The Effect of HSV-2 Infection on the Expression of Cellular Mitochondrial Aspartate Aminotransferase

Collins, Terry Cordell (1998) The Effect of HSV-2 Infection on the Expression of Cellular Mitochondrial Aspartate Aminotransferase. PhD thesis, University of Glasgow.

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Herpes simplex virus has long been implicated in the development of human genital cancers, especially carcinoma of the cervix. Recent epidemiological and biological studies suggest that a cooperative interaction between herpes simplex virus and human papillomavirus leads to an increased risk of developing cervical neoplasia. A set of cell coded polypeptides has been detected in a transformed rat cell line (Bn5T) produced by transformation of rat embryo fibroblasts with a fragment of HSV-2. This group of polypeptides (200kDa, 90kDa (a doublet U90 and L90), 40kDa and 32kDa) were immunoprecipitated specifically from transformed cells by sera from tumour bearing rats, and from rats immunised with non-transformed HSV-2 infected cells. This suggested that HSV-2 might be inducing polypeptides with a function in transformed cells. This thesis describes studies on the 40kDa polypeptide from this group. The 40kDa polypeptide is specifically induced in transformed cells and is upregulated by HSV-2 infection. The 40kDa polypeptide was purified from Bn5T cells by ammonium sulphate fractionation and fast protein liquid chromatography in an attempt to raise an antiserum to this protein. A poor immune response was observed in rabbits injected with this immunogen, an observation previously and independently reported in our laboratory. However, amino acid sequence data and subsequent immunological studies (Lucasson, 1992) demonstrated that the 40kDa polypeptide was related to mitochondrial aspartate aminotransferase (mAspAT). The steady state level of mAspAT mRNA was examined to determine whether HSV-2 infection increased the amount of mRNA in transformed cells, as the protein level and enzyme activity had previously been shown to be increased. This study demonstrated an increase of approximately 80% in the level of mAspAT mRNA following HSV-2 infection of Bn5T cells. This conclusion was obtained from Northern blotting and reverse transcriptase PCR experiments. During Western blotting experiments to confirm the increased mAspAT protein level following HSV-l/HSV-2 infection (previously observed by slot blot immunoblotting), a second polypeptide was identified specifically in the HSV infected samples. This polypeptide was found to have a slightly greater molecular weight in cells infected with HSV-1 than in HSV-2 infected cells. This information was used to map the region of the HSV genome encoding this polypeptide, by analysis of the polypeptide produced following infection of cells with a panel of intertypic recombinant herpes simplex viruses. The polypeptide was identified as the HSV dUTPase enzyme, although no obvious reason for the cross-reactivity could be found. A plasmid was constructed with the mAspAT cDNA under the control of the HCMV major immediate early promotor. This plasmid expressed the mAspAT polypeptide in transfected cells, as determined by immunofluorescence studies. Transfection of this plasmid into normal rat kidney cells led to an increase in the number of cells which formed colonies in soft agar medium, in comparison to cells transfected with the parental control plasmid. This experiment demonstrated a direct biological transforming effect of increased mAspAT expression in normal cells in vitro. Transformation assays in vivo using these cells injected into nude mice were inconclusive, as tumours also developed in animals injected with control cells. During RT-PCR experiments to quantify the level of mAspAT mRNA following HSV infection, an additional PCR product was observed in HSV-2 infected BnST cell samples. This product was slightly smaller than the normal sized product, and might explain the appearance of a 40kDa mAspAT polypeptide (in addition to the consitutive cellular 44kDa enzyme) and the increased membrane mAspAT immunofluorescence observed in transformed cells (J.C.M. Macnab, unpublished results). The smaller PCR product was cloned into pGEM-T and partial DNA sequence was obtained. This demonstrated that the mitochondrial targetting presequence was present in the smaller clones, and did not therefore explain the altered localisation of mAspAT to the cell surface observed in the transformed cell. My results demonstrate an increase in the level of mAspAT mRNA in transformed cells following infection with HSV-2, and that increased expression of mAspAT can induce transformation in normal cells. The possible effects of increased mAspAT activity on tumour cell metabolism are discussed, along with the function of mAspAT as a plasma membrane long chain fatty acid binding protein (FABP(pm)) and a possible role for the dUTPase-mAspAT cross reactivity in promoting cervical neoplasia.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Joan MacNab
Keywords: Virology
Date of Award: 1998
Depositing User: Enlighten Team
Unique ID: glathesis:1998-75872
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Dec 2019 09:15
Last Modified: 19 Dec 2019 09:15

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