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REPLICATION

PU.1 Binding to ets Motifs within the Equine Infectious Anemia Virus Long Terminal Repeat (LTR) Enhancer: Regulation of LTR Activity and Virus Replication in Macrophages

Robert Hines, Brenda R. Sorensen, Madeline A. Shea, Wendy Maury
Robert Hines
1Division of Basic Biomedical Science, University of South Dakota, Vermillion, South Dakota 57069
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Brenda R. Sorensen
2Department of Biochemistry
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Madeline A. Shea
2Department of Biochemistry
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Wendy Maury
3Department of Microbiology, University of Iowa, Iowa City, Iowa 52242
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  • For correspondence: wendy-maury@uiowa.edu
DOI: 10.1128/JVI.78.7.3407-3418.2004
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ABSTRACT

Binding of the transcription factor PU.1 to its DNA binding motif regulates the expression of a number of B-cell- and myeloid-specific genes. The long terminal repeat (LTR) of macrophage-tropic strains of equine infectious anemia virus (EIAV) contains three PU.1 binding sites, namely an invariant promoter-proximal site as well as two upstream sites. We have previously shown that these sites are important for EIAV LTR activity in primary macrophages (W. Maury, J. Virol. 68:6270-6279, 1994). Since the sequences present in these three binding motifs are not identical, we sought to determine the role of these three sites in EIAV LTR activity. While DNase I footprinting studies indicated that all three sites within the enhancer were bound by recombinant PU.1, reporter gene assays demonstrated that the middle motif was most important for basal levels of LTR activity in macrophages and that the 5′ motif had little impact. The impact of the 3′ site became evident in Tat transactivation studies, in which the loss of the site reduced Tat-transactivated expression 40-fold. In contrast, elimination of the 5′ site had no effect on Tat-mediated activity. Binding studies were performed to determine whether differences in PU.1 binding affinity for the three sites correlated with the relative impact of each site on LTR transcription. While small differences were observed in the binding affinities of the three sites, with the promoter-proximal site having the strongest binding affinity, these differences could not account for the dramatic differences observed in the transcriptional effects. Instead, the promoter-proximal position of the 3′ motif appeared to be critical for its transcriptional impact and suggested that the PU.1 sites may serve different roles depending upon the location of the sites within the enhancer. Infectivity studies demonstrated that an LTR containing an enhancer composed of the three PU.1 sites was not sufficient to drive viral replication in macrophages. These findings indicate that while the promoter-proximal PU.1 site is the most critical site for EIAV LTR activity in the presence of Tat, other elements within the enhancer are needed for EIAV replication in macrophages.

  • Copyright © 2004 American Society for Microbiology
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PU.1 Binding to ets Motifs within the Equine Infectious Anemia Virus Long Terminal Repeat (LTR) Enhancer: Regulation of LTR Activity and Virus Replication in Macrophages
Robert Hines, Brenda R. Sorensen, Madeline A. Shea, Wendy Maury
Journal of Virology Mar 2004, 78 (7) 3407-3418; DOI: 10.1128/JVI.78.7.3407-3418.2004

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PU.1 Binding to ets Motifs within the Equine Infectious Anemia Virus Long Terminal Repeat (LTR) Enhancer: Regulation of LTR Activity and Virus Replication in Macrophages
Robert Hines, Brenda R. Sorensen, Madeline A. Shea, Wendy Maury
Journal of Virology Mar 2004, 78 (7) 3407-3418; DOI: 10.1128/JVI.78.7.3407-3418.2004
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KEYWORDS

Infectious Anemia Virus, Equine
macrophages
Proto-Oncogene Proteins
Response Elements
Terminal Repeat Sequences
Trans-Activators
transcription factors
virus replication

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