HLA单倍型与HTLV-1相关疾病

日本鹿儿岛大学医学院病毒学教授园田俊郎于1991年4月5日在病毒学研究所介绍了他们在Ⅰ型嗜人T细胞病毒(HTLV-1)方面的研究进展.     

HTLV-1 TaX蛋白与T细胞的细胞周期调变

人1型T细胞白血病病毒(HTLV-1)能引发成人急性T细胞白血病(ATL)以及一种慢性渐进性的中枢神经系统疾病——热性痉挛性下身截瘫/白血病病毒相关脊髓病(TSP/HAM)。成人T细胞白血病(ATL)表现为成熟T淋巴细胞恶性增生。由于HTLV-1 Tax蛋白与T细胞增殖调控有重要关系,本文将主要综述HTLV-1 Tax蛋白如何参与调变T细胞细胞周期从而探讨Tax在T淋巴细胞转化中的作用。     

HBZ在人类T淋巴细胞白血病1型病毒HTLV-1致癌中的功能

人类T淋巴细胞白血病1型病毒(Human T-cell leukemia virus type 1,HTLV-1)是与人类疾病发生密切相关的逆转录病毒,HTLV-1的感染可引起成人T细胞白血病(Adult T-cell leukemia,ATL)。HBZ(HTLV-1bZIP factor)是由HTLV-1前病毒反义链编码的病毒蛋白。在HTVL-1所编码的病毒基因中,HBZ是唯一一个在所有ATL病人样品中持续、稳定表达的病毒基因。而且,HBZ在HTLV-1诱发肿瘤的过程中发挥着极其重要的作用。近十年来对HBZ结构及其功能的研究成为白血病研究领域的热点。因此,本文就HBZ在HTLV-1致癌机制方面的相关研究成果作一综述。     

世界卫生组织推荐的蛋白印迹法用于HlV-1,HlV-2和HTLV-Ⅰ/HTLV-Ⅱ检测结果的判断标准说明

<正>蛋白印迹技术(WB)是最常用的鉴定HlV-1,HlV-2和HTLV-Ⅰ/HTLV-Ⅱ特异性抗体的验证技术,但是否作为常规诊断方法尚有争议。试剂的准备和应用缺乏标准化程序,而且就制定统一的WB     

Role of post-translational modifications of HTLV-1 Tax in NF-κB activation

Human T-cell leukemia virus type 1 (HTLV-1), the first human retrovirus discovered, is the etiological agent of adult-T-cell leukemia/lymphoma. The HTLV-1 encoded Tax protein is a potent oncoprotein that deregulates gene expression by constitutively activating nuclear factor-κB (NF-κB). Tax activation of NF-κB is critical for the immortalization and survival of HTLV-1-infected T cells. In this review, we summarize the present knowledge on mechanisms underlying Tax-mediated NF-κB activation, with an emphasis on post-translational modifications of Tax.     

Interferon-γ Promotes Inflammation and Development of T-Cell Lymphoma in HTLV-1 bZIP Factor Transgenic Mice

Human T-cell leukemia virus type 1 (HTLV-1) is an etiological agent of several inflammatory diseases and a T-cell malignancy, adult T-cell leukemia (ATL). HTLV-1 bZIP factor (HBZ) is the only viral gene that is constitutively expressed in HTLV-1-infected cells, and it has multiple functions on T-cell signaling pathways. HBZ has important roles in HTLV-1-mediated pathogenesis, since HBZ transgenic (HBZ-Tg) mice develop systemic inflammation and T-cell lymphomas, which are similar phenotypes to HTLV-1-associated diseases. We showed previously that in HBZ-Tg mice, HBZ causes unstable Foxp3 expression, leading to an increase in regulatory T cells (Tregs) and the consequent induction of IFN-γ-producing cells, which in turn leads to the development of inflammation in the mice. In this study, we show that the severity of inflammation is correlated with the development of lymphomas in HBZ-Tg mice, suggesting that HBZ-mediated inflammation is closely linked to oncogenesis in CD4⁺ T cells. In addition, we found that IFN-γ-producing cells enhance HBZ-mediated inflammation, since knocking out IFN-γ significantly reduced the incidence of dermatitis as well as lymphoma. Recent studies show the critical roles of the intestinal microbiota in the development of Tregs in vivo. We found that even germ-free HBZ-Tg mice still had an increased number of Tregs and IFN-γ-producing cells, and developed dermatitis, indicating that an intrinsic activity of HBZ evokes aberrant T-cell differentiation and consequently causes inflammation. These results show that immunomodulation by HBZ is implicated in both inflammation and oncogenesis, and suggest a causal connection between HTLV-1-associated inflammation and ATL.     

