The human cytomegalovirus immediate early proteins, IE72 and IE86 are believed to play a key role in the viral life cycle. Both proteins are able to activate individually and synergistically a number of viral and cellular promoters. However, the mechanisms of activation by the two proteins are not clearly defined despite the identification of a number of viral and cellular proteins with which both proteins can interact. In this study I have tried to identify novel factors which can interact directly with the viral immediate early proteins. The original approach taken was the screening of a λgt11 cDNA expression library with recombinant IE72 and IE86. The screening using IE72 was unsuccessful, however, I was able to identify a number of putative interaction partners for IE86. Amongst these were two proteins which are believed to play a role in gene expression in the context of chromatin, histone deacetylase 1 (hdac1) and human brahma (hbrm). I have demonstrated that both proteins are able to bind to IE86 in vitro and have identified regions of IE86 which are important for this binding. In addition I have shown that IE86 is able to interact with hbrm in transfection studies giving synergistic activation of a cellular and a viral promoter. To identify IE72 binding proteins, a large-scale affinity column analysis was carried out using HeLa cell extract. This yielded a protein of approximately 100kDa which was able to interact with IE72. Mass spectrometry analysis of fragments of this protein suggested that the protein may be elongation factor (EF2), however, IE72 had no effect on protein translation in a rabbit reticulocyte lysate assay and an antibody to EF2 did not react with the 100kDa protein. The identify of this 100kDa is therefore still not clear. During this analysis I also examined the interaction of IE72 with the viral DNA polymerase and found that IE72 was able to interact in vitro with HCMV DNA polymerase.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:598927 |
| Date | January 1999 |
| Creators | Fairley, J. A. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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