Inactivity of purified reovirus RNA as a template for E. coli polymerases in vitro.

Abstract

The properties of reovirus RNA isolated by equilibrium density gradient centrifugation closely resemble those previously reported from reovirus RNA not purified In C32SO4. Both preparations contain two or more components with sedimentation coefficients at 11-14S. The RNA''s are relatively resistant to RNase digestion, and undergo a sharp thermal denaturation in the range 80-100[degree]C. Furthermore, reovirus RNA has a buoyant density of 1.61 in CS2SO4, similar to other double-stranded RNA''s. Although centrifugation in CS2SO4 does not detectably affect the physical properties of reovirus RNA, this procedure abolished any activity for polynucleotlde synthesis associated with this RNA. The experiments described above indicate that the loss of activity does not result from exposure of the RNA to CS2SO4 as such, and Is not due to the presence of an Inhibitor of the polymerases. Instead, it appears more likely that the activity before CS2SO4 purification can be ascribed to the presence of some host mouse cell DNA in the RNA preparations. This is in accord with the failure of MS2 replicative form to serve as a template for RNA poly-merase. It is of interest that the individual nearest-neighbor determinations of RNA polymerase product in a "reovirus RNA-primed synthesis" are, within the limits of precision of the method, indistinguishable from those reported for mouse DNA, and that the ratio (A + U)/ (G + C) derived from these data yields a value of 1.43. This value is identical with that obtained from mouse DNA itself, and differs from the chemically determined ratio, 1.27, reported for reovirus RNA. If reovirus RNA does not serve as a template for the normal cell polymerase, then it is reasonable to expect that a new virus-specific replication system is produced in the course of infection. The inhibitory effect of actinomycin on reovirus multiplication suggests that the antibiotic is affecting cellular activities required for virus development.