Chou-Zen Giam

An exposed KID-like domain in human T-cell lymphotropic virus type 1 Tax is responsible for the recruitment of coactivators CBP/p300.

ABSTRACT Human T-cell lymphotropic virus type 1 (HTLV-1) transcriptional activation is mediated by the viral transactivator, Tax, and three 21-bp repeats (Tax response element [TxRE]) located in the U3 region of the viral long terminal repeat (LTR). Each TxRE contains a core cyclic AMP response element (CRE) flanked by 5′ G-rich and 3′ C-rich sequences. The TxRE binds CREB (CRE-binding protein) and Tax to form a ternary complex and confers Tax-dependent transactivation. Recent data indicate that Tax functions as a specific link to connect CREB-binding protein (CBP)/p300 in a phosphorylation-independent manner to CREB/ATF-1 assembled on the viral 21-bp repeats. GlutathioneS-transferase pull-down performed with Tax deletion mutants and peptide competition have localized the site in Tax critical for binding CBP/p300 to a highly protease-sensitive region around amino acid residues 81 to 95 (81QRTSKTLKVLTPPIT95) which lies between the domains previously proposed to be important for CREB binding and Tax subunit dimerization. Amino acid residues around the trypsin- and chymotrypsin-sensitive sites (88KVL90) of Tax bear resemblance to those in the kinase-inducible domain of CREB (129SRRPSYRKILNE140) surrounding Ser-133, which undergoes signal-induced phosphorylation to recruit CBP/p300. Site-directed mutagenesis of residues in this domain (R82A, K85A, K88A, and V89A) resulted in proteins which failed to transactivate from the HTLV-1 LTR in vivo. These mutants (K85A, K88A, and V89A) bind CREB with similar affinities as wild-type Tax, yet interaction with CBP/p300 is abrogated in various biochemical assays, indicating that the recruitment of CBP/p300 is crucial for Tax transactivation. A Tax mutant, M47, defective in the COOH-terminal transactivation domain, continued to interact with CBP/p300, suggesting that interactions with additional cellular factors are required for proper Tax function.

A human suppressor of c-Jun N-terminal kinase 1 activation by tumor necrosis factor alpha

Tumor necrosis factor α (TNFα) has pleiotropic effects on cellular metabolism. One of the signaling paths from the TNFα receptor induces a stress-activated protein kinase cascade. Components within this TNFα kinase cascade include mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1) and stress-activated protein kinase/extracellular signal-regulated kinase kinase (SEK), which regulate the activity of c-Jun N-terminal kinase 1 (JNK1). Currently, molecules upstream of MEKK1 that link TNFα receptor to downstream kinases are not well understood. Besides TNFα, many other stimuli including several oncoproteins can activate JNK1. In most cases, the signaling cascade(s) leading from oncoproteins to JNK1 is poorly elucidated. We report here that the human T-cell lymphotrophic virus, type I (HTLV-I) oncoprotein, Tax, can activate JNK1. We isolated a novel human cell factor, G-protein pathway suppressor 2 (GPS2), by its ability to bind the HTLV-I oncoprotein, and we show that this factor can potently suppress Tax activation of JNK1. In trying to understand the mechanism of GPS2 activity, we found that it also suppressed TNFα activation of JNK1 but not TNFα activation of p38 kinase nor phorbol activation of extracellular signal-regulated kinase 2. Because GPS2 has minimal effect on MEKK1- or SEK-regulated JNK1 activity, it could act at a point between the TNFα receptor and MEKK1 in the initial step(s) of this kinase cascade. Alternatively, it is not excluded that GPS2 could work in a parallel pathway that leads from TNFα to JNK1. GPS2 represents a new molecule that could contribute important insights toward how cytokine- and oncoprotein-mediated signal transduction might converge.

