Upstream Stimulatory Factors Binding to an E Box Motif in the R Region of the Bovine Leukemia Virus Long Terminal Repeat Stimulates Viral Gene Expression

Abstract

The bovine leukemia virus (BLV) promoter is located in its 5-long terminal repeat and is composed of the U3, R, and U5 regions. BLV transcription is regulated by cis-acting elements located in the U3 region, including three 21-bp enhancers required for transactivation of the BLV promoter by the virus-encoded transactivator Tax BLV . In addition to the U3 cis-acting elements, both the R and U5 regions contain stimulatory sequences. To date, no transcription factor-binding site has been identified in the R region. Here sequence analysis of this region revealed the presence of a potential E box motif (5-CACGTG-3). By competition and supershift gel shift assays, we demonstrated that the basic helix-loop-helix transcription factors USF1 and USF2 specifically interacted with this R region E box motif. Mutations abolishing upstream stimulatory factor (USF) binding caused a reproducible decrease in basal or Tax-activated BLV promoter-driven gene expression in transient transfection assays of B-lymphoid cell lines. Cotransfection experiments showed that the USF1 and USF2a transactivators were able to act through the BLV R region E box. Taken together, these results physically and functionally characterize a USF-binding site in the R region of BLV. This E box motif located downstream of the transcription start site constitutes a new positive regulatory element involved in the transcriptional activity of the BLV promoter and could play an important role in virus replication. Bovine leukemia virus (BLV)1 is a B-lymphotropic oncogenic retrovirus that infects cattle and is associated with enzootic bovine leukosis, a neoplastic proliferation of B-cells (1-6). BLV is closely related to human T-lymphotropic viruses HTLV-I and -II. The majority of BLV-infected cattle are asymptomatic carriers of the virus. Only about 30% of BLV-infected animals develop a preneoplastic condition termed persistent lymphocytosis, with 2-5% developing B-cell leukemia and/or lymphoma after a long latency period. The virus can be experimentally transmitted to sheep, in which it causes similar pathologies, providing a helpful model to understand BLV and HTLV-induced leukemogenesis. BLV infection is characterized by viral latency in the large majority of infected cells and by the absence of viremia. These features are thought to be due to the transcriptional repression of viral expression in vivo (7-9). The latency is likely to be a viral strategy to escape the host immune response and allow tumor development.BLV transcription initiates at the unique promoter located in the 5Ј-long terminal repeat (5Ј-LTR) of the BLV genome. The 5Ј-LTR is composed of the U3, R, and U5 regions and transcription initiates at the U3-R junction. The U3 region contains the main sites that regulate viral transcription (Fig. 1) as follows: the promoter CAAT and TATA boxes (10, 11), a glucocorticoidresponsive element (12-15), and a large segment protected in DNase footprinting assays containing NF-B-related sites (16,17). Among the most important sites are three copies of an imp...

Deacetylase Inhibitors and the Viral Transactivator TaxBLV Synergistically Activate Bovine Leukemia Virus Gene Expression via a cAMP-responsive Element- and cAMP-responsive Element-binding Protein-dependent Mechanism

“…The p53 expression vector was kindly provided by Dr. Bert Volgestein. The pGEM-LTR BLV used in RNase protection analysis contains a 201-bp fragment from the BLV 5ЈLTR (nt Ϫ118 to ϩ83) and was described previously (37). The dominant-negative inhibitor A-CREB cytomegalovirus expression vector was a kind gift of Dr. Charles Vinson (40).…”

“…Plasmid Constructs-The pLTRwt-luc, containing the luciferase gene under the control of the complete 5ЈLTR of the 344 BLV provirus, was described previously (37). This construct was used as a substrate for mutagenesis by the QuikChange site-directed mutagenesis method (Stratagene).…”

“…RNase Protection Assays-RNase protection assays were performed as described previously (37). The BLV-specific 32 P-labeled antisense riboprobe was synthesized in vitro by transcription of XbaI-restricted pGEM-LTR BLV with SP6 polymerase according to the protocol provided with the Riboprobe in vitro Transcription Systems (Promega).…”

“…The human T-lymphoid cell line SupT1 was obtained from J. Hoxie through the AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, National Institutes of Health). Raji and Sup T1 cell lines were grown as reported previously (37,41). The adherent cell line COS-7, a simian fibroblastic cell line, was cultured in Dulbecco's modified Eagle's-Glutamax I medium containing 5% fetal bovine serum, 50 units of penicillin/ml, and 50 g of streptomycin/ml.…”

“…Electrophoretic Mobility Shift Assays-Nuclear extracts were prepared and EMSAs and supershift assays were performed as described previously (37). The DNA sequence of the coding strand of the doublestranded TxRE2 probe (nt Ϫ139 to Ϫ133) is 5Ј-AAGCTGGTGACG-GCAGCTGGT-3Ј (the CRE-like site is underlined).…”