Analysis of potential phosphorylation sites in human T cell leukemia virus type 1 Tax

The human T cell leukemia virus type 1 (HTLV-1) Tax is a phosphoprotein, however, the contribution of phosphorylation to Tax activity is unknown. Previous studies have shown that phosphorylation of Tax occurs on serine residue(s), within one tryptic fragment, in response to 4-phorbol-12-myristate-13-acetate, in both mouse and human cells. Studies were conducted in multiple cell lines to identify the specific phosphorylated serines as a prelude to functional analysis. The phosphorylation pattern of Tax was found to be different in 293T and COS-7 cells in comparison with MT-4 and Px-1 cells. However, one tryptic fragment remained consistent in comigration analyses among all cell lines. Using selected Tax serine mutants a tryptic fragment containing a serine at residue 113 believed to be the site of phosphorylation of Tax did not comigrate with the common phosphorylated tryptic fragment. Analysis of selected Tax mutants for ability totrans-activate the cytomegalovirus promoter demonstrated mutation of serine 77 to alanine reducedtrans-activation by 90% compared to wild-type Tax. However, examination of the phosphorylation pattern of the serine 77 mutant demonstrated that it is not the site of phosphorylation. These studies demonstrate the importance of using relevant cell lines to characterize the role of phosphorylation in protein function.     

Reduced frequency, diversity, and function of human T cell leukemia virus type 1-specific CD8+ T cell in adult T cell leukemia patients

Human T cell lymphotropic virus type 1 (HTLV-1)-specific CTL are thought to be immune effectors that reduce the risk of adult T cell leukemia (ATL). However, in vivo conditions of anti-HTLV-1 CTL before and after ATL development have yet to be determined. To characterize anti-HTLV-1 CTL in asymptomatic HTLV-1 carriers (AC) and ATL patients, we analyzed the frequency and diversity of HTLV-1-specific CD8+ T cells in PBMC of 35 AC and 32 ATL patients using 16 distinct epitopes of HTLV-1 Tax or Env/HLA tetramers along with intracellular cytolytic effector molecules (IFN-gamma, perforin, and granzyme B). Overall frequency of subjects possessing Tax-specific CD8+ T cells was significantly lower in ATL than AC (53 vs 90%; p = 0.001), whereas the difference in Env-specific CD8+ T cells was not statistically significant. AC possessed Tax11-19/HLA-A*0201-specific tetramer+ cells by 90% and Tax301-309/HLA-A*2402-specific tetramer+ cells by 92%. Some AC recognized more than one epitope. In contrast, ATL recognized only Tax11-19 with HLA-A*0201 and Tax301-309 with HLA-A*2402 at frequencies of 30 and 55%. There were also significant differences in percentage of cells binding Tax11-19/HLA-A*0201 and Tax301-309/HLA-A*2402 tetramers between AC and ATL. Anti-HTLV-1 Tax CD8+ T cells in AC and ATL produced IFN-gamma in response to Tax. In contrast, perforin and granzyme B expression in anti-HTLV-1 CD8+ T cells of ATL was significant lower than that of AC. Frequency of Tax-specific CD8+ T cells in AC was related to proviral load in HLA-A*0201. These results suggest that decreased frequency, diversity, and function of anti-HTLV-1 Tax CD8+ T cell clones may be one of the risks of ATL development.     

Regulation of human T cell leukemia virus expression

Retroviruses of the type C morphology have been implicated in a wide variety of diseases in animals and humans. The human T cell leukemia viruses types I (HTLV-I) and II (HTLV-II), the prototypic human-type C retroviruses, have been identified as the causative agents of some forms of human leukemia and neurological disorders. The genetic structure and regulation of the HTLVs are more complex than their avian and murine leukemia virus counterparts. In addition to the gag, pol, and env genes that encode the characteristic virion proteins of all replication competent retroviruses, the genomes of HTLV encode the non-structural proteins, Tax and Rex, which are required for regulating viral gene expression. To understand what appears to be a complex mechanism of disease induction by HTLV, elucidating the regulation and function of the viral gene products and the interaction of these products with each other, as well as with cellular factors, will be critical. This review focuses primarily on regulation of HTLV gene expression in the infected human T lymphocyte, but also discusses analogous gene regulation by the human immunodeficiency virus (HIV). It concentrates specifically on the role these gene products play in virus replication and, ultimately, pathogenesis.     

