The polycythemic strain of the spleen focus-forming virus (SFFVp) contains the 
most potent murine retroviral enhancer configuration known so far for gene 
expression in myeloerythroid hematopoietic cells. In the present study, we 
mapped two crucial elements responsible for the high activity of the SFFVp 
enhancer to an altered upstream control region (UCR) containing a GC-rich motif 
(5'-GGGCGGG-3') and to a unique enhancer core (5'-TGCGGTC-3'). Acquisition of 
these motifs accounts for half of the activity of the complete retroviral 
enhancer in hematopoietic cells, irrespective of the developmental stage or 
lineage. Furthermore, the UCR motif contains the major determinant for the 
enhancer activity of SFFVp in embryonic stem (ES) cells. Using electrophoretic 
mobility shift assays, we show that the UCR of SFFVp, but not of Friend murine 
leukemia virus, is targeted by the ubiquitous transcriptional activator, Sp1. 
The core motif of SFFVp creates a specific and high-affinity target for 
polyomavirus enhancer binding protein/core binding factor (PEBP/CBF) and 
excludes access of CAAT/enhancer binding protein. Cotransfection experiments 
with ES cells imply that PEBP/CBF cooperates with the neighboring element, LVb 
(the only conserved Ets consensus in the SFFVp enhancer), and that the Sp1 motif 
in the UCR stimulates transactivation through the Ets-PEBP interaction. Putative 
secondary structures of the retroviral enhancers are proposed based on these 
data.