Molecular Analysis of the Interaction of Herpes Simplex Virus and Steroid Hormones in Epithelial Cells

Offord, Elizabeth Ann (1988) Molecular Analysis of the Interaction of Herpes Simplex Virus and Steroid Hormones in Epithelial Cells. PhD thesis, University of Glasgow.

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Herpes simplex virus (HSV) is associated with the development of cervical cancer. Steroid hormones play a role in the normal development and in the pathology of the cervix. In view of the increased risk of developing cervical cancer in people taking the steroid - based contraceptive pill, possible interactions between steroid hormones and HSV were investigated. The growth of HSV-2 in either primary ectocervical cells or in the breast cancer cell line, ZR-75-1, was shown to be independent of either oestradiol (E2) or progesterone (Pg), in the concentration range 10-9M to 10-7M. The HSV IE activator sequence TAATGARAT (R = purine), which responds to the virion trans-inducing factor, Vmw 65, is partially homologous to the consensus binding site for PR found upstream of chicken egg-white genes. The ability of the hormones, E2 and Pg to modulate gene expression via the HSV TAATGARAT element was tested. MCF-7 or ZR-75-1 cells were transfected with plasmids encoding the CAT gene under the control of an HSV IE promoter and upstream TAATGARAT elements, in the presence and absence of physiological concentrations of E2 or Pg. No significant differences in the level of CAT activity with or without hormones were seen. Vmw 65 interacts with the TAATGARAT sequence in association with cellular factors. Specific complexes between nuclear protein(s) from MCF-7 and ZR-75-1 cells and TAATGARAT DNA were detected by gel retardation assays. Interestingly, the two cell lines tested showed different binding patterns, indicating differences in the cellular factors which recognize TAATGARAT. E2 treatment of cells did not result in any modification of the complexes formed with TAATGARAT. No virus induced protein, specific for TAATGARAT, was observed under the conditions used for these experiments. Thus no effect of E2 or Pg on the HSV IE activator sequence, TAATGARAT, was seen in the gene expression and DNA-binding studies described here. Biochemical and immunological steroid receptor assays were used to test the levels of ER and PR in MCF-7 and ZR-75-1 cells after E2 treatment and/or HSV (-1 or -2) infection. E2 treatment induced a 4- to 7-fold stimulation of detectable PR, indicating that the cells were responding as expected to E2. In contrast, both E2 and HSV caused the level of ER to fall to about 10% of the value measured in control, untreated cells. In vivo, E2 is known to stimulate the level of ER. A drop in detectable receptor may be due to modification of the receptor which prevents ligand binding or recognition by the monoclonal antibody. HSV also caused the level of PR to fall, indicating that E2 and HSV have different mechanisms of action. Attempts to in vitro translate and immunoprecipitate the ER protein from total cytoplasmic RNA were unsuccessful, despite the increase in ER mRNA levels detected in these samples in response to E2 treatment or HSV infection (see later). The failure to detect ER by in vitro translation and immunoprecipitation was probably due to either the low abundance of the ER mRNA population or to the inability of the monoclonal antibody to recognize newly synthesized ER. The possibility that HSV could influence steroid gene expression at the transcriptional level was tested by Northern blot and slot-blot analysis of ER mRNA levels. Changes in ER mRNA levels were correlated with the level of actin mRNA, taken as a standard message. E2 stimulation (10-8 M E2, 24h) resulted in a 50% increase in ER mRNA levels. HSV-1 infection resulted in a time-dependent increase of ER mRNA, reaching up to 250% of the control level by 8h post-infection (10 pfu/cell). HSV-2 infection (10 pfu/cell, 6h) stimulated the ER mRNA level by only 30%. The lower stimulation by HSV-2 may reflect the fact that HSV-2 infection induced earlier detachment from the substrate than HSV-1. The combination of E2 together with HSV gave a greater response than with either agent alone, showing that HSV and E2 stimulate the level of the ER message by different mechanisms. Stimulation of the ER message may be by transcriptional activation or by message stabilization. ER mRNA levels were 10 - 50% lower than the control value if cells were treated with cycloheximide for 30 minutes prior to and throughout the course of infection. This result suggests the involvement of both viral and cellular proteins in stimulation of the ER mRNA level. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biochemistry, Virology
Date of Award: 1988
Depositing User: Enlighten Team
Unique ID: glathesis:1988-77667
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 11:53
Last Modified: 14 Jan 2020 11:53

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