experiments except one in which BHK21 cells were employed. The latter cells were obtained from T. Wiktor of The Wistar Institute. The medium was as previously described (10, 18). Cultures were infected by allowing virus, at a multiplicity of 20 to 30 plaque-forming units per cell, to adsorb for 2 hr at room temperature to cells in suspension at a concentration of 106 cells/ml. The cells were then centrifuged and resuspended to a concentration of 5 X 105/ml in medium; they were placed at 37 C and 289 on May 9, 2020 by guest
doi:10.1128/jvi.2.4.289-297.1968 fatcat:rld4rb5a4jbmdfohffzri5dj5y