HTLV-1病毒蛋白Tax和HBZ协同促进成人T细胞白血病发生的机制

人类T细胞白血病1型病毒(Human T-cell leukemia virus type 1,HTLV-1)是与人类疾病发生密切相关的逆转录病毒。该病毒的感染可引起成人T细胞白血病(Adult T-cell leukemia,ATL)等多种疾病的发生。Tax和HBZ(HTLV-1 basic zipper protein)是由HTLV-1前病毒编码的两个关键病毒蛋白,它们被认为在HTLV-1病毒的复制、生存和致癌过程中发挥了至关重要的作用。本文就Tax和HBZ的蛋白结构以及它们在调控病毒转录、细胞生长和凋亡、病毒潜伏期,最终协同促进成人T细胞白血病发生过程中发挥的作用作一综述。     

HTLV-Ⅰ tax反义RNA逆转小鼠神经纤维瘤

来源于转基因小鼠 (HTLV ⅠLTRtax基因 )的神经纤维瘤细胞系的细胞中 ,外源基因HTLV I tax高表达产生mRNA和蛋白质 ,使细胞出现转化表型 .当将反向插入了HTLV I taxcDNA的逆转录病毒导入这种细胞后 ,转录生成的反义RNA可抑制tax基因的表达 ,mRNA和蛋白质均减少 40 %强 ;细胞的形态和生长特征也随之发生明显变化 ;原先由Tax蛋白质激活的基因如GM -CSF ,IL -6,LT/TNF (蛋白质水平 ) ,c -myc和LIF(mRNA水平 )等的表达均下调 ;M -CSF(蛋白质水平 )和原癌基因c src(mRNA水平 )的表达上升 ;β 肌纤蛋白mRNA则不受影响 .这些变化可能是由于tax反义RNA降低了细胞内Tax蛋白质浓度的缘故 .这表明HTLV ⅠTax蛋白质维持转化细胞的形态、生长和增殖起关键性作用 ;由Tax蛋白质激活的细胞内源基因的表达受阻 ,则是转基因小鼠发生神经纤维瘤的原因 .     

Bcl-3, induced by Tax and HTLV-1, inhibits NF-κB activation and promotes autophagy

The human T cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes an aggressive leukemia known as adult T cell leukemia (ATL). The HTLV-1-encoded oncoprotein Tax induces persistent activation of the nuclear factor-κB (NF-κB) pathway, which is perceived as the primary cause of ATL. Bcl-3, a member of the NF-κB inhibitor (IκB) family, is highly expressed in many HTLV-1-infected T cell lines and ATL cells. However, the role of Bcl-3 in Tax-induced NF-κB activation has not been fully elucidated. Here, we show that Tax induces Bcl-3 expression, which in turn negatively regulates the Tax-induced NF-κB activation. Interestingly, both Bcl-3 up-regulation and NF-κB inhibition promote the autophagy process in HTLV-1-infected cells. Consistent with this, over-expression of Bcl-3 also results in enhancement of rapamycin-, pifithrin-α- or starvation-induced autophagy in control cells. Together, these data demonstrate that Bcl-3 acts as a negative regulator of NF-κB activation and promotes autophagy in HTLV-1-infected cells.     

