Previous experiments have shown that STAT-induced STAT inhibitor-1 (SSI-1; also named suppressors of cytokine signaling-1 (SOCS-1) or Janus kinase binding protein) is predominantly expressed in lymphoid organs and functions in vitro as a negative regulator of cytokine signaling. To determine the function of SOCS-1 in vivo, we generated SSI-1 transgenic mice using the lck proximal promoter that drives transgene expression in T cell lineage. In thymocytes expressing SSI-1 transgene, tyrosine phosphorylation of STATs in response to cytokines such as IFN-γ, IL-6, and IL-7 was inhibited, suggesting that SSI-1 suppresses cytokine signaling in primary lymphocytes. In addition, lck-SSI-1 transgenic mice showed a reduction in the number of thymocytes as a result of the developmental blocking during triple-negative stage. They also exhibited a relative increase in the percentage of CD4+ T cells, a reduction in the number of γδ T cells, as well as the spontaneous activation and increased apoptosis of peripheral T cells. Thus, enforced expression of SSI-1 disturbs the development of thymocytes and the homeostasis of peripheral T cells. All these features of lck-SSI-1 transgenic mice strikingly resemble the phenotype of mice lacking common γ-chain or Janus kinase-3, suggesting that transgene-derived SSI-1 inhibits the functions of common γ-chain-using cytokines. Taken together, these results suggest that SSI-1 can also inhibit a wide variety of cytokines in vivo.

The pX sequcncc of human T cell Icukcmia virus type I (HTLV-I) has been thought to bc cxprcsscd as a doubly spliced mRNA thal codes for p40lax. p27rcx and p21X. However. WC identified a novel allcrnativcly spliced mRNA in the HTLV-I infccvd cells by using rcvcrsc transcription rollowed by the polymcrasc chain rcaction. This mRNA conlains only the firs1 and third cxons of the doubly spliced mRNA and encodes only p2lX. Our data thaw this mRNA is rcsponsiblc for expressing p2lX exists in most or HTLV-I infcclcd cells strongly suggcs~s that p2lX may play a crucial role for HTLV .I rcplicalion.

We have shown that human T-cell leukemia virus type I (HTLV-I) gene expression is negatively regulated by the U5 repressive element (U5RE) of its long terminal repeat (LTR). To isolate factors binding to U5RE, we screened a cDNA expression library by south-western blotting with a U5RE probe. Screening 2 x10(6) clones gave a positive clone with a 3.8 kb insert encoding a novel 671 residue polypeptide, named HTLV-I U5RE binding protein 1 (HUB1), with five zinc finger domains and a Krüppel-associated box like domain; HUB1 may be related to a repressor belonging to the Krüppel type zinc finger protein. A 4.0 kb mRNA for HUB1 is ubiquitously expressed among all human tissues tested. HUB1 recognizes the TCCACCCC sequence as a core motif and exerts a strong repressive effect on HTLV-I LTR-mediated expression. A new repressive domain, named HUB1 repressive (HUR) domain, was identified, rather than the Krüppel-associated box like domain. The N-terminal region upstream of HUR domain seemed to be also indispensable to the repression. Thus, we propose that HUB1 is a new type repressor and plays an important role in the HTLV-I U5-mediated repression.

We have identified several nuclear proteins binding to the U5 repressive element (U5RE) at the U5 region of the human T cell leukemia virus type I (HTLV-I) long terminal repeat (LTR). In gel mobility shift assays with the U5RE DNA probe, Jurkat T cell nuclear proteins generated five different complexes, named U5RE binding protein complexes (U5RP)-A1, -A2, -A3, -B, and -C. Only U5RP-C was affected by pretreatment with an excess of poly(dI-dC) and was immunodepressed by anti-Ku/p80 antibodies, suggesting that U5RP-C is a nonspecific complex involving Ku antigen. UV cross-linking showed at least six nuclear proteins involved in the other complexes, including U5RP-A1, -A2, -A3, and -B. The sequence of the binding core element of these specific complexes, determined by competition assays and gel mobility shift assays using a series of the U5RE mutants, is CACCC which is identical to that for the Sp1 transcription factor. LTR with a mutant U5RE, which has no ability to bind with the nuclear proteins, showed stronger promoter activity than LTR with the wild U5RE, suggesting that the specific interaction of these U5RE-binding proteins might result in the U5-mediated repression. U5RP-A1 was supershifted by anti-Sp1 antibodies and U5RP-A2 and -B were supershifted by anti-Sp3 antibodies, suggesting that Sp1 or Sp3 is involved in U5RP-A1 or U5RP-A2 and -B, respectively. Although the other nuclear proteins remain to be characterized, these findings suggest that U5RE-binding proteins in U5RP-A1, -A2, -A3, and -B are involved in HTLV-I gene repression.

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...

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We recently cloned a gene encoding a new mitogenic factor (MF) from Streptococcus pyogenes NY-5. In the present study, we determined the distribution of this MF gene (ml) by PCR based upon its sequence. Of 371 streptococcal group A strains isolated from clinical specimens, 370 (99.7%) were positive for mf. The strain that was negative for the MF gene was also negative for the streptolysin 0 gene (slo). Some streptococcal strains belonging to groups C and G were negative for mf but positive for slo. Group B strains were negative for both. Furthermore, we examined the presence of mf in 54 strains belonging to 28 families and found mfonly in group A streptococci. These results indicate that mf is distributed specifically in group A streptococci and the presence of mf in clinical samples strongly suggests infection with group A streptococci.