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Journal of Virology, September 2000, p. 8277-8285, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

In Vivo Genomic Footprinting of the Human T-Cell Leukemia Virus Type 1 (HTLV-1) Long Terminal Repeat Enhancer Sequences in HTLV-1-Infected Human T-Cell Lines with Different Levels of Tax I Activity

Shoibal Datta, Nayantara H. Kothari, and Hung Fan*

Cancer Research Institute and Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697

Received 19 April 2000/Accepted 16 June 2000

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) enhances viral gene expression through sequences in the U3 region of the viral long terminal repeat. These sequences consist of three imperfect 21-bp repeats (TRE-1s) and a region between the promoter-central and promoter-proximal 21-bp repeats (TRE-2). The TRE-1s contain a core cyclic AMP response element (CRE) motif and can be bound by CREB, ATF-1, ATF-2, and other members of the CREB-ATF superfamily of transcription factors. Tax enhances CREB binding to TRE-1 in vitro, and it promotes dimerization of CREB as well as other bZIP proteins. Using ligation-mediated PCR on in vivo dimethyl sulfate-treated HTLV-1-infected cell lines MT-2 and MT-4, we have compiled a profile of protein occupancy in the HTLV-1 enhancer sequences in the presence of high (MT-2) and low (MT-4) levels of biologically active Tax I. The in vivo footprinting showed that all three TRE-1s were bound by protein(s), but only in MT-2 cells. In MT-2 cells, all TRE-1s showed strong protection of the G residues in the central CRE, and the footprints extended to differing degrees into the GC-rich flanking sequences. This indicated Tax I-dependent loading of transcription factors onto the HTLV-1 TRE-1s in vivo. In vivo footprinting on TRE-2 indicated that this region was bound by proteins regardless of the Tax I status of the cell line. However, the presence of Tax I increased the extent and altered the profile of proteins binding TRE-2 in vivo.

* Corresponding author. Mailing address: Cancer Research Institute, Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697. Phone: (949) 824-5554. Fax: (949) 824-4023. E-mail: hyfan{at}uci.edu.

Journal of Virology, September 2000, p. 8277-8285, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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