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The highly conserved coadapters CREB binding protein (CBP) and p300 form complexes with CREB as well as other DNA binding transcription factors to modulate chromatin remodeling and thus transcription. Human T-lymphotropic virus type 1 (HTLV-1) transcription is controlled, in part, by the CREB/ATF family of transcription factors which bind promoter sequences and function as complexes with the viral oncogenic protein Tax. We have reported that the nuclear localizing protein p30 II of HTLV-1 functions as a transcription factor, differentially modulates CREB-responsive promoters, and is critical for maintenance of proviral loads in rabbits. In this study, we tested whether p30 II directly interacts with CBP/p300 to modulate gene transcription. Gal4(BD)-p30II -mediated transactivation was enhanced following exogenous expression of p300 and was competitively repressed by the p300 binding protein, adenovirus E1A, and E1ACR2 (mutated for retinoblastoma binding but retaining p300 binding). In contrast, E1ACR1 (mutated for p300 binding) failed to alter Gal4(BD)-p30 II -mediated transactivation. In addition, Gal4(BD)-p30 II -mediated transactivation was competitively inhibited by the cotransfection of CMV-p30 II -HA and CMV-Tax but could be rescued by exogenous p300. Importantly, we demonstrate that p30 II colocalizes with p300 in cell nuclei and directly binds to CBP/p300 in cells. Deletion mutants of CBP/p300 were used to localize the site critical for binding p30 II to a highly conserved KIX region. DNA binding assays confirmed the interference of p30 II with the assembly of CREB-Tax-p300/CBP multiprotein complexes on 21-bp repeat oligonucleotides in vitro. Collectively, our results demonstrate that CBP/p300 is a cellular protein target for HTLV-1 p30 II and mediates its transcriptional effects in vivo.The coactivators CREB binding protein (CBP) and p300 mediate transcriptional control of various cellular and viral DNA binding transcription factors. These coactivators are highly similar in nucleotide sequence, are evolutionarily conserved, and are often referred to together as CBP/p300, despite evidence of divergent functions (10, 25). These proteins bridge transcription factors to relevant promoters, have intrinsic histone acetyltransferase (HAT) activity, and form complexes with proteins such as CBP/p300 binding protein-associated factor, which also exhibits HAT activity (26). Recent reviews provide a growing list of cellular and viral proteins that interact with either CBP or p300, including steroid and retinoid hormone receptors, CREB, c-Jun, c-Myb, Sap-1a, c-Fos, MyoD, p53, Stat-1/2, NF-B, pp90rsk , TATA-binding protein, and TFIIB (4, 25, 29, 30). Among viral regulatory proteins, human T-lymphotropic virus type 1 (HTLV-1) Tax, adenovirus E1A, Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor protein, and simian virus 40 large T antigen also target and affect CBP and p300 functions (1-3, 17, 32, 38, 41, 52).Complex retroviruses, like HTLV-1, must regulate their gene expression in cooperation...

Human T-lymphotropic virus type 1 (HTLV-1) is a complex retrovirus encoding regulatory and accessory genes in four open reading frames (ORF I to IV) of the pX region. Emerging evidence indicates an important role for the pX ORF I-encoded accessory protein p12 I in viral replication, but its contribution to viral pathogenesis remains to be defined. p12I is a conserved, membrane-associated protein containing four SH3-binding motifs (PXXP). Its interaction with the interleukin-2 (IL-2) receptor ␤-and ␥-chains implies an involvement of p12 I in intracellular signaling pathways. In addition, we have demonstrated that expression of pX ORF I p12 I is essential for persistent infection in rabbits. In contrast, standard in vitro systems have thus far failed to demonstrate a contribution of p12 I to viral infectivity and ultimately cellular transformation. In this study we developed multiple in vitro coculture assays to evaluate the role of p12 I in viral infectivity in quiescent peripheral blood mononuclear cells to more accurately reflect the virus-cell interactions as they occur in vivo. Using these assays, we demonstrate a dramatic reduction in viral infectivity in quiescent T lymphocytes for a p12 mutant viral clone (ACH.p12) in comparison to the wild-type clone ACH. Moreover, addition of IL-2 and phytohemagglutinin during the infection completely rescued the ability of ACH.p12 to infect primary lymphocytes. When newly infected primary lymphocytes are used to passage virus, ACH.p12 also exhibited a reduced ability to productively infect activated lymphocytes. Our data are the first to demonstrate a functional role for pX ORF I in the infection of primary lymphocytes and suggest a role for p12 I in activation of host cells during early stages of infection.