Human T-lymphotropic Virus Type I Tax Activates I-κB Kinase by Inhibiting I-κB Kinase-associated Serine/Threonine Protein Phosphatase 2A

I-κB kinase (IKK) is a serine/threonine kinase that phosphorylates I-κBα and I-κBβ and targets them for polyubiquitination and proteasome-mediated degradation. IKK consists of two highly related catalytic subunits, α and β, and a regulatory γ subunit, which becomes activated after serine phosphorylation of the activation loops of the catalytic domains. The human T-lymphotropic retrovirus type-I trans-activator, Tax, has been shown to interact directly with IKKγ and activates IKK via a mechanism not fully understood. Here we demonstrate that IKK binds serine/threonine protein phosphatase 2A (PP2A), and via a tripartite protein-protein interaction, Tax, IKKγ, and PP2A form a stable ternary complex.In vitro, PP2A down-regulates active IKK prepared from Tax-producing MT4 cells. In the presence of Tax, however, the ability of PP2A to inactivate IKK is diminished. Despite their interaction with IKKγ, PP2A-interaction-defective Tax mutants failed to activate NF-κB. Our data support the notion that IKKγ-associated PP2A is responsible for the rapid deactivation of IKK, and inhibition of PP2A by Tax in the context of IKK·PP2A·Tax ternary complex leads to constitutive IKK and NF-κB activation.

HTLV-1 Tax and adult T-cell leukemia.

Human T-lymphotropic virus type I (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 viral transactivator/oncoprotein, Tax, activates viral transcription and usurps regulatory mechanisms that are critical for cell growth and division to facilitate viral replication. The effects that Tax exerts on cells include potent NF-k B activation, cell cycle perturbation and cell transformation. How Tax influences ATL development is incompletely understood at present. While Tax-expression is needed at the early stages of cellular transformation, at later times most ATL cells do not express tax; therefore, genetic and epigenetic changes in HTLV-1-infected cells are believed to play an important role in the etiology of ATL. This review attempts to integrate recent literature on the biological activities of Tax and the properties of HTLV-1 transformed T-cells and ATL cells, and speculate on what cellular changes may collaborate with Tax to effect cell transformation and ATL development.

NF-κB Hyper-Activation by HTLV-1 Tax Induces Cellular Senescence, but Can Be Alleviated by the Viral Anti-Sense Protein HBZ

Activation of I-κB kinases (IKKs) and NF-κB by the human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, is thought to promote cell proliferation and transformation. Paradoxically, expression of Tax in most cells leads to drastic up-regulation of cyclin-dependent kinase inhibitors, p21CIP1/WAF1 and p27KIP1, which cause p53-/pRb-independent cellular senescence. Here we demonstrate that p21CIP1/WAF1-/p27KIP1-mediated senescence constitutes a checkpoint against IKK/NF-κB hyper-activation. Senescence induced by Tax in HeLa cells is attenuated by mutations in Tax that reduce IKK/NF-κB activation and prevented by blocking NF-κB using a degradation-resistant mutant of I-κBα despite constitutive IKK activation. Small hairpin RNA-mediated knockdown indicates that RelA induces this senescence program by acting upstream of the anaphase promoting complex and RelB to stabilize p27KIP1 protein and p21CIP1/WAF1 mRNA respectively. Finally, we show that down-regulation of NF-κB by the HTLV-1 anti-sense protein, HBZ, delay or prevent the onset of Tax-induced senescence. We propose that the balance between Tax and HBZ expression determines the outcome of HTLV-1 infection. Robust HTLV-1 replication and elevated Tax expression drive IKK/NF-κB hyper-activation and trigger senescence. HBZ, however, modulates Tax-mediated viral replication and NF-κB activation, thus allowing HTLV-1-infected cells to proliferate, persist, and evolve. Finally, inactivation of the senescence checkpoint can facilitate persistent NF-κB activation and leukemogenesis.