Activation of the IκB kinase complex by HTLV-1 Tax requires cytosolic factors involved in Tax-induced polyubiquitination

Activation of NF-κB by human T cell leukaemia virus type 1 Tax is thought to be crucial in T-cell transformation and the onset of adult T cell leukaemia. Tax activates NF-κB through activation of the IκB kinase (IKK) complex, similar to cytokine-induced NF-κB activation, which involves active signalling complex formation using polyubiquitin chains as a platform. Although polyubiquitination of Tax was reported to be required for IKK activation, most studies have been performed using intact cells, in which secondary NF-κB activation can be induced by various cytokines that are secreted due to Tax-mediated primary NF-κB activation. Therefore, a cell-free assay system, in which IKK can be activated by adding highly purified recombinant Tax to cytosolic extract, was used to analyse Tax-induced IKK activation. In contrast to the cytosolic extract, the purified IKK complex was not activated by Tax, whereas, it was efficiently activated by MEKK1, that does not require polyubiquitination to activate IKK. Moreover, Tax-induced IKK activation was blocked when the cytosolic extract was mixed with either lysine-free, methylated or K63R ubiquitin. These results obtained through our cell-free assay suggest that K63-linked polyubiquitination is critical, but linear polyubiquitination is dispensable or insufficient for Tax-induced IKK activation.     

Ascorbic Acid Has Superior Ex Vivo Antiproliferative, Cell Death-Inducing and Immunomodulatory Effects over IFN-α in HTLV-1-Associated Myelopathy

Background

Clear therapeutic guidelines for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are missing due to the lack of randomized double-blind controlled clinical trials. Moderate yet similar clinical benefit has been demonstrated for IFN-α and high-dose ascorbic acid (AA) monotherapy in a large open clinical trial. However, there is a lack of in vivo and in vitro studies exploring and comparing the effects of high-dose AA and IFN-α treatment in the context of HAM/TSP. Therefore, we performed the first comparative analysis of the ex vivo and in vitro molecular and cellular mechanisms of action of IFN-α and high-dose AA in HAM/TSP.

Principal Findings

Through thymidine incorporation and quantification of Th1/Th2/Th17 cytokines, we demonstrate that high-dose AA displays differential and superior antiproliferative and immunomodulatory effects over IFN-α in HAM/TSP PBMCs ex vivo. In addition, high-dose AA, but not IFN-α, induced cell death in both HAM/TSP PBMCs and HTLV-1-infected T-cell lines MT-2 and MT-4. Microarray data combined with pathway analysis of MT-2 cells revealed AA-induced regulation of genes associated with cell death, including miR-155. Since miR-155 has recently been demonstrated to up-regulate IFN-γ, this microRNA might represent a novel therapeutic target in HAM/TSP, as recently demonstrated in multiple sclerosis, another neuroinflammatory disease. On the other hand, IFN-α selectively up-regulated antiviral and immune-related genes.

Conclusions

In comparison to IFN-α, high-dose AA treatment has superior ex vivo and in vitro cell death-inducing, antiproliferative and immunomodulatory anti-HTLV-1 effects. Differential pathway activation by both drugs opens up avenues for targeted treatment in specific patient subsets.     

Is unphosphorylated Rex,as multifunctional protein of HTLV-1, a fully intrinsically disordered protein? An in silico study

