Akt Pathway Activation by Human T-cell Leukemia Virus Type 1 Tax Oncoprotein.

Human T-cell leukemia virus (HTLV) type 1, the etiological agent of adult T-cell leukemia, expresses the viral oncoprotein Tax1. In contrast, HTLV-2, which expresses Tax2, is non-leukemogenic. One difference between these homologous proteins is the presence of a C-terminal PDZ domain-binding motif (PBM) in Tax1, previously reported to be important for non-canonical NFκB activation. In contrast, this study finds no defect in non-canonical NFκB activity by deletion of the Tax1 PBM. Instead, Tax1 PBM was found to be important for Akt activation. Tax1 attenuates the effects of negative regulators of the PI3K-Akt-mammalian target of rapamycin pathway, phosphatase and tensin homologue (PTEN), and PHLPP. Tax1 competes with PTEN for binding to DLG-1, unlike a PBM deletion mutant of Tax1. Forced membrane expression of PTEN or PHLPP overcame the effects of Tax1, as measured by levels of Akt phosphorylation, and rates of Akt dephosphorylation. The current findings suggest that Akt activation may explain the differences in transforming activity of HTLV-1 and -2.

Inducible nitric oxide synthase mediates DNA double strand breaks in Human T-Cell Leukemia Virus Type 1-induced leukemia/lymphoma.

BACKGROUND: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive and fatal malignancy of CD4(+) T-lymphocytes infected by the Human T-Cell Virus Type 1 (HTLV-1). The molecular mechanisms of transformation in ATLL have not been fully elucidated. However, genomic instability and cumulative DNA damage during the long period of latency is believed to be essential for HTLV-1 induced leukemogenesis. In addition, constitutive activation of the NF-κB pathway was found to be a critical determinant for transformation. Whether a connection exists between NF-κB activation and accumulation of DNA damage is not clear. We recently found that the HTLV-1 viral oncoprotein, Tax, the activator of the NF-κB pathway, induces DNA double strand breaks (DSBs). RESULTS: Here, we investigated whether any of the NF-κB target genes are critical in inducing DSBs. Of note, we found that inducible nitric oxide synthase (iNOS) that catalyzes the production of nitric oxide (NO) in macrophages, neutrophils and T-cells is over expressed in HTLV-1 infected and Tax-expressing cells. Interestingly, we show that in HTLV-1 infected cells, iNOS expression is Tax-dependent and specifically requires the activation of the classical NF-κB and JAK/STAT pathways. A dramatic reduction of DSBs was observed when NO production was inhibited, indicating that Tax induces DSBs through the activation of NO synthesis. CONCLUSIONS: Determination of the impact of NO on HTLV-1-induced leukemogenesis opens a new area for treatment or prevention of ATLL and perhaps other cancers in which NO is produced.

HTLV-I tax increases genetic instability by inducing DNA double strand breaks during DNA replication and switching repair to NHEJ.

BACKGROUND: Appropriate responses to damaged DNA are indispensible for preserving genome stability and preventing cancer. Tumor viruses often target DNA repair machinery to achieve transformation. The Human T-cell leukemia virus type I (HTLV-I) is the only known transforming human retrovirus and the etiological agent of Adult T-cell Leukemia (ATLL). Although HTLV-I-transformed leukemic cells have numerous genetic lesions, the precise role of the viral tax gene in this process is not fully understood. RESULTS: Our results show a novel function of HTLV-I oncoprotein Tax as an inducer of genomic DNA double strand breaks (DDSB) during DNA replication. We also found that Tax acts as a potent inhibitor of homologous recombination (HR) DNA repair through the activation of the NF-kB pathway. These results were confirmed using HTLV-I molecular clones expressing Tax at physiological levels in a natural context. We further found that HTLV-I- and Tax-transformed cells are not more susceptible to DNA damaging agents and repair DNA lesions at a rate similar to that of normal cells. Finally, we demonstrated that during S phase, Tax-associated DDSB are preferentially repaired using the error-prone non-homologous end joining (NHEJ) pathway. CONCLUSIONS: This study provides new insights in Tax effects on DNA repair and genome instability. Although it may not be self sufficient, the creation of DNA breaks and subsequent abnormal use of the non-conservative NHEJ DNA repair during the S phase in HTLV-I-infected Tax-expressing cells may cooperate with other factors to increase genetic and genome instability and favor transformation.