Human T-Lymphotropic Virus Type 1 Oncoprotein Tax Promotes Unscheduled Degradation of Pds1p/Securin and Clb2p/Cyclin B1 and Causes Chromosomal Instability

ABSTRACT Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia. The HTLV-1 transactivator, Tax, is implicated as the viral oncoprotein. Naïve cells expressing Tax for the first time develop severe cell cycle abnormalities that include increased DNA synthesis, mitotic arrest, appearance of convoluted nuclei with decondensed DNA, and formation of multinucleated cells. Here we report that Tax causes a drastic reduction in Pds1p/securin and Clb2p/cyclin B levels in yeast, rodent, and human cells and a loss of cell viability. With a temperature-sensitive mutant of the CDC23 subunit of the anaphase-promoting complex (APC), cdc23ts; a temperature-sensitive mutant of cdc20; and a cdh1-null mutant, we show that the diminution of Pds1p and Clb2p brought on by Tax is mediated via the Cdc20p-associated anaphase-promoting complex, APCCdc20p. This loss of Pds1p/securin and Clb2p/cyclin B1 occurred before cellular entry into mitosis, caused a G2/M cell cycle block, and was accompanied by severe chromosome aneuploidy in both Saccharomyces cerevisiae cells and human diploid fibroblasts. Our results support the notion that Tax aberrantly targets and activates APCCdc20p, leading to unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1, a delay or failure in mitotic entry and progression, and faulty chromosome transmission. The chromosomal instability resulting from a Tax-induced deficiency in securin and cyclin B1 provides an explanation for the highly aneuploid nature of adult T-cell leukemia cells.

Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 Transcriptional Activator Rta Is an Oligomeric DNA-Binding Protein That Interacts with Tandem Arrays of Phased A/T-Trinucleotide Motifs

ABSTRACT Kaposis sarcoma associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) encodes an immediate early transcriptional activator, Rta, which mediates viral reactivation from latency and lytic viral replication. Here we report the purification and characterizations of HHV-8 Rta and its interaction with Rta-responsive DNA elements. The Rta response element (RtaRE) in the promoter of the KSHV/HHV-8 K8 open reading frame was mapped to a 47-bp sequence (RtaRE1) and a 60-bp sequence (RtaRE2) upstream of the TATA motif. A comparison of the K8 RtaREs with other viral RtaREs revealed a pattern of multiple A/T triplets spaced with a periodicity of 10 or 20 bp. Substitutions of the in-phase A/T trinucleotides of the RtaRE1 with G/C bases greatly diminished Rta responsiveness and Rta binding. By contrast, base substitutions in an out-of-phase A/T-trinucleotide sequence had no effect. Importantly, multimers of (A/T)3N7 and N5(A/T)5N6(A/T)4 motifs supported a strong Rta response in a copy number-dependent manner. No specific sequence motifs in the spacer regions could be discerned. Potent Rta response, however, was obtained with phased A/T trinucleotides with 7-bp spacers of arbitrary sequences with high G/C content. Lengthening of the phased A/T motifs or lowering of the G/C content of the spacers resulted in a reduction in Rta response. Finally, Escherichia coli-derived Rta is an oligomer of 440 kDa in molecular size and binds RtaRE as an oligomer. These results support a model of Rta transactivation wherein the subunits of the Rta oligomer make multiple contacts with a tandem array of phased A/T triplets in the configuration of (A/T)3(G/C)7 repeats.

Human T-Cell Leukemia Virus Type 1 Infection Leads to Arrest in the G1 Phase of the Cell Cycle

