The in vitro transcription and translation of bluetongue virus mRNA

DSc (Biochemistry), North-West University, Potchefstroom Campus

This investigation was primarily aimed at studying the in vitro synthesis of bluetongue virus proteins. Both BTV mRNAs and denatured dsRNAs were used as templates for translation. Double-stranded RNAs could be purified from infected cells, but the only means of obtaining pure BTV mRNAs was to synthesise them in vitro. Therefore, some of the factors that influence the in vitro transcription reaction were also investigated. The transcriptase of BTV can be activated in vitro by removal of the outer capsid proteins, which results in the conversion of virions to core particles. It was found that the concentration of the core particles has a marked effect on the efficiency of the transcription reaction. At high core concentrations the transcription reaction is severely inhibited, resulting in a complete termination of transcription after a relatively short incubation period. It was found that it is possible to counteract this inhibition by lowering the incubation temperature. Consequently, at high core concentrations the in vitro transcription reaction is much more efficient at a lower incubation temperature (28°C), than at a higher incubation temperature (37°C). At low core concentrations, this low temperature optimum is not observed. It was furthermore found that the core-mediated inhibition of in vitro transcription is completely reversible. Reactions in which the synthesis of mRNAs is completely terminated at 37°C. can be reactivated by lowering the incubation temperature. Another important finding was that the core- mediated inhibition can be counteracted by including compounds such as sucrose and glycerol in the reaction mixture. The BTV mRNA species synthesised during the in vitro transcription reaction were analysed by agarose gel electrophoresis. It was found that the 10 different BTV mRNA species are not synthesised in vitro in equal amounts. The results confirm those previously obtained by much more indirect methods. The purpose of studying in vitro translation of BTV was to identify all the proteins that are specified by BTV mRNA species, and determine the RNA- protein coding assignments of the genome segnents. All 9 BTV proteins previously identified in BTV - infected cells can be synthesised in vitro using BTV mRNAs as templates, a result that confirms that the two non-structural proteins, NS1 and NS2, are indeed virus-specific. Apart from these proteins, three new virus-specific non - structural proteins, NS3A, NS3B and protein C were identified by means of in vitro translation. They were subsequently also detected in infected cells, where they seem to be synthesised only in very small amounts. The relative amounts in which the BTV proteins are synthesised in vitro, differ from that observed in infected cells. The RNA-protein coding assignments for seven of the ten genome segments, the three medium-sized and the four small- sized segments, were determined by in vitro translation of denatured dsRNAs. It correlates with results published for BTV types and 17 respectively. Genome segment 10 codes for both proteins N53A and N538. The peptide maps of these proteins are virtually identical, indicating that they may be translated from overlapping in - phase reading frames. Preliminary results indicate that genome segment 9, which codes for protein 6, also codes for protein C. It was also investigated whether in vitro synthesised N52 is phosphorylated and has affinity for single- stranded RNA, as has been reported for N52 synthesised in infected cells . It was found that in vitro synthesised N52 has affinity for ssRNA, similar to N52 present in infected cells, but no phosphorylation of in vitro synthesised N52 has as yet been observed. On the other hand, it was found that N52 from infected cells can be phosphorylated in vitro. The kinase responsible for phosphorylating N52 was found to be present in all preparations of in vivo synthesised N52 - even in those purified by means of affinity column chromatography.

Doctoral