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Journal of Virology, July 2000, p. 5776-5787, Vol. 74, No. 13
Cancer Research Institute and Department of
Molecular Biology and Biochemistry, University of California at Irvine,
Irvine, California 92697
Received 10 February 2000/Accepted 10 March 2000
Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of a
contagious bronchioloalveolar carcinoma of sheep known as sheep pulmonary adenomatosis (SPA; ovine pulmonary carcinoma). JSRV is unique
among retroviruses because it transforms the alveolar type II cells and
the nonciliated bronchiolar cells (Clara cells) of the lungs; these
cells are where JSRV is specifically expressed in both naturally and
experimentally SPA-affected sheep. In this study, we investigated the
cell specificity of JSRV expression. By transient-transfection assays
of 23 different cell lines with a reporter plasmid driven by the JSRV
long terminal repeat (LTR), pJS21-luc, we found that the JSRV LTR is
preferentially active in cell lines derived from type II pneumocytes
and Clara cells (MLE-15 and mtCC1-2 mouse cell lines). Reporter assays
using progressive 5' deletions of pJS21-luc allowed us to establish
that the JSRV enhancers are able to activate the JSRV proximal promoter
in MLE-15 and mtCC1-2 cells, but they have very low activity in mouse
cells of other lineages (e.g., NIH 3T3). The JSRV enhancers are able to
activate heterologous promoters in both MLE-15 and 3T3 cells, although
optimal activity is achieved in MLE-15 cells only with the homologous
JSRV promoter. Thus, JSRV cell-specific LTR activity appears to result
from an interaction between the enhancer elements and the JSRV proximal
promoter elements. By mutation analysis, we established that an
upstream NF-
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The Long Terminal Repeat of Jaagsiekte Sheep
Retrovirus Is Preferentially Active in Differentiated Epithelial
Cells of the Lungs
B-like element appears to be responsible for
approximately 50% of the JSRV LTR transcriptional activity in MLE-15
cells. Electrophoretic mobility shift assays showed evidence of a
factor(s) that binds to this sequence. Antibody supershift experiments
indicated that the factor(s) is not related to NF-
B component p50 or
p52. This factor also appeared to be present in cells that do not
support a high level of JSRV expression. Finally the JSRV21
LTR contains putative enhancer binding motifs for transcription factors
such as hepatocyte nuclear factor 3 (HNF-3) that are involved in
lung-specific gene expression. Cotransfection experiments demonstrated
that exogenous HNF-3 is able to enhance the expression of pJS21-luc in
NIH 3T3 cells, which normally show minimal enhancer activity for the
JSRV LTR.
*
Corresponding author. Mailing address: Cancer Research
Institute, Bio. Sci. II, University of California Irvine, Irvine, CA 92697. Phone: (949) 824-6631. Fax: (949) 824-4023. E-mail:
hyfan{at}uci.edu.
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