The long terminal repeat of Moloney murine leukemia virus (MuLV) contains the upstream conserved region (UCR). The UCR core sequence, CGCCATTTT, binds a ubiquitous nuclear factor and mediates negative regulation of MuLV promoter activity. We have isolated murine cDNA clones encoding a protein, referred to as UCRBP, that binds specifically to the UCR core sequence. Gel mobility shift assays demonstrate that the UCRBP fusion protein expressed in bacteria binds the UCR core with specificity identical to that of the UCR-binding factor in the nucleus of murine and human cells. Analysis of full-length UCRBP cDNA reveals that it has a putative zinc finger domain composed of four C2H2 zinc fingers of the GLI subgroup and an N-terminal region containing alternating charges, including a stretch of 12 histidine residues. The 2.4-kb UCRBP message is expressed in all cell lines examined (teratocarcinoma, B-and T-cell, macrophage, fibroblast, and myocyte), consistent with the ubiquitous expression of the UCR-binding factor. Expression of type C retroviruses, including the murine leukemia viruses (MuLV), is regulated by various sequence motifs in the long terminal repeat (LTR), which are conserved in both the replication-defective endogenous and the exogenous/infectious viruses (14, 46, 47, 53). The 3' end of the LTR contains the promoter elements, the CCAAT and the TATA motifs. The middle part of the LTR contains at least six known cis motifs, and these function as a strong enhancer (27,46). This middle region may also be involved in negative regulation of viral transcription in undifferentiated embryonal carcinoma (EC) cells (17). The 5' part of the LTR of both the infectious and the MuLV-related endogenous defective retroviruses contains the upstream conserved region (UCR), whose core motif is CGCCATTTT (the location of which is shown in Fig. 1). The UCR and the other downstream motifs are conserved in over 90% of more than 35 mammalian type C retrovirus isolates (13,15,24).We have previously shown that the UCR binds a ubiquitous nuclear factor(s) and that in murine L cells, the UCR and its binding factor(s) are involved in negative regulation of MuLV promoter activity (13). In addition to our work, the UCR-binding activity also has been reported in a study of developmental regulation of MuLV in undifferentiated F9 EC cells (51). These authors found that the UCR-binding activity occurs irrespective of retinoic acid-induced differentiation of F9 cells and that developmental control of MuLV is mediated by an element independent of the UCR.With the aim of further studying the structure and function of the UCR-binding factor, we have cloned a gene encoding a UCR-binding protein (UCRBP) by screening several * Corresponding author. expression libraries with multimerized UCR. Here we report the isolation and initial characterization of a full-length UCRBP cDNA that encodes a C2H2 zinc finger protein and is capable of specifically binding to the UCR core motif. We show that transient transfection of an expressible UCRBP cDNA leads ...