Sp family members preferentially interact with the promoter proximal repeat within the HTLV-I enhancer.

Human T cell lymphotropic virus type I (HTLV-I) encodes the transactivator protein, Tax, which facilitates viral transcription from three 21 bp repeated elements within the U3 region of the long terminal repeat (LTR). Examination of the basal factors interacting with the 21 bp repeat elements through electrophoretic mobility shift (EMS) analyses has demonstrated the formation of DNA-protein complexes common to each of the 21 bp repeats (C1-C3) as well as three DNA-protein complexes specific to the promoter proximal (pp) repeat (U1 (U1A/U1B) and U2; 1-4). These studies have indicated that the individual repeats are not identical with respect to the cellular factors with which they interact. EMS analyses utilizing a series of mutated pp repeat elements demonstrate that the nucleotide sequence requirements for U1 (U1A/U1B) and U2 formation are separable from those required for C1-C3 formation. Competition EMS analyses utilizing Sp1 and CREB binding site oligonucleotides demonstrate that Sp family members are critical components of U1 (U1A/U1B) and U2 and that ATF/CREB family members are critical components of C1-C3. EMS supershift analyses have demonstrated that Sp1 is involved in U1A formation while Sp3 is involved in U1B and U2 formation. EMS analyses performed with nuclear extracts from Tax-expressing Jurkat cells and HTLV-I-transformed peripheral blood mononuclear cells demonstrate that Tax prevents the formation of U1 (U1A/U1B) and U2 DNA-protein complexes. Therefore, Tax appears to inhibit the interaction of Sp family members with the pp repeat. Based on these observations, it is possible that the interaction of Sp and ATF/CREB family members with the pp repeat during basal and Tax-mediated transcription may play a critical role in viral gene expression during the initial stages of virus infection or during activation of a latent infection.

AP-1 derived from mature monocytes and astrocytes preferentially interacts with the HTLV-I promoter central 21 bp repeat.

Characterization of the cellular transcription factors interacting with the human T cell lymphotropic virus type I (HTLV-I) long terminal repeat (LTR) is essential to dissecting the mechanisms involved in viral transcription that may be pertinent to the oncogenic and neuropathogenic processes associated with HTLV-I infection in both the immune and nervous systems. Electrophoretic mobility shift (EMS) analyses utilizing oligonucleotides homologous to each of the 21 bp repeat elements reacted with nuclear extracts derived from cell lines of lymphocytic, monocytic, neuronal, and glial cell origin have demonstrated differential binding of cellular factors to the three 21 bp repeats (1-4). ATF/CREB and Sp family members interacted with the 21 bp repeats to form DNA-protein complexes common to all cell types examined. However, a unique DNA-protein complex was detected when the promoter central 21 bp repeat was reacted with nuclear extracts derived from either the U-373 MG glioblastoma cell line or the THP-1 mature monocytic cell line. Based on nucleotide sequence requirements and immunoreactivity, we demonstrate that this DNA-protein complex is comprised of the AP-1 components, Fos and Jun.

Characterization of a glial cell-specific DNA-protein complex formed with the human T cell lymphotropic virus type I (HTLV-I) enhancer.

Human T cell lymphotropic virus type I (HTLV-I) encodes the trans-activator, Tax, which facilitates viral transcription from three 21 bp repeated elements within the U3 region of the long terminal repeat (LTR). Electrophoretic mobility shift (EMS) analyses utilizing double-stranded (ds) oligonucleotides homologous to each of the 21 bp repeats and nuclear extracts derived from selected cell lines of lymphocytic, neuronal, and glial origin have demonstrated the differential binding of cellular factors to each of the three 21 bp repeats. Specifically, both a glial cell-specific DNA-protein complex (designated GCS) and 21 bp repeat-specific DNA-protein complexes (designated U1 and U2) were detected. The formation of the GCS DNA-protein complex may involve activating transcription factor (ATF)/cAMP-response element (CRE) binding protein (CREB) family member(s) while the formation of the U1 and U2 DNA-protein complexes may involve an Sp1-related factor. In addition, three ATF-CREB-related DNA-protein complexes common to each individual 21 bp repeat (designated C1-C3) were also detected. However, we demonstrated that the abundance of the C1 and C2 DNA-protein complexes detected with the individual 21 bp repeats and glial cell nuclear extract was relatively low compared to that obtained with lymphocyte, monocyte, or neuronal nuclear extracts. We also have demonstrated that the ATF-CREB factors participating formation of the GCS DNA-protein complex are distinct from those participating in formation of the C1-C3 DNA-protein complexes. Based on nucleotide sequence requirements and immunoreactivity, we suggest that the GCS DNA-protein complex may contain a novel glial cell type specific ATF-CREB-related factor(s). Furthermore, we demonstrate that the CRE modulator (CREM) protein in conjunction with the ATF/CREB factor, CREBP1, interact with each of the three 21 bp repeats to form the C3 DNA-protein complex. However, the abundance of the C3 DNA-protein complex formed utilizing the promoter proximal repeat is dramatically lower compared to either of the other two 21 bp repeat elements. Based on these observations, we suggest that the differential binding of cellular factors to each of the three 21 bp repeat elements may play a role in basal as well as Tax-mediated LTR-directed transcription within cell populations of either immune or nervous system origin.

