The study of retroviruses has been of pivotal significance to the field of biomedical
science, where it has provided fundamental insights into the processes underlying both
viral and non-viral carcinogenesis. Ovine pulmonary adenocarcinoma (OP A), a contagious
lung cancer of sheep and goats, has emerged over the past three decades as an invaluable
model of human epithelial cancers. It is one of the very few animal models of retrovirus induced neoplasia of epithelial tissues,
whereas most other such animal models of human
cancers pertain to the haematopoietic system. OP A represents a unique, naturally
occurring, inducible, outbred animal model of peripheral lung carcinomas, and is caused
by a betaretrovirus - jaagsiekte sheep retrovirus (JSRV) - that is receiving increasing
attention in the fields of retrovirology and lung cancer research.
JSRV exists in two highly homologous, yet molecularly distinct forms. The first is an
exogenous form of the virus that is transmitted horizontally from one animal to another.
This form is infectious and the direct cause of OP A. The other is an endogenous form, 15
to 20 proviral copies of which reside benignly in the genome of sheep and are transmitted
vertically from one generation to the next. At the time this study commenced, no
knowledge existed regarding the underlying pathogenic mechanism by which JSRV causes
OPA. Even though the nucleotide sequence of exogenous JSRV had been elucidated seven
years earlier, only limited sequence information was available on endogenous JSRVs. With
a view towards identifying genetic regions or elements within exogenous JSRV that could
potentially be implicated in its pathogenic function, this study began with the cloning of
the first three full-length endogenous JSRV loci ever isolated from sheep. The DNA
sequences of these full-length endogenous JSRV loci were determined and
comprehensively analysed. Comparison with exogenous JSRV isolates revealed that the
two forms of the virus are highly homologous, yet can be consistently distinguished in
three short regions within the coding genes. Two of these reside in the gag gene, and one at
the end of the env gene. These regions were named the variable regions (VRs) of sheep
betaretroviruses.
The JSRV VR3 in env was linked by our collaborators to the virus's ability to transform
cells in tissue culture. The effects and biological significance of VRI and VR2 in gag are
subtler and more difficult to determine. After identifying these regions, it became the
objective of this study to develop relevant molecular tools that could be used to discern the
significance of these variable regions in vivo, and to characterise these tools in vitro to
assess their suitability for in vivo studies. The development of these tools entailed the
design of a novel strategy that was implemented to precisely substitute the endogenous
VRI and VR2 (individually and in combination) into an infectious molecular clone of
exogenous JSRV. These chimeric constructs were shown to support retroviral particle
release into the supernatant of transiently transfected cells in tissue culture. These particles
were confirmed by independent experiments to have arisen specifically from transfection
with the chimeric clones. Finally, the particles were shown to be capable of infecting
cultured cells and of productively integrating their genomes into those of their host cells,
rendering these particles fully competent retroviruses that can be used in the context of in
vivo studies to determine the biological significance of VRI and VR2.
This study has made a significant contribution to the further development of the OP A /
JSRV model system of human epithelial lung cancers. It has also led to the design of a
molecular substitution strategy that can be adapted to introduce any genetic region into a
cloned DNA construct, regardless of the degree - or lack of interrelation - of the two DNA
sequences, thereby creating a highly versatile molecular biological tool. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2007.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/9730 |
| Date | January 2007 |
| Creators | Hallwirth, Claus Volker. |
| Contributors | York, Denis F., Fan, Hung Y. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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