Intracellularlocation of a viral unspliced mRNA in host cell is a crucial factor for normal life of the virus. Rex is a neucleo-cytoplasmic shuffling protein of Human T-cell Leukemia Virus-1(HTLV-1)which has important role in active transport of cargo-containing RNA from nucleus to cytoplasm. Therefore, it plays a crucial role in the disease development by the virus. In spite of its importance, the 3d-structurephosphorylated and unphosphorylated of this protein has not been determined. In this study, first we predicted whether Rex protein is an ordered or disordered protein. In second step protein 3Dstructure of Rex was obtained. The content of disorder-promoting amino acids, flexibility, hydrophobicity, short linear motifs (SLiMs) and protein binding regions and probability of Rex crystallization were calculated by various In Silico methods. The3D models of Rex protein were obtained by various In Silico methods, such as homology modeling, threading and ab initio, including; I-TASSER, LOMETS, SPARSKS, ROBBETA and QUARK servers. By comparing and analyzing Qmean, z-scores and energy levels of selected models, the best structures with highest favored region in Ramachandran plot (higher than 90%) was refined with MODREFINER software. In silico analysis of Rex physicochemical properties and also predicted SLiMs and binding regions sites confirms that unphosphorylated Rex protein in HTLV-1 as Rev protin in HIV is wholly disordered protein belongs to the class of intrinsically disordered proteins with extended disorder (native coils, native pre-molten globules).     

HTLV-1 propels thymic human T cell development in "human immune system" Rag2⁻/⁻ gamma c⁻/⁻ mice

Alteration of early haematopoietic development is thought to be responsible for the onset of immature leukemias and lymphomas. We have previously demonstrated that Tax(HTLV-1) interferes with ?-selection, an important checkpoint of early thymopoiesis, indicating that human T-cell leukemia virus type 1 (HTLV-1) infection has the potential to perturb thymic human αβ T-cell development. To verify that inference and to clarify the impact of HTLV-1 infection on human T-cell development, we investigated the in vivo effects of HTLV-1 infection in a "Human Immune System" (HIS) Rag2?/?γ(c)?/? mouse model. These mice were infected with HTLV-1, at a time when the three main subpopulations of human thymocytes have been detected. In all but two inoculated mice, the HTLV-1 provirus was found integrated in thymocytes; the proviral load increased with the length of the infection period. In the HTLV-1-infected mice we observed alterations in human T-cell development, the extent of which correlated with the proviral load. Thus, in the thymus of HTLV-1-infected HIS Rag2?/?γc?/? mice, mature single-positive (SP) CD4? and CD8? cells were most numerous, at the expense of immature and double-positive (DP) thymocytes. These SP cells also accumulated in the spleen. Human lymphocytes from thymus and spleen were activated, as shown by the expression of CD25: this activation was correlated with the presence of tax mRNA and with increased expression of NF-kB dependent genes such as bfl-1, an anti-apoptotic gene, in thymocytes. Finally, hepato-splenomegaly, lymphadenopathy and lymphoma/thymoma, in which Tax was detected, were observed in HTLV-1-infected mice, several months after HTLV-1 infection. These results demonstrate the potential of the HIS Rag2?/?γ(c)?/? animal model to elucidate the initial steps of the leukemogenic process induced by HTLV-1.     

Effects of human T-cell leukemia virus type 1 (HTLV-1) p13 on mitochondrial K permeability: A new member of the viroporin family?

Human T-cell leukemia virus type-1 (HTLV-1) encodes a mitochondrial protein named p13. p13 mediates an inward K⁺ current in isolated mitochondria that leads to mitochondrial swelling, depolarization, increased respiratory chain activity and reactive oxygen species (ROS) production. These effects trigger the opening of the permeability transition pore and are dependent on the presence of K⁺ and on the amphipathic alpha helical domain of p13. In the context of cells, p13 acts as a sensitizer to selected apoptotic stimuli. Although it is not known whether p13 influences the activity of endogenous K⁺ channels or forms a channel itself, it shares some structural and functional analogies with viroporins, a class of small integral membrane proteins that form pores and alter membrane permeability.     

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, p21(CIP1/WAF1) and p27(KIP1), which cause p53-/pRb-independent cellular senescence. Here we demonstrate that p21(CIP1/WAF1)-/p27(KIP1)-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 p27(KIP1) protein and p21(CIP1/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.