Model for studying virus attachment. II. Binding of biotinylated human T cell leukemia virus type I to human blood mononuclear cells potential targets for human T cell leukemia virus type I infection.

Purified human T cell leukemia virus type I (HTLV-I) was biotinylated and used to study its attachment to human PBMC. The use of biotinylated HTLV-I (biot-HTLV-I) in conjunction with mouse mAb specific for selected cell-surface molecules and flow cytometric analysis allowed us to positively identify virus-binding cells among a heterogeneous blood mononuclear cell population. Biot-HTLV-I efficiently bound not only to T cells, but also to B cells and monocytes. Preincubation of monocytes with excess of unlabeled HTLV-I significantly reduced the attachment of biot-HTLV-I. HTLV-I not only bound to, but also infected, B cells, as suggested by: i) in situ hybridization of a 35S-labeled full length HTLV-I DNA probe with EBV-transformed B cells, previously cocultured with HTLV-I-producing (G11MJ) T cells, and ii) hybridization of the same nick-translated 32P-labeled DNA probe with blotted DNA from similar HTLV-I-infected EBV-transformed B cells. HTLV-I infection did not affect the ability of B cells to secrete IgG. These findings suggest that HTLV-I cannot only infect cells of the T lineage, but can also infect B cells.     

A milk protein lactoferrin enhances human T cell leukemia virus type I and suppresses HIV-1 infection

Human T cell leukemia virus type I (HTLV-I) and HIV-1, causative agents of adult T cell leukemia/lymphoma and AIDS, respectively, are transmitted vertically via breast milk. Here we demonstrate that lactoferrin, a milk protein that has a variety of antimicrobial and immunomodulatory activities, facilitates replication of HTLV-I in lymphocytes derived from asymptomatic HTLV-I carriers and transmission to cord blood lymphocytes in vitro. Transient expression assays revealed that lactoferrin can transactivate HTLV-I long terminal repeat promoter. In contrast, lactoferrin inhibits HIV-1 replication, at least in part, at the level of viral fusion/entry. These results suggest that lactoferrin may have different effects on vertical transmission of the two milk-borne retroviruses.     

Retroviral interference on STAT activation in individuals coinfected with human T cell leukemia virus type 2 and HIV-1

Human T cell leukemia virus (HTLV) type-2 is a human retrovirus whose infection has not been tightly linked to human diseases. However, the fairly high prevalence of this infection among HIV-1-positive individuals indicates the importance of better understanding the potential interference of HTLV-2 infection on HIV-1 infection and AIDS. We previously demonstrated that one signature of PBMC freshly derived from HIV-1-infected individuals is the constitutive activation of a C-terminal truncated STAT5 (STAT5Delta). Therefore, we analyzed the potential activation of STATs in HTLV-2 monoinfected and HTLV-2/HIV-1 dually infected individuals. We observed that PBMC of HTLV-2-infected individuals do not show STAT activation unless they are cultivated ex vivo, in the absence of any mitogenic stimuli, for at least 8 h. The emergence of STAT activation, namely of STAT1, in culture was mostly related to the secretion of IFN-gamma. Of note, this phenomenon is not only a characteristic feature of HTLV-2-infected individuals but also occurred with PBMC of HIV-1(+) individuals. Surprisingly, HTLV-2/HIV-1 coinfection resulted in low/absent STAT activation in vivo that paralleled a diminished secretion of IFN-gamma after ex vivo cultivation. Our findings indicate that both HTLV-2 and HIV-1 infection prime T lymphocytes for STAT1 activation, but they also highlight an interference exerted by HTLV-2 on HIV-1-induced STAT1 activation. Although the nature of such a phenomenon is unclear at the present, these findings support the hypothesis that HTLV-2 may interfere with HIV-1 infection at multiple levels.     

