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Journal of Virology, January 2006, p. 332-341, Vol. 80, No. 1
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.1.332-341.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

In Vivo and In Vitro Analysis of Factor Binding Sites in Jaagsiekte Sheep Retrovirus Long Terminal Repeat Enhancer Sequences: Roles of HNF-3, NF-I, and C/EBP for Activity in Lung Epithelial Cells

Kathleen McGee-Estrada and Hung Fan*

Department of Molecular Biology and Biochemistry and Cancer Research Institute, University of California, Irvine, California 92697-3905

Received 30 June 2005/ Accepted 9 October 2005

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma, a contagious lung cancer of sheep that arises from type II pneumocytes and Clara cells of the lung epithelium. Studies of the tropism of this virus have been hindered by the lack of an efficient system for viral replication in tissue culture. To map regulatory regions important for transcriptional activation, an in vivo footprinting method that couples dimethyl sulfate treatment and ligation-mediated PCR was performed in murine type II pneumocyte-derived MLE-15 cells infected with a chimeric Moloney murine leukemia virus driven by the JSRV enhancers ({Delta}Mo+JS Mo-MuLV). In vivo footprints were found in the JSRV enhancers in two regions previously shown to be important for JSRV long terminal repeat (LTR) activity: a binding site for the lung-specific transcription factor HNF-3ß and an E-box element in the distal enhancer adjacent to an NF-{kappa}B-like binding site. In addition, in vivo footprints were detected in two downstream motifs likely to bind C/EBP and NF-I. Mutational analysis of a JSRV LTR reporter construct (pJS21luc) revealed that the C/EBP binding site is critical for LTR activity, while the putative NF-I binding element is less important; elimination of these sites resulted in 70% and 40% drops in LTR activity, respectively. Electrophoretic mobility shift assays using nuclear extracts from MLE-15 murine Clara cell-derived mtCC1-2 cells with probes corresponding to the NF-I or C/EBP sites revealed several complexes. Antiserum directed against NF-IA, C/EBP{alpha}, or C/EBPß supershifted the corresponding protein-DNA complexes, indicating that these isoforms, which are also important for the expression of several cellular lung-specific genes, may be important for JSRV expression in lung epithelial cells.

* Corresponding author. Mailing address: Cancer Research Institute, University of California, Irvine, California 92697-3905. Phone: (949) 824-5554. Fax: (949) 824-4023. E-mail: hyfan{at}uci.edu.

Journal of Virology, January 2006, p. 332-341, Vol. 80, No. 1
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.1.332-341.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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