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.