Human T cell leukemia virus type I induces DNA synthesis and immunoglobulin secretion in human lymphocyte cultures

Viral particles obtained from HTLV-I (human T cell leukemia virus, type I)-transformed T cell lines induced immunoglobulin production by normal peripheral blood lymphocytes. Conversely, no immunoglobulin could be detected in the supernatant medium in purified B cells cultivated with HTLV-I, suggesting that the presence of T cells is mandatory for HTLV-I to induce B cell polyclonal activation. The T cell help was mediated by soluble factors, as indicated in experiments showing that cell-free conditioned medium from T lymphocytes activated by HTLV-I was able to induce B cell proliferation and differentiation. Furthermore, a direct effect of HTLV-I on B cell proliferation was demonstrated when viral particles were added to purified B cells together with suboptimal doses of Staphylococcus aureus Cowan strain I (SAC). These observations show that an immediate early effect of HTLV-I infection was exerted on B cells, mainly in a T cell-dependent manner. Such an effect may account for the hypergammaglobulinemia observed in HTLV-I seropositive individuals, and in patients with HTLV-I-associated neurological disorders.     

Ubiquitin-specific peptidase 20 targets TRAF6 and human T cell leukemia virus type 1 tax to negatively regulate NF-kappaB signaling

NF-κB plays a key role in innate and acquired immunity. Its activity is regulated through intricate signaling networks. Persistent or excessive activation of NF-κB induces diseases, such as autoimmune disorders and malignant neoplasms. Infection by human T cell leukemia virus type 1 (HTLV-1) causes a fatal hematopoietic malignancy termed adult T cell leukemia (ATL). The HTLV-1 viral oncoprotein Tax functions pivotally in leukemogenesis through its potent activation of NF-κB. Recent findings suggest that protein ubiquitination is crucial for proper regulation of NF-κB signaling and for Tax activity. Here, we report that ubiquitin-specific peptidase USP20 deubiquitinates TRAF6 and Tax and suppresses interleukin 1β (IL-1β)- and Tax-induced NF-κB activation. Our results point to USP20 as a key negative regulator of Tax-induced NF-κB signaling.     

The PDZ domain-binding motif of the human T cell leukemia virus type 1 tax protein induces mislocalization of the tumor suppressor hScrib in T cells

Interactions with cellular PDZ domain-containing proteins obviously contribute to the tumorigenic potential of several viral oncoproteins. In this regard, the oncogenic potential of the human T cell leukemia virus type 1 Tax protein correlates with its binding capacity to the tumor suppressor hDlg. Recent results show that hDlg in T cells is associated to a network of scaffolding proteins including another PDZ domain-containing protein termed hScrib. Interestingly, previous studies have revealed complementary activities of both proteins in the control of epithelial cell polarity. Here, we demonstrate that Tax can bind to hScrib and that the resulting Tax/hScrib complex is present in human T cell leukemia virus type 1-infected T cells. By confocal microscopy, we show that Tax modifies the localization of hScrib in transfected COS cells as well as in infected T cell lines and targets hScrib to particular spots exhibiting a granular distribution, mainly distributed in the cytoplasm. Given that Tax sequesters hScrib to these particular structures, we postulate that Tax might inhibit hScrib activity. Providing further support to this idea, we find that transient overexpression of hScrib attenuates T cell receptor-induced NFAT activity but that the presence of Tax counteracts this negative effect on the NFAT pathway. The fact that hDlg and hScrib are both targeted by Tax underlies their importance in T cell function.     

