Cis and Trans Regulation of Feline Immunodeficiency Virus

Thompson, Fiona Jane (1994) Cis and Trans Regulation of Feline Immunodeficiency Virus. PhD thesis, University of Glasgow.

Full text available as:
[img]
Preview
PDF
Download (13MB) | Preview

Abstract

The DNA sequence of the long terminal repeat (LTR) of several U.K. isolates of feline immunodeficiency virus (FIV) were determined and compared with those of other world-wide strains. These studies revealed a highly conserved structure with > 80% sequence identity between the different isolates. Nuclear protein binding sites in the LTR of FIV were identified by the method of DNase I footprinting. Using nuclear protein extracts from a feline T-lymphoma cell line, several discrete footprints were generated upstream of the transcriptional initiation site (-50 to -150). The specificity of protein binding was examined by competition with oligonucleotides representing consensus DNA binding sites for known transcription factors. Binding to AP-1 (-124) and ATF (-58) motifs was observed, with cross competition between these sites. A strong footprint was also detected over a tandemly repeated C/EBP motif (-94 to -86) while an adjacent weaker footprint was found to be specific for a NF-1 motif (-72 to -63). The effect on FIV LTR promoter activity of progressively deleting these nuclear factor binding sites was examined by linking LTR deletion mutants to the chloramphenicol acetyl transferase gene. Deletion of the AP-1 site caused a 10-25 fold loss of CAT activity while deletion past the ATF site reduced activity virtually to background levels. The effects of deleting the C/EBP and NF-1 sites were less marked and varied according to cell type. Trans-activation of the LTR was assayed using constructs linked to a CAT reporter gene. The full length FIV LTR was not significantly trans-activated. However the expression of a deleted LTR construct lacking the AP-1 / AP-4 site but retaining the C/EBP and ATF sites was partially restored by co- infection with FIV or by co-transfection with an infectious molecular clone of nv (PPR). The ORF-2 gene of FIV-G8 was cloned and sequenced and used in co-transfection studies to try to analyse viral trans-activation. This region of the genome is highly conserved among FIV isolates (-80%) and it has been suggested that it may be involved in trans-activation however the co-transfection experiments which were carried out during this study did not show any evidence of such a function. These results show that host transcription factors responsive to cellular activation have a major role in regulating FIV expression and suggest that virus-coded trans-activators may play a cell-type specific accessory role via U3 sequences.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Jim Neil
Keywords: Veterinary science, Virology
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-75669
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Dec 2019 09:15
Last Modified: 19 Dec 2019 09:15
URI: http://theses.gla.ac.uk/id/eprint/75669

Actions (login required)

View Item View Item

Downloads

Downloads per month over past year