Die in vitro aktivering van transkipsieaktiwiteit by bloutongvirus

Abstract

The investigation into the in vitro transcription of the BTV dsRNA genome focussed on the activation of the virion-associated transcriptase, the determination of the optimal conditions for the transcription reaction and a preliminary analysis of the synthesized product . The following are the most important conclusions: 1. A method has been developed for the selective removal of the two outer capsid proteins of BTV. The first outer capsid polypiptide , P2, can be removed by means of enzymatic digestion with chymotrypsin. The other polypeptide , PS, can be dissociated from the remaining ISVP by treatment with high concentrations (600 mM) of a divalent cation such as Mg++_ 2. The outer capsid proteins of two other orbiviruses, namely EHDV and AHSV can be removed in a similar way. 3 . The method can be applied to partially purified virus material and is suitable for the large scale preparation of BTV and other orbivirus c ore particles. 4. The removal of both outer capsid polypeptides of BTV, P2 and PS, is required for the in vitro activation of the virion-associated transcriptase . 5. The transcriptase activity is dependent on divalent cations such as Mg++ and Mn++. The stimulation with Mg++ is much higher than with Mn++. The effect of these two cations is probably additative and it appears as if they stimulate the BTV- transcriptase in two different ways. 6 . The addition of a methyl donor to the transcription mixture causes a twofold stimulation in transcriptase activity. 7 . The transcriptase activity is dependent on the concentration of core particles. At high core concentrations (> 4 A 260 -uni ts/ml) the in vitro transcription reaction is almost fully inhibited . The cause

of this inhibition is as yet unknown. 8. The i n vitro transcription reaction displays a sharp temperature optimum at 28 °c-3i° c. 9. Analysis of the product of the in vitro transcription reaction reveals that all ten double-stranded RNA genome segments are transcribed. Evidence has been found indicating regulation of transcription. Some parts of this investigation were recited at the Congress on Virology and Class 4 Agents which was presented by the Poliomyelitis Research Foundation and the Department of Health in Johannesburg from the 17th to the 19th September 1979. This work has been submit ,ted for publication in "Virology".