The in Vitro activation and further characterization of the bluetongue virus-associated transcriptase

Abstract The bluetongue virus capsid consists of seven polypeptides of which two, polypeptides P2 and P5, form an outer layer (D. W. Verwoerd, H. J. Els, E.- M. De Villiers, and H. Huismans, 1972 , J. Virol. 10 , 783–794. We found that these two polypeptides can be removed selectively from the virion by a combination of chymotrypsin and specific ionic conditions. Treatment of the virion with chymotrypsin at cation concentrations below 400 m M results in the removal of polypeptide P2 from the outer layer. When the concentration of divalent cations is raised to above 400 m M during this treatment both P2 and P5 are removed. With monovalent cations at concentrations of 800 m M or more, the dissociation of the outer layer is only observed when the pH is reduced to 6.0. Under these conditions the core particles themselves also appear to be unstable and there is a marked reduction in the relative amount of some of the core polypeptides. The removal of both P2 and P5 from the virion activates the core-associated transcriptase. The transcriptase reaction has a low temperature optimum, a Mg 2+ dependency, and is stimulated by Mn 2+ . A twofold stimulation of the reaction was also observed by the methyl donor S -adenosyl- l -methionine. The reaction is furthermore strongly affected by the core concentration. This is reflected by a rapid decline in the incorporation of [ 3 H]UMP per unit of core particles with increasing core concentration. The in vitro synthesized product of transcription was analyzed by hybridization and found to contain all 10 species of mRNA.