ABSTRACT Infection by the human T-cell leukemia virus type 1 (HTLV-1) is thought to cause dysregulated T-cell proliferation, which in turn leads to adult T-cell leukemia/lymphoma. Early cellular changes after HTLV-1 infection have been difficult to study due to the poorly infectious nature of HTLV-1 and the need for cell-to-cell contact for HTLV-1 transmission. Using a series of reporter systems, we show that HeLa cells cease proliferation within one or two division cycles after infection by HTLV-1 or transduction of the HTLV-1 tax gene. HTLV-1-infected HeLa cells, like their tax-transduced counterparts, expressed high levels of p21CIP1/WAF1 and p27KIP1, developed mitotic abnormalities, and became arrested in G1 in senescence. In contrast, cells of a human osteosarcoma lineage (HOS) continued to divide after HTLV-1 infection or Tax expression, albeit at a reduced growth rate and with mitotic aberrations. Unique to HOS cells is the dramatic reduction of p21CIP1/WAF1 and p27KIP1 expression, which is in part associated with the constitutive activation of the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) pathway. The loss of p21CIP1/WAF1 and p27KIP1 in HOS cells apparently allows HTLV-1- and Tax-induced G1 arrest to be bypassed. Finally, HTLV-1 infection and Tax expression also cause human SupT1 T cells to arrest in the G1 phase of the cell cycle. These results suggest that productive HTLV-1 infection ordinarily leads to Tax-mediated G1 arrest. However, T cells containing somatic mutations that inactivate p21CIP1/WAF1 and p27KIP1 may continue to proliferate after HTLV-1 infection and Tax expression. These infected cells can expand clonally, accumulate additional chromosomal abnormalities, and progress to cancer.

Regulation of human T-lymphotropic virus type I latency and reactivation by HBZ and Rex.

Human T lymphotropic virus type I (HTLV-I) infection is largely latent in infected persons. How HTLV-1 establishes latency and reactivates is unclear. Here we show that most HTLV-1-infected HeLa cells become senescent. By contrast, when NF-κB activity is blocked, senescence is averted, and infected cells continue to divide and chronically produce viral proteins. A small population of infected NF-κB-normal HeLa cells expresses low but detectable levels of Tax and Rex, albeit not Gag or Env. In these “latently” infected cells, HTLV-1 LTR trans-activation by Tax persists, but NF-κB trans-activation is attenuated due to inhibition by HBZ, the HTLV-1 antisense protein. Furthermore, Gag-Pol mRNA localizes primarily in the nuclei of these cells. Importantly, HBZ was found to inhibit Rex-mediated export of intron-containing mRNAs. Over-expression of Rex or shRNA-mediated silencing of HBZ led to viral reactivation. Importantly, strong NF-κB inhibition also reactivates HTLV-1. Hence, during HTLV-1 infection, when Tax/Rex expression is robust and dominant over HBZ, productive infection ensues with expression of structural proteins and NF-κB hyper-activation, which induces senescence. When Tax/Rex expression is muted and HBZ is dominant, latent infection is established with expression of regulatory (Tax/Rex/HBZ) but not structural proteins. HBZ maintains viral latency by down-regulating Tax-induced NF-κB activation and senescence, and by inhibiting Rex-mediated expression of viral structural proteins.

Induction of p21CIP1/WAF1 expression by human T-lymphotropic virus type 1 Tax requires transcriptional activation and mRNA stabilization

HTLV-1 Tax can induce senescence by up-regulating the levels of cyclin-dependent kinase inhibitors p21CIP1/WAF1 and p27KIP1. Tax increases p27KIP1 protein stability by activating the anaphase promoting complex/cyclosome (APC/C) precociously, causing degradation of Skp2 and inactivation of SCFSkp2, the E3 ligase that targets p27KIP1. The rate of p21CIP1/WAF1 protein turnover, however, is unaffected by Tax. Rather, the mRNA of p21CIP1/WAF1 is greatly up-regulated. Here we show that Tax increases p21 mRNA expression by transcriptional activation and mRNA stabilization. Transcriptional activation of p21CIP1/WAF1 by Tax occurs in a p53-independent manner and requires two tumor growth factor-β-inducible Sp1 binding sites in the -84 to -60 region of the p21CIP1/WAF1 promoter. Tax binds Sp1 directly, and the CBP/p300-binding activity of Tax is required for p21CIP1/WAF1 trans-activation. Tax also increases the stability of p21CIP1/WAF1 transcript. Several Tax mutants trans-activated the p21 promoter, but were attenuated in stabilizing p21CIP1/WAF1 mRNA, and were less proficient in increasing p21CIP1/WAF1 expression. The possible involvement of Tax-mediated APC/C activation in p21CIP1/WAF1 mRNA stabilization is discussed.