New Intermediate Subviral Particles in the In Vitro Uncoating of Reovirus Virions by Chymotrypsin

Abstract

Reovirus virions, grown in suspension cultures of L cells and extensively purified by density gradient and velocity gradient centrifugation after their release from cell debris by fluorocarbon extraction, are characterized by a mean particle diameter of 73 nm and a density in CsCl of 1.36 to 1.37 g/cm 3 . Treatment of intact virions by chymotrypsin (CHT) digestion in vitro converts them to subviral particles (SVP) having characteristics which are determined by the species of monovalent cation present during the digestion. In the presence of Cs + ions, CHT converts the virions to SVP of mean diameter 51 nm and density 1.43 to 1.44 g/cm 3 . In the presence of K + ions, the conversion is to SVP of diameter 51 nm and density 1.39 to 1.40 g/cm 3 . The SVP made in the presence of either Cs + or K + possess an extremely active RNA polymerase and nucleoside triphosphate phosphohydrolase (NTPase) activity in vitro and are resistant to further digestion by CHT. Treatment of intact virions with CHT in the presence of Na + or Li + ions results in their conversion to SVP of mean diameter 64 nm and density 1.37 to 1.38 g/cm 3 . Such SVP are not active in in vitro RNA synthesis or NTP hydrolysis and are resistant to further digestion by CHT even during prolonged exposure to high concentrations of enzyme. Addition of Cs + or K + ions to the digestion mixture allows conversion of the 64-nm diameter SVP to 51-nm diameter SVP in which the RNA polymerase and NTPase are active in vitro. Analysis of the proteins present in intact virions and in the different SVP reveals clear differences which indicate that the conversions are accomplished by removal or cleavage of particular species of polypeptides.