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We have identified and analyzed a 27-nucleotide sequence (U5 repressive element, designated as USRE) at the U5 region of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) which is required for HTLV-I basal transcriptional repression. The basal promoter strength of constructs that contained deletions in the U5 region of the LTR was analyzed by chloramphenicol acetyltransferase (CAT) assays following transfection of HeLa cells or Jurkat T-cells in the presence or absence of viral transactivator tax protein. We consistently observed a 2-to 5-fold increase in basal promoter activity when sequences between +277 to +306 were deleted. In vivo competition experiments suggested that the U5 DNA fragment from +269 to +295 contains a functional repressive element (USRE). Using gel mobility shift assays, we have purifled a highly enriched fraction that could specifically bind USRE. This DNA afbnity column fraction contained three major detectable proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining: llO-, 80-and 'IO-kDa proteins. The llO-kDa protein appeared to be a novel DNA-binding protein whose characteristics are still obscure, while the 70-and IO-kDa proteins were shown to be related to the human autoantigen Ku, the Ku (p701p80) complex, as demonstrated by amino acid sequencing and immunological analyses. As Ku is known to be involved in transcriptional regulation, the specific interaction of Ku with USRE raises intriguing possibilities for its function in Hl?_V-I basal transcriptional repression.Key words: Human T-cell leukemia virus type I (HTLV-I); DNA binding protein; Ku protein; Transcriptional repression IutroductionHuman T cell leukemia virus type I (HTLV-I) is an exogenous human retrovirus that has been shown to be the etiologic agent of a type of acute T-cell leukemia, known as adult T-cell leukemia (ATL), as well as a neurological disorder, known as HTLV-I-associated myelopathy or tropical spastic paraparesis (HAMfTSP) [l-6], and an eye disease, HTLV-I uveitis [7]. The latter two diseases are considered to be outcomes of immune disorders caused by HTLV-I infection. After infection into humans, the virus requires a long latent period until the onset of such diseases [8]. The full mechanism of the viral latency has not been uncovered yet. Analysis of HTLV-I gene expression in vitro, however, provides some important information on the mechanisms of the viral latent infection and activation from the latent state.After integrating into host chromosomal DNA, the expression of the viral genes is known to be regulated by various viral and host nuclear factors through the viral 5' long terminal repeat (LTR). The 21-bp repeat elements, TRE-1 (HTLV-I-tax protein responsive element l), are required for the transactivation of the HTLV-I tax protein that is reported to bind indirectly to the enhancer elements through host cell nuclear factors [9-l 11. The cellular nuclear factors such as SPl, TIF-1, Ets and Myb interact with the LTR at the region located...