Human T-lymphotropic type 1 virus p30 inhibits homologous recombination and favors unfaithful DNA repair.

Whereas oncogenic retroviruses are common in animals, human T-lymphotropic virus 1 (HTLV-1) is the only transmissible retrovirus associated with cancer in humans and is etiologically linked to adult T-cell leukemia. The leukemogenesis process is still largely unknown, but relies on extended survival and clonal expansion of infected cells, which in turn accumulate genetic defects. A common feature of human tumor viruses is their ability to stimulate proliferation and survival of infected pretumoral cells and then hide by establishing latency in cells that have acquired a transformed phenotype. Whereas disruption of the DNA repair is one of the major processes responsible for the accumulation of genomic abnormalities and carcinogenesis, the absence of DNA repair also poses the threat of cell-cycle arrest or apoptosis of virus-infected cells. This study describes how the HTLV-1 p30 viral protein inhibits conservative homologous recombination (HR) DNA repair by targeting the MRE11/RAD50/NBS1 complex and favors the error-prone nonhomologous-end-joining (NHEJ) DNA-repair pathway instead. As a result, HTLV-1 p30 may facilitate the accumulation of mutations in the host genome and the cumulative risk of transformation. Our results provide new insights into how human tumor viruses may manipulate cellular DNA-damage responses to promote cancer.

HTLV-I p30 inhibits multiple S phase entry checkpoints, decreases cyclin E-CDK2 interactions and delays cell cycle progression.

BACKGROUND: Human T-cell leukemia virus type I (HTLV-I) has efficiently adapted to its host and establishes a persistent infection characterized by low levels of viral gene expression and slow proliferation of HTLV-I infected cells over decades. We have previously found that HTLV-I p30 is a negative regulator of virus expression. RESULTS: In this study we show that p30 targets multiple cell cycle checkpoints resulting in a delayed entry into S phase. We found that p30 binds to cyclin E and CDK2 and prevents the formation of active cyclin E-CDK2 complexes. In turn, this decreases the phosphorylation levels of Rb and prevents the release of E2F and its transcriptional activation of genes required for G1/S transition. Our studies also show that HTLV-II p28 does not bind cyclin E and does not affect cell cycle progression. CONCLUSIONS: In contrast to HTLV-I, the HTLV-II-related retrovirus is not oncogenic in humans. Here we report that the HTLV-I p30 delays cell cycle progression while its homologue, HTLV-II p28, does not, providing evidence for important differences between these two related retrovirus proteins.

Notch signaling contributes to proliferation and tumor formation of human T-cell leukemia virus type 1-associated adult T-cell leukemia.

The Notch signaling pathway plays an important role in cellular proliferation, differentiation, and apoptosis. Unregulated activation of Notch signaling can result in excessive cellular proliferation and cancer. Human T-cell leukemia virus type 1 (HTLV-I) is the etiological agent of adult T-cell leukemia (ATL). The disease has a dismal prognosis and is invariably fatal. In this study, we report a high frequency of constitutively activated Notch in ATL patients. We found activating mutations in Notch in more than 30% of ATL patients. These activating mutations are phenotypically different from those previously reported in T-ALL leukemias and may represent polymorphisms for activated Notch in human cancers. Compared with the exclusive activating frameshift mutations in the proline, glutamic acid, serine, and threonine (PEST) domain in T-ALLs, those in ATLs have, in addition, single-substitution mutations in this domain leading to reduced CDC4/Fbw7-mediated degradation and stabilization of the intracellular cleaved form of Notch1 (ICN1). Finally, we demonstrated that inhibition of Notch signaling by γ-secretase inhibitors reduced tumor cell proliferation and tumor formation in ATL-engrafted mice. These data suggest that activated Notch may be important to ATL pathogenesis and reveal Notch1 as a target for therapeutic intervention in ATL patients.