Identification of human T-cell lymphotropic virus type I 21-base-pair repeat-specific and glial cell-specific DNA-protein complexes.

The human T-cell lymphotropic virus type I (HTLV-I)-encoded protein, Tax, is capable of trans-activating HTLV-I transcription by interacting with specific sequences in the HTLV-I long terminal repeat (LTR) which comprise an inducible enhancer containing three imperfect tandem repeats of a 21-bp sequence. There is no evidence that purified Tax can bind to DNA in the absence of cellular factors, suggesting that Tax most likely regulates transcription via interaction with cellular factors. Since HTLV-I is a documented agent of adult T-cell leukemia and tropical spastic paraparesis, disorders of the immune and nervous systems, respectively, characterization of cellular factors of lymphoid and neuroglial origin which interact with the 21-bp repeat elements is essential to understanding of the mechanisms involved in basal and Tax-mediated transcription in cells of immune and nervous system origin. Utilizing electrophoretic mobility shift (EMS) analyses, we have detected both 21-bp repeat-specific and glial cell-specific DNA-protein complexes. Several 21-bp repeat-specific DNA-protein complexes were detected when nuclear extracts derived from cells of lymphoid (Jurkat, SupT1, and H9), neuronal (IMR-32 and SK-N-MC), and glial (U-373 MG, Hs683, and U-118) origin were used in reactions with each of the three 21-bp repeat elements. In addition, a glial cell-specific DNA-protein complex was detected when nuclear extracts derived from U-373 MG, Hs683, and U-118 glial cell lines reacted with the promoter-distal and central 21-bp repeat elements. Furthermore, EMS analyses performed with nuclear extracts derived from lymphocytic and glial cell origin and a 223-bp fragment of the HTLV-I long terminal repeat encompassing the three 21-bp repeat elements (designated Tax-responsive elements 1 and 2, TRE-1/-2) have also resulted in the detection of glial cell type-specific DNA-protein complexes. Competition EMS analyses with oligonucleotides containing transcription factor binding site sequences indicate the involvement of a cyclic AMP response element binding protein in the formation of DNA-protein complexes which form with all three 21-bp repeat elements and the glial cell-specific DNA-protein complex as well as the involvement of Sp1 or an Sp1-related factor in the formation of the 21-bp repeat III-specific DNA-protein complexes.

Neuroglial-specific factors and the regulation of retrovirus transcription.

Retroviruses have been implicated as causative agents of a variety of human diseases including malignancy, immune system dysfunction, and neurologic disorders. Despite the isolation of various retroviral agents from patients suffering from malignant neoplasias and neurologic disorders, only the human T-cell lymphotropic virus type I (HTLV-I) and the human immunodeficiency virus (HIV) have been definitively accepted as etiologic agents of human disease (Hjelle, 1991; Gessain and Gout, 1992; Rosenblatt, 1993). Because of their increasingly defined roles in disease progression, the replication of HTLV-I and HIV is an important focus for understanding the pathogenic processes resulting from viral infection. Of particular interest are the molecular mechanisms by which expression of retroviral genomes is regulated by their regulatory units, the long terminal repeats (LTR), in a manner specific to the cellular targets which they infect.