Ribozymes and rotavirus replication

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dc.contributor.advisor Bellamy, Richard en
dc.contributor.author Anthony, Indumathy Dileeni en
dc.date.accessioned 2007-06-30T01:19:58Z en
dc.date.available 2007-06-30T01:19:58Z en
dc.date.issued 2000 en
dc.identifier.citation Thesis (PhD--Biochemistry and Molecular Biology (Biological Sciences))--University of Auckland, 2000 en
dc.identifier.uri http://hdl.handle.net/2292/604 en
dc.description Full text is available to authenticated members of The University of Auckland only. en
dc.description.abstract 1. Among all viruses, the group most potentially suited for ribozyme-mediated interference are the RNA viruses, i.e. those that replicate without the involvement of a DNA intermediate. Rotavirus is a dsRNA virus that causes severe gastroenteritis in infants world-wide. The ability of ribozymes to suppress rotavirus replication in cell culture has been investigated using transformed cell lines constitutively expressing ribozymes targeted against the mRNA transcript of the virus-encoded, intracellular receptor, NSP4. NSP4 is a key component in the maturation of rotavirus in which the immature virus particle (ICP) is transferred across the ER membrane. 2. Three 'hammerhead' ribozymes were chosen that target the mRNA transcript of the rotavirus intracellular receptor protein NSP4. Two of these ribozymes targeted a cleavage site at nucleotide 308 in the coding region of the transcript. The third ribozyme was an inactive version in which a point mutation had been inserted into the catalytic domain. All the ribozymes were subcloned into a high level eukaryotic expression vector (pRc/CMV) downstream of the cytomegalovirus immediate-early promoter. Both ribozyme and target RNAs were synthesised in vitro in 'run-off' transcription reactions. Cleavage reactions performed using these transcripts confirmed that the catalytically-active ribozymes were able to cleave the target NSP4 mRNA specifically and produce the anticipated cleavage products. 3. The pRc/CMV-ribozyme constructs were used to transform MA104 monkey kidney cells to generate transformed cell lines that constitutively synthesised ribozyme transcripts. Integration of the cassettes was confirmed by PCR and Southern analysis. Transcription of the ribozymes was demonstrated by RT-PCR. The transformed cell lines were assessed for effects on the rotavirus replicative cycle by performing plaque assays on each of the isolated clonal cell lines. Approximately 50% of the transformed cell lines exhibited a small plaquing phenotype indicating that rotavirus replication might be down-regulated. The small plaque morphology was reproducible and the reduction in size was both consistent and statistically significant. However, the small plaquing phenotype was also observed in the control cell lines transformed with the plasmid lacking the ribozyme cassette, indicating that the apparent down-regulation of virus replication could not be attributed to ribozyme activity. 4. Further investigation of the transformed cell lines demonstrated that the small plaquing effect was rotavirus specific. These cell lines were able to support the replication of polio virus, vaccinia virus and reovirus and the morphology of the plaques produced by these viruses was the same as that produced on wild-type cells. The rotavirus progeny isolated from small plaques were able to produce 'normal' plaques on wild-type MA104 cells, indicating that the small plaque phenotype was a result of some property of the cell lines isolated and not of the rotavirus isolate employed in the assay. The frequency with which clones of cells yielding the small plaque phenotype were generated was relatively high and ranged between 3.3x10-4 for cells that incorporated only plasmid sequences and 4.4x l0-4 for cells that incorporated the ribozyme cassette. 5. Investigation of the possible factors that might affect plaque morphology revealed that neither the expression of the selection marker, nor the presence of selection agent (G418), was responsible for the reduced plaque area. Attempts made to 'cure' cell lines of the integrated plasmid were inconclusive, because repeated passaging of the cell lines produced cells incapable of surviving under agar. Transformation of MA104 cells with a number of different high-level eukaryotic expression vectors produced isolates with the same small plaquing phenotype, indicating that the effect was not specific to pRc/CMV. There were no differences in the adsorption of rotavirus to small plaquing and wild-type cells, nor were there differences in the replication rates or final yields of rotavirus released from cells. However a difference was detected in the integrity of the plasma membrane post-infection between transformed and wild-type cells. Preliminary investigations of the morphology of the transformed cells revealed that the cells were smaller and more closely packed; electron microscopy suggested that there was a predominance of dome shaped cells in the small plaquing cell lines. 6. The marked reduction of rotavirus plaque size which occurs on cells transformed by eukaryotic vectors expressing antibiotic selectable markers is best attributed to a difference in packing of the cells in the monolayer accompanied by an increased stability of the plasma membrane. In this circumstance, it is proposed that the ability of virus to diffuse away from the point of infection is impaired because the close packing of the cells limits the expansion of the plaque. The reason why such a heritable and stable phenotypic change occurs at such a high frequency amongst MA104 cells transformed by vectors remains unknown. en
dc.language.iso en en
dc.publisher ResearchSpace@Auckland en
dc.relation.ispartof PhD Thesis - University of Auckland en
dc.relation.isreferencedby UoA9991294414002091 en
dc.rights Restricted Item. Available to authenticated members of The University of Auckland. en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. en
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.title Ribozymes and rotavirus replication en
dc.type Thesis en
thesis.degree.discipline Biochemistry and Molecular Biology (Biological Sciences) en
thesis.degree.grantor The University of Auckland en
thesis.degree.level Doctoral en
thesis.degree.name PhD en
dc.rights.holder Copyright: The author en

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