Developing a System for the Application of Reverse Genetics to Bunyaviruses

Pritlove, David Charles (1993) Developing a System for the Application of Reverse Genetics to Bunyaviruses. PhD thesis, University of Glasgow.

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For many positive strand RNA viruses, genomic RNA can initiate productive infection in the absence of preformed viral proteins when introduced into susceptible cells. RNA transcribed in vitro from cDNA copies of positive strand virus genomes is also infectious. This property has enabled the powerful techniques of recombinant DNA technology to be applied to produce viruses carrying defined mutations by manipulation of cDNA clones. In contrast, deproteinised genomes of negative strand RNA viruses are non infectious. The study of negative strand viruses has therefore been hampered by the absence of methodology enabling the manipulation of their genomes. The non infectious nature of negative strand genomes is due to the requirement of viral proteins to transcribe translation competent positive strand mRNA. The template for transcription by the RNA dependent RNA polymerases of negative strand viruses is a ribonucleoprotein complex (RNP). Production of infectious RNA from cDNA of negative strand RNA virus genomes is therefore thought to require correct assembly of the synthetic RNA into RNP which can then be expressed and replicated by viral RNA polymerase. This thesis describes work carried out with the overall aim of establishing a system to produce Bunyamwera virus, a segmented negative strand RNA virus, carrying defined nucleotide alterations to its genome. Towards this goal, two broad approaches were investigated to attempt to present replicating virus with synthetic RNA in a form in which it could be recognised as a transcription template by the viral polymerase. The first approach involved transcribing viral-like RNA in vivo from transfected cDNA constructs in the presence of replicating Bunyamwera virus in the hope that the intracellular pool of viral proteins would enable encapsidation and replication of the synthetic RNA. The second approach involved attempts to reconstitute RNP in vitro by incubating synthetic RNA with nucleocapsid protein derived from Bunyamwera virus. For in vivo transcription of bunyavirus-like RNA from transfected plasmids, a recombinant vaccinia virus (vTF7-3) which expresses T7 RNA polymerase was investigated to assess its suitability for producing a Bunyamwera vims small (S) segment RNA from transfected cDNA in the presence of a replicating helper bunyavirus. The helper virus used was an existing reassortant virus (Bun/Bun/Mag) containing Bunyamwera virus large (L) and middle (M) genome segments and a Maguari vims S segment. Successful replication of the plasmid derived Bunyamwera S segment by the Bun/Bun/Mag helper virus would result in a proportion of progeny virus having all three gene segments of Bunyamwera virus. Dual infection experiments were performed to investigate the compatibility of the component viruses of the rescue strategy. Metabolic labelling and Northern analysis suggested that the general strategy of rescuing a Bunyamwera virus S segment RNA, transcribed in vivo by vTF7-3, using the Bun/Bun/Mag reassortant virus, was feasible with regard to interactions of the component viruses. Transcription of Bun S segment RNA in vivo from transfected cDNA by T7 RNA polymerase supplied by vTF7-3 was investigated by Northern blot analysis. It was not possible to detect RNA of the expected size. Transcription did occur since N protein could be detected in vTF7-3 infected cells transfected with a cDNA construct designed to produce positive strand S segment RNA from a T7 promoter. In an attempt to rescue plasmid derived Bunyamwera virus S segment RNA into a Bun/Bun/Mag reassortant virus, supernatants were retained from cells which had been infected with vTF7-3 and the Bun/Bun/Mag reassortant virus and transfected with Bunyamwera virus S segment cDNA. 200 individual plaques of progeny vims were screened by metabolic labelling to determine the origin of their N protein. No Bun/Bun/Bun virus was recovered indicating that if rescue occured, it did so at a frequency of <1/200. As an alternative to screening larger numbers of progeny virus, a counter- selectable rescue virus was sought to enable selection of any progeny virus containing plasmid derived (wild type) S segment RNA. Temperature sensitive (ts) mutants of Maguari vims were further characterised in attempt to identify a vims with a S segment lesion. To enable the S segments of mutants from each of the three reassortment groups to be sequenced, a rapid PCR-based procedure was developed which allowed full-length S segment cDNA to be amplified in a single step from RNA isolated from crude preparations of virus. Using this method, full-length S segment cDNAs were amplified and cloned from a representative of each of the three reassortment groups(ts6,ts17 and ts23) and from wild type Maguari virus. Three cDNA clones of each segment were sequenced by the Sanger di-deoxy chain termination method. A single point mutation was found in the S segment cDNA of Maguari virusts23. This virus was used as a helper virus in attempt to replicate RNA transcribed in vivo from a Bunyamwera virus S segment cDNA by the vTF7-3 expression system. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: J H Sharpe-Subak
Keywords: Virology, Genetics
Date of Award: 1993
Depositing User: Enlighten Team
Unique ID: glathesis:1993-74780
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 16:32
Last Modified: 27 Sep 2019 16:32

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