Activation and characterization of the reovirus transcriptase: genetic analysis

Abstract

We studied the ability of chymotrypsin to activate the transcriptases of the three serotypes of reovirus. When we used conditions that reproducibly caused the activation of type 3 transcriptase by chymotrypsin alone, type 2 transcriptase was sometimes activated, and type 1 transcriptase was never activated. Using intertypic recombinants containing various combinations of genome segments from reovirus types 3 and 1, we showed that the M2 segment determined this difference. Biochemical experiments indicated that the digestion of reovirus type 1 by chromotrypsin was blocked at an intermediate stage in uncoating. We found conditions which reproducibly activated the transcriptases of all three serotypes. This allowed us to compare the biochemical properties of the three transcriptases. Although the monovalent cation preferences, divalent cation preferences and optima, and temperature optima of type 1, 2, and 3 transcriptases were indistinguishable, the pH activity curves were reproducibly different. The largest difference was between type 2 and 3 transcriptases; the pH optimum of type 2 transcriptase was lower than the pH optimum of type 3 transcriptase. Using intertypic recombinants containing various combinations of genome segments from reovirus types 2 and 3, we demonstrated that the L1 segment specified this difference.