The p13 protein of human T cell leukemia virus type 1 (HTLV-1) modulates mitochondrial membrane potential and calcium uptake

Human T cell leukemia virus type 1 (HTLV-1) encodes p13, an 87-amino-acid protein that accumulates in the inner mitochondrial membrane. Recent studies performed using synthetic p13 and isolated mitochondria demonstrated that the protein triggers an inward potassium (K⁺) current and inner membrane depolarization. The present study investigated the effects of p13 on mitochondrial inner membrane potential (Δψ) in living cells. Using the potential-dependent probe tetramethyl rhodamine methyl ester (TMRM), we observed that p13 induced dose-dependent mitochondrial depolarization in HeLa cells. This effect was abolished upon mutation of 4 arginines in p13's α-helical domain that were previously shown to be essential for its activity in in vitro assays. As Δψ is known to control mitochondrial calcium (Ca²⁺) uptake, we next analyzed the effect of p13 on Ca²⁺ homeostasis. Experiments carried out in HeLa cells expressing p13 and organelle-targeted aequorins revealed that the protein specifically reduced mitochondrial Ca²⁺ uptake. These observations suggest that p13 might control key processes regulated through Ca²⁺ signaling such as activation and death of T cells, the major targets of HTLV-1 infection.     

Human T cell leukemia virus type 1 Tax inhibits innate antiviral signaling via NF-kappaB-dependent induction of SOCS1

Human T cell leukemia virus type 1 (HTLV-1) inhibits host antiviral signaling pathways although the underlying mechanisms are unclear. Here we found that the HTLV-1 Tax oncoprotein induced the expression of SOCS1, an inhibitor of interferon signaling. Tax required NF-κB, but not CREB, to induce the expression of SOCS1 in T cells. Furthermore, Tax interacted with SOCS1 in both transfected cells and in HTLV-1-transformed cell lines. Although SOCS1 is normally a short-lived protein, in the presence of Tax, the stability of SOCS1 was greatly increased. Accordingly, Tax enhanced the replication of a heterologous virus, vesicular stomatitis virus (VSV), in a SOCS1-dependent manner. Surprisingly, Tax required SOCS1 to inhibit RIG-I-dependent antiviral signaling, but not the interferon-induced JAK/STAT pathway. Inhibition of SOCS1 by RNA-mediated interference in the HTLV-1-transformed cell line MT-2 resulted in increased IFN-β expression accompanied by reduced HTLV-1 replication and p19(Gag) levels. Taken together, our results reveal that Tax inhibits antiviral signaling, in part, by hijacking an interferon regulatory protein.     

Persistent T cell anergy in human type 1 diabetes

An anergic phenotype has been observed in nonobese diabetic (NOD) mice and some autoreactive T cells from patients with type I diabetes. To better understand this phenomenon, we measured T cell proliferative responses to 10 diabetes-associated and up to 9 control Ags/peptides in 148 new diabetic children, 51 age- and MHC (DQ)-matched siblings (sibs), 31 patients with longstanding diabetes, and 40 healthy controls. Most (78-91%) patient and sib responses to glutamate decarboxylase of 65 kDa (GAD65), islet cell cytoplasmic autoantibody (ICA) 69, diabetes-associated T cell epitopes in ICA69 (Tep69), and heat shock protein (Hsp) 60 involved anergic T cells that required exogenous IL-2 to proliferate. Responses to proinsulin, IA-2 (and tetanus toxoid) required no IL-2 and generated sufficient cytokine to rescue anergic T cell responses. Most new patients (85%) had autoreactive T cells, three quarters targeting more than half of the diabetes Ags. Only 7.8% of the sibs and none of the controls had such multiple T cell autoreactivities, which thus characterize overt disease. Multiple anergic and nonanergic T cell autoreactivities were sustained during 2 yr follow-up after onset and in patients with longstanding (3-26 yr) diabetes. Activated patient T cells survived severe IL-2 deprivation, requiring 20-100 times less IL-2 than normal T cells to escape apoptosis. Diabetic T cell anergy thus persists for decades and is Ag and host specific but not related to disease course. Rescue by IL-2 from bystander T cells and high resistance to apoptosis may contribute to this persistence. These data explain some of the difficulties in the routine detection of disease-associated T cells, and they emphasize challenges for immunotherapy and islet transplantation.