HTLV-I Tax-dependent and -independent events associated with immortalization of human primary T lymphocytes.

Human T-cell leukemia virus type I (HTLV-I)-associated malignancies are seen in a small percentage of infected persons. Although in vitro immortalization by HTLV-I virus is very efficient, we report that Tax has poor oncogenic activity in human primary T cells and that immortalization by Tax is rare. Sustained telomerase activity represents one of the oncogenic steps required for Tax-mediated immortalization. Tax expression was required for the growth of primary T cells, but was not sufficient to propel T cells into cell cycle in the absence of exogenous interleukin-2 (IL-2). Tax was sufficient to activate the phosphoinositide-3 kinase (PI3K)/Akt pathway as shown by down regulation of Src homology phosphatase-1 and increased phosphorylation of Akt. We also found disruption of putative tumor suppressors IL-16 and translocated promoter region (TPR) in Tax-immortalized and HTLV-I-transformed cell lines. Our results confirmed previous observations that Tax activates the anaphase-promoting complex. However, Tax did not affect the mitotic spindle checkpoint, which was also functional in HTLV-I-transformed cells. These data provide a better understanding of Tax functions in human T cells, and highlight the limitations of Tax, suggesting that other viral proteins are key to T-cell transformation and development of adult T-cell leukemia.

HTLV-1 Yin and Yang: Rex and p30 master regulators of viral mRNA trafficking.

Human retroviruses are associated with a variety of malignancies including Kaposi's sarcoma and Epstein-Barr virus-associated lymphoma in HIV infection, T-cell leukemia/lymphoma and a neurologic disorder in human T-cell lymphotropic virus type 1 (HTLV-1) infection. Both HIV and human T-cell lymphotropic virus type 1 have evolved a complex genetic organization for optimal use of their limited genome and production of all necessary structural and regulatory proteins. Use of alternative splicing is essential for balanced expression of multiple viral regulators from one genomic polycistronic RNA. In addition, nuclear export of incompletely spliced RNA is required for production of structural and enzymatic proteins and virus particles. Decisions controlling these events are largely guarded by viral proteins. In human T-cell lymphotropic virus type 1, Rex and p30 are both nuclear/nucleolar RNA binding regulatory proteins. Rex interacts with a Rex-responsive element to stimulate nuclear export of incompletely spliced RNA and increase production of virus particles. In contrast, human T-cell lymphotropic virus type 1 p30 is involved in the nuclear retention of the tax/rex mRNA leading to inhibition of virus expression and establishment of viral latency. How these two proteins, with apparently opposite functions, orchestrate virus replication and ensure vigilant control of viral gene expression is discussed.

Platelet-derived growth factor protects neurons against gp120-mediated toxicity.

The human immunodeficiency virus (HIV)-1 envelope glycoprotein gp120 has been implicated in mediating neuronal apoptosis, a hallmark feature of HIV-associated dementia (HAD). Mitigation of the toxic effects of gp120 could thus be a potential mechanism for reducing HIV toxicity in the brain. In this study the authors hypothesized that neurotrophic factor, such as platelet-derived growth factor (PDGF), could protect the neurons against gp120-mediated apoptosis. SH-SY5Y cells treated with gp120 exhibited increased cell death when measured by lactate dehydrogenase (LDH) and deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay, with concomitant loss of neurites and increased cell rounding. Pretreatment with PDGF-BB, however, reduced gp120-associated neurotoxicity and rescued the neurite outgrowth. Additionally, gp120-mediated activation of caspase-3 was also significantly reduced in cells pretreated with PDGF-BB. Antiapoptotic effects of PDGF-BB were also confirmed by monitoring levels of anti- and proapoptotic genes, Bcl-xL and Bax, respectively. Furthermore, PDGF-mediated protection against gp120 involved the phosphoinositide (PI) 3-kinase/Akt pathway. Taken together these findings lead us to suggest that PDGF-BB could be considered as a therapeutic agent that can mitigate gp120-mediated neurotoxicity in HAD.

