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Title: Development of a reverse genetics system to investigate Bunyamwera virus RNA synthesis
Author: Dunn, Ewan Frazer
ISNI:       0000 0001 3435 6732
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2000
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Bunyamwera (BUN) virus is the prototype type virus of the family Bunyaviridae. The virus has a genome composed of three single stranded RNA segments of negative polarity termed large (L), medium (M), and small (S). Described in this thesis, is the development of a reverse genetics reporter system to study the synthesis of BUN viral RNA and the proteins involved. The system utilises a recombinant RNA template, BUNSCAT, which comprises an antisense CAT gene flanked by the authentic BUN S 5' and 3' untranslated regions (UTRs). Transfection of in vitro transcribed BUNSCAT RNA into cells transiently expressing recombinant BUN viral proteins resulted in CAT activity only when both the L segment and the S segment proteins were expressed, indicating that the negative sense template had been transcribed into a positive sense, translatable RNA. Further analysis revealed that just BUN N and L proteins were sufficient for the transcription of BUNSCAT RNA. It was also demonstrated that the BUN L protein was able to transcribe BUNSCAT RNA in concert with the N proteins of certain heterologous bunyavirus S segments. Modifications to the BUNSCAT UTRs indicated that these terminal regions play an important role in transcription of BUNSCAT RNA. The addition of nucleotides to both the 3' and 5' termini were tolerated to an extent. While deletion of one nucleotide at the 3' terminus gave near wildtype CAT activity, any further deletions resulted in a significant drop in CAT activity, as did any deletion of the 5' terminus. Analysis of a series of single point and complimentary base-pair mutations to the 5' and 3' termini determined that sequence specificity for nucleotides 2-12 of the termini was essential while maintenance of base-pairing between nucleotides 13-15 of the termini was also important. Construction and comparison of BUNLCAT and BUNMCAT RNAs with the BUNSCAT reporter determined that the L UTRs gave a higher CAT activity than M UTRs which in turn were higher than S UTRs. Construction and analysis of a series of chimeric BUN segment UTR reporters (e.g. BUNL 3'-CAT-BUNM 5') showed that none of the chimeric RNAs were transcribed. A significant level of CAT activity was restored, however, to the BUNL/M-CAT reporter when the BUN L 5' UTR terminus was mutated to recreate partly M 5' UTR sequence, thereby restoring complementarity between the 18 terminal nucleotides. Collectively, these results demonstrate that the reporter assay is a useful, reliable and convenient tool to study the molecular processes of Bunyamwera virus. Using this system, it is expected that further mutational analysis of the BUN RNA segments and replication proteins will provide insight into transcription and replication processes, and the cis-acting signals which control and orchestrate these important events.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Bunyaviridae diseases; BUN