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Title: U90, a tumour associated polypeptide altered by HSV infection
Author: Grassie, Morag Anne
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1993
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Herpes simplex virus (HSV) has been associated with cervical neoplastic disease for a number of years and has been shown to induce morphological transformation of rodent cells. HSV infection alone is not thought to induce cancer but rather it is proposed to act as a co-factor initiating one of the many steps towards a potentially cancerous phenotype. A number of possible mechanisms of HSV transformation have been proposed, but the oncogenic properties of the virus are still not fully understood. The subject matter of this thesis will be concerned with one of these mechanisms, namely the postulate that transformation may occur by altering the expression of host cell polypeptides. A set of polypeptides called transformation associated polypeptides was previously found to be recognised in immunoprecipitation reactions by both sera from tumour bearing animals and antisera raised against HSV infected cells. This observation implicated the induction of such cellular polypeptides by HSV infection in the changes involved in the progression from a normal cell to that of a transformed phenotype. One of these transformation associated polypeptides a 90,000MW polypeptide called the U90, has been investigated. The U90 polypeptide was previously thought to be specific to the transformed state as it could only be immunoprecipitated from radiolabelled transformed cells and not from primary control cells. Characterization studies have now shown that the U90 is, in fact, a highly conserved polypeptide and as such is likely to play an important role in the cell. HSV infection was found to increase the expression of the U90 by up to 8 fold (at 3 hours post-infection) and resulted in the accumulation of the U90 in the cytoplasmic fraction - as opposed to the membrane associated fraction from where it is normally isolated in the uninfected tumour cell. The accumulation of the U90 in the cytoplasm following HSV infection, enabled the protein to be purified with much greater ease as a consequence of (1) the increased amount of the protein and (2) the soluble nature of the U90. The purified U90 was subsequently used to generate partial internal amino acid sequence (after enzymatic digestion) and also used to generate a polyclonal mono-specific antibody. Comparison of the amino acid sequences of the seven different U90 peptides with sequences entered in the NBRF databases resulted in no significant homology being found with any of the entries and confirmed that the U90 was not a stress protein such as hsp90 or GRP94 which had previously been suggested. This indicated that the U90 is a novel transformation associated polypeptide. The sequence data generated was insufficient to allow searches for functional domains to be completed and therefore the function of the U90 is still not known. By applying the U90 purification strategy to control RE cells and using the U90 monospecific antibody it was found that control cells did in fact express a homologue of the U90, which was recognised by the monospecifc U90 antibody. This 90K polypeptide behaved in an highly similar manner to the U90 - HSV infection resulted in the accumulation of the polypeptide in the cytoplasmic fraction and it could be purified m an identical manner to the U90 using the U90 purification strategy. Comparison of the U90 isolated from the Bn5T tumour cells to the U90 homologue isolated from control primary RE cells showed that in the transformed cells the U90 had a much slower turnover than in the control cells (approximately 13 hours as opposed to 31 minutes). If the U90 is implicated in regulation of cell growth or differentiation as suggested previously by the detection of the U90 in rat embryos up to 14 days gestation, increasing the total amount of the protein in the cell by HSV infection could have a significant affect on the cell regulation. A similar affect is seen in the transformed cell where the increased half life of the U90 means that more of this protein is present in the cell for extended periods. During these studies HSV-1 but not HSV-2 infection, was found to increase the levels of a second host polypeptide, a glucose regulated polypeptide GRP94. The increase in the total amount of GRP94 in the cell following HSV-1 infection appears to be a consequence of a translational or post-translational change, but in contrast to HSV induction of U90, immediate early gene expression was found to be inadequate to increase GRP94. Due to time limitations studies were concentrated on the U90. However the increase of GRP94 by HSV type 1 but not type 2, marks an interesting distinction between the host cell interactions of the two viruses.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available