Regulation of the human T-cell leukemia virus gene expression depends on the localization of regulatory proteins Tax, Rex and p30II in specific nuclear subdomains.

The human T-cell leukemia virus HTLV-1 encodes regulatory proteins, Tax, Rex and p30(II), which are involved in the control of viral gene expression at the transcriptional and post-transcriptional levels. Tax localizes in unique nuclear bodies that contain components of the transcription and splicing complexes. In this work, we studied the relative intracellular localizations of Tax, Rex and p30(II). Run-on transcription assays and immunocytochemistry at light and electron microscopy levels indicated that the Tax nuclear bodies included both de novo transcribed RNA and the RNA polymerase II form that is phosphorylated on its carboxy-terminal domain whereas contacts with chromatin were observed at the periphery of these nuclear bodies. Rex first accumulated in nucleolar foci and then spread across the whole nucleus to display a diffuse and punctuate nucleoplasmic distribution. This distribution of Rex was observed in HTLV-1 transformed lymphocytes and in COS cells expressing the HTLV-1 provirus. Rex colocalized with the cellular export factor CRM-1 in the nucleolar foci as well as in the nucleoplasmic foci that did not overlap with Tax nuclear bodies but were found at the boundaries of the Tax bodies. In addition, we demonstrate that p30(II) interacts with Rex and colocalizes with the Rex/CRM-1 complexes in the nucleoli leading to their clearance from the nucleoplasm. Our results suggest that transcripts originating from Tax-induced activation of gene expression at the boundaries of the Tax bodies are transported out of the nucleus by nucleoplasmic Rex/CRM-1 complexes that are first assembled in nucleolar foci. In addition, p30(II) might exert its negative effect on viral RNA transport by preventing the release of the Rex/CRM-1 complexes from sequestration in nucleolar foci. These data support the idea that the transcriptional and post-transcriptional regulation of HTLV-1 gene expression depends on the concentration of select regulatory complexes at specific area of the nucleus.

Critical role of hnRNP A1 in HTLV-1 replication in human transformed T lymphocytes.

BACKGROUND: In this study, we have examined the role of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in viral gene expression in T lymphocytes transformed by HTLV-1. RESULTS: We have previously observed that hnRNP A1 (A1) down-modulates the post transcriptional activity of Rex protein of HTLV-1. Here, we tested whether the ectopic expression of a dominant negative mutant (NLS-A1-HA) defective in shuttling activity or knockdown of the hnRNPA1 gene using RNA interference could inhibit Rex-mediated export of viral mRNAs in HTLV-1 producing C91PL T-cells. We show that the expression of NLS-A1-HA does not modify the export of Rex-dependent viral mRNAs. Conversely, inhibiting A1 expression in C91PL cells by RNA interference provoked an increase in the Rex-dependent export of unspliced and singly spliced mRNAs. Surprisingly, we also observed a significant increase in proviral transcription and an accumulation of unspliced mRNAs, suggesting that the splicing process was affected. Finally, A1 knockdown in C91PL cells increased viral production by these cells. Thus, hnRNP A1 is implicated in the modulation of the level of HTLV-1 gene expression in T cells transformed by this human retrovirus. CONCLUSIONS: These observations provide an insight into a new cellular control of HTLV-1 replication and suggest that hnRNP A1 is likely part of the regulatory mechanisms of the life cycle of this human retrovirus in T cells.