Cis- and trans-regulation of feline immunodeficiency virus: identification of functional binding sites in the long terminal repeat.
Nuclear protein binding sites in the long terminal repeat (LTR) of feline immunodeficiency virus (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 signal was also detected over a tandemly repeated C/EBP motif (-94, -86) and an adjacent weaker footprint was found to be specific for an NF1 motif (-72/-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 acetyltransferase (CAT) gene. Deletion of the AP-1 site caused a 10- to 25-fold loss of CAT activity whereas deletion past the ATF site reduced activity virtually to background levels. The effects of deleting the C/EBP and NF1 sites were less marked and varied according to cell type. Transactivation 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-4/AP-1 site but retaining C/EBP and ATF sites was partially restored by co-infection with FIV or by co-transfection with an infectious molecular clone of FIV (FIV-PPR). 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 acting through U3 may play a role in some cellular environments.
Thompson
F J
FJ
Department of Veterinary Pathology, University of Glasgow, Bearsden, U.K.
Elder
J
J
Neil
J C
JC
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D002415:Cats; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D017353:Gene Deletion; D015967:Gene Expression Regulation, Viral; D016089:Immunodeficiency Virus, Feline / Q000235:genetics*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D015500:Chloramphenicol O-Acetyltransferase