IN-VIVO PROTEIN-BINDING AND FUNCTIONAL-ANALYSIS OF CIS-ACTING ELEMENTS IN THE U3 REGION OF THE BOVINE LEUKEMIA-VIRUS LONG TERMINAL REPEAT

Bovine leukemia virus (BLV) is a member of the human T-cell leukemia v irus (HTLV)/BLV group of retroviruses, These viruses regulate their ow n transcription by producing Tax, a protein which activates the virus promoter region, the long terminal repeat (LTR), To explore the molecu lar mechanisms involved in the transactivation, we identified protein binding elements by in vivo footprinting and analyzed their function b y site-directed mutagenesis, We used in vivo dimethyl sulfate footprin ting by ligation-mediated PCR to detect constitutive in vivo protein-D NA interactions in a BLV-producing cell line, Bat(2)Cl(6). The U3 regi on and part of the R region of the LTR were footprinted. In addition t o the cis-acting elements (three cyclic AMP-responsive elements [CREs] and two AP4 sites) reported by others to be important for Tax-mediate d activation of the BLV LTR, we found footprints in regions flanking t hese elements and in the core promoter region. The importance of these sites for transcriptional activation was studied by site-directed mut agenesis followed by promoter function analysis of the mutants with a chloramphenicol acetyltransferase reporter system. Our data corroborat e those of others showing that the CREs are necessary for transactivat ion of the LTR, and they identify two new functional sites not previou sly reported by others. We show that the middle region of the BLV U3 c ontains multiple dual-functioning cia-acting elements which act as eit her positive or negative regulatory elements depending on the cell typ e tested, This is the first report of a functional mapping of the cis- acting elements of a virus of the HTLV/BLV group.