One of the early events in O2 chemoreception is inhibition of O2‐sensitive K+ (KO2) channels. Characterization of the molecular composition of the native KO2 channels in chemosensitive cells is important to understand the mechanism(s) that couple O2 to the KO2 channels. The rat phaeochromocytoma PC12 clonal cell line expresses an O2‐sensitive voltage‐dependent K+ channel similar to that recorded in other chemosensitive cells. Here we examine the possibility that the Kv1.2 α‐subunit comprises the KO2 channel in PC12 cells. Whole‐cell voltage‐clamp experiments showed that the KO2 current in PC12 cells is inhibited by charybdotoxin, a blocker of Kv1.2 channels. PC12 cells express the Kv1.2 α‐subunit of K+ channels: Western blot analysis with affinity‐purified anti‐Kv1.2 antibody revealed a band at ≈80 kDa. Specificity of this antibody was established in Western blot and immunohystochemical studies. Anti‐Kv1.2 antibody selectively blocked Kv1.2 current expressed in the Xenopus oocyte, but had no effect on Kv2.1 current. Anti‐Kv1.2 antibody dialysed through the patch pipette completely blocked the KO2 current, while the anti‐Kv2.1 and irrelevant antibodies had no effect. The O2 sensitivity of recombinant Kv1.2 and Kv2.1 channels was studied in Xenopus oocytes. Hypoxia inhibited the Kv1.2 current only. These findings show that the KO2 channel in PC12 cells belongs to the Kv1 subfamily of K+ channels and that the Kv1.2 α‐subunit is important in conferring O2 sensitivity to this channel.

Human T lymphotropic virus type 1 (HTLV-1) is a complex retrovirus containing regulatory and accessory genes encoded in four open reading frames (ORF I-IV) of the pX region. It is not clear what role pX ORFs I and II-encoded proteins have in the pathogenesis of the lymphoproliferative diseases associated with HTLV-1 infection. The conserved ORF I encodes for a hydrophobic 12-kDa protein, p12, (I) that contains four SH3 binding motifs (PXXP) that localizes to cellular endomembranes when overexpressed in cultured cells. Differential splicing of pX ORF II results in the production of two nuclear proteins, p13(II) and p30(II). p13(II) also localizes to mitochondria. p30(II) shares homology with the POU family of transcription factors. We have identified functional roles of pX ORF I and ORF II in establishment and maintenance of infection in a rabbit model. To functionally study p12(I) we have tested a proviral clone with selective ablation of ORF I (ACH.p12(I)) for its ability to infect quiescent peripheral blood mononuclear cells (PBMC). Our data indicate that T cells infected with the wild-type clone of HTLV-1 (ACH) are more efficient than ACH.p12(I) in infecting quiescent PBMC. These findings parallel our animal model data and suggest a role for p12(I) in the activation of quiescent lymphocytes, a prerequisite for effective viral replication in vivo. To test the ability of p30(II) to function as a transcription factor we have constructed p30(II) as a Gal4-fusion protein. When transfected with Gal4-driven luciferase reporter genes, the p30(II)-Gal4-fusion protein induces transcriptional activity up to 50-fold in both 293 and HeLa-Tat cells. These systems will be useful to identify molecular mechanisms that explain the functional role of pX ORF I and ORF II-encoded proteins in HTLV-1 replication.