Human adenoviruses (Ads) are DNA viruses believed to hold potential as gene therapy vectors. However, Ad vectors often express residual late structural proteins, which stimulate immune responses resulting in rapid clearance of the vector. Therefore a greater understanding of the mechanisms controlling Ad late gene expression is required. During the late phase of adenovirus 5 (Ad5) infection it was previously shown that a nuclear compartment involved in RNA metabolism known as the Cajal body (CB) is disassembled from 1-6 punctate domains per cell into numerous microfoci. Furthermore, the marker protein of CBs, p80 coilin, was suggested to play a role in the expression of Ad late phase proteins. However, the exact function of coilin in Ad late protein expression is unknown. The aim of this investigation was to determine the roles of coilin and additional CB proteins during Ad infection. Immunofluorescence microscopy was utilised to investigate the redistribution of key CB proteins following Ad5 infection of A549 cells. Whilst coilin was redistributed from CBs into microfoci, another CB protein, termed survival of motor neuron (SMN), was redistributed from CBs into the nucleoplasm. To assess the roles of coilin and SMN during Ad5 infection, coilin was depleted in A549 cells by RNA interference (RNAi). Cells were infected with Ad5 and the virus yield, Ad proteins levels and Ad mRNA levels were assessed. Depletion of coilin reduced the virus yield and decreased the synthesis of Ad early, intermediate and late phase proteins. Although Ad mRNA expression was mostly unaffected, nuclear export of Ad mRNAs was abrogated in coilin-depleted cells. This indicated that coilin plays a role in Ad mRNA transport. Similar to coilin, SMN depletion significantly reduced the virus yield. However, in contrast to coilin, SMN depletion resulted in significant decreases in the levels of Ad capsid proteins, whilst non-structural proteins were either increased or unaffected. SMN depletion was found to reduce early gene transcription and altered alternative splicing patterns of Ad mRNAs. This suggested that SMN plays a role in Ad transcription and mRNA splicing. This investigation uncovered the involvement of two CB proteins in two very distinct roles during Ad infection. This is the first report suggesting a role for CBs in mRNA export. Further study is now required to identify the exact function of coilin in Ad mRNA export. Furthermore, investigation of a role for coilin in cellular mRNA export and mRNA export during infection with other viruses is also warranted. Although SMN was known to play a role in cellular mRNA splicing, this is the first report indicating that SMN may be involved in splicing of virus mRNAs. Additional work is required to define the precise function of SMN in transcription and mRNA splicing during Ad infection and during infection with other virus species.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:589153 |
| Date | January 2012 |
| Creators | White, Laura |
| Contributors | Blair, Eric |
| Publisher | University of Leeds |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.whiterose.ac.uk/4926/ |
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