Claudine Pique

Pathways of cell-cell transmission of HTLV-1

The deltaretroviruses human T cell lymphotropic virus type 1 (HTLV-1) and human T cell lymphotropic virus type 2 (HTLV-2) have long been believed to differ from retroviruses in other genera by their mode of transmission. While other retroviruses were thought to primarily spread by producing cell-free particles that diffuse through extracellular fluids prior to binding to and infecting target cells, HTLV-1 and HTLV-2 were believed to transmit the virus solely by cell–cell interactions. This difference in transmission was believed to reflect the fact that, relative to other retroviruses, the cell-free virions produced by HTLV-infected cells are very poorly infectious. Since HTLV-1 and HTLV-2 are primarily found in T cells in the peripheral blood, spread of these viruses was believed to occur between infected and uninfected, T cells, although little was known about the cellular and viral proteins involved in this interaction. Recent studies have revealed that the method of transmission of HTLV is not unique: other retroviruses including human immunodeficiency virus (HIV) are also transmitted from cell-to-cell, and this method is dramatically more efficient than cell-free transmission. Moreover, cell–cell transmission of HTLV-1, as well as HIV, can occur following interactions between dendritic cells and T cells, as well as between T cells. Conversely, other studies have shown that cell-free HTLV-1 is not as poorly infectious as previously thought, since it is capable of infecting certain cell types. Here we summarize the recent insights about the mechanisms of cell–cell transmission of HTLV-1 and other retroviruses. We also review in vitro and in vivo studies of infection and discuss how these finding may relate to the spread of HTLV-1 between individuals.

Alteration of Blood-Brain Barrier Integrity by Retroviral Infection

The blood–brain barrier (BBB), which forms the interface between the blood and the cerebral parenchyma, has been shown to be disrupted during retroviral-associated neuromyelopathies. Human T Lymphotropic Virus (HTLV-1) Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) is a slowly progressive neurodegenerative disease associated with BBB breakdown. The BBB is composed of three cell types: endothelial cells, pericytes and astrocytes. Although astrocytes have been shown to be infected by HTLV-1, until now, little was known about the susceptibility of BBB endothelial cells to HTLV-1 infection and the impact of such an infection on BBB function. We first demonstrated that human cerebral endothelial cells express the receptors for HTLV-1 (GLUT-1, Neuropilin-1 and heparan sulfate proteoglycans), both in vitro, in a human cerebral endothelial cell line, and ex vivo, on spinal cord autopsy sections from HAM/TSP and non-infected control cases. In situ hybridization revealed HTLV-1 transcripts associated with the vasculature in HAM/TSP. We were able to confirm that the endothelial cells could be productively infected in vitro by HTLV-1 and that blocking of either HSPGs, Neuropilin 1 or Glut1 inhibits this process. The expression of the tight-junction proteins within the HTLV-1 infected endothelial cells was altered. These cells were no longer able to form a functional barrier, since BBB permeability and lymphocyte passage through the monolayer of endothelial cells were increased. This work constitutes the first report of susceptibility of human cerebral endothelial cells to HTLV-1 infection, with implications for HTLV-1 passage through the BBB and subsequent deregulation of the central nervous system homeostasis. We propose that the susceptibility of cerebral endothelial cells to retroviral infection and subsequent BBB dysfunction is an important aspect of HAM/TSP pathogenesis and should be considered in the design of future therapeutics strategies.

Current concepts regarding the HTLV-1 receptor complex

The identity of the Human T lymphotropic Virus type 1 (HTLV-1) receptor remained an unsolved puzzle for two decades, until the recent demonstration that three molecules, Glucose Transporter 1, Neuropilin-1 and Heparan Sulfate Proteoglycans are involved in HTLV-1 binding and entry. Despite these advances, several questions remain unanswered, including the precise role of each of these molecules during virus entry. In light of the most recent data, we propose a model of the HTLV-1 receptor complex and discuss its potential impact on HTLV-1 infection.

Molecular aspects of HTLV-1 entry: functional domains of the HTLV-1 surface subunit (SU) and their relationships to the entry receptors.

The initial step in retroviral infection involves specific interactions between viral envelope proteins (Env) and specific receptors on the surface of target cells. For many years, little was known about the entry receptors for HTLV-1. During this time, however, functional domains of the HTLV-1 Env were identified by analyzing the effects of neutralizing antibodies and specific mutations in Env on HTLV-1 infectivity. More recent studies have revealed that HTLV-1 infectivity involves interactions with three different molecules: heparan sulfate proteoglycans (HSPG), the VEGF-165 receptor Neuropilin 1 (NRP-1) and glucose transporter type 1 (GLUT1). Here, we revisit previously published data on the functional domains of Env in regard to the recent knowledge acquired about this multi-receptor complex. We also discuss the similarities and differences between HTLV-1 and other deltaretroviruses in regards to receptor usage.

Human Discs Large is a new negative regulator of human immunodeficiency virus-1 infectivity.

Human immunodeficiency virus (HIV)-1 replication is positively or negatively regulated through multiple interactions with host cell proteins. We report here that human Discs Large (Dlg1), a scaffold protein recruited beneath the plasma membrane and involved in the assembly of multiprotein complexes, restricts HIV-1 infectivity. The endogenous Dlg1 and HIV-1 Gag polyprotein spontaneously interact in HIV-1-chronically infected T cells. Depleting endogenous Dlg1 in either adherent cells or T cells does not affect Gag maturation, production, or release, but it enhances the infectivity of progeny viruses five- to sixfold. Conversely, overexpression of Dlg1 reduces virus infectivity by approximately 80%. Higher virus infectivity upon Dlg1 depletion correlates with increased Env content in cells and virions, whereas the amount of virus-associated Gag or genomic RNA remains identical. Dlg1 knockdown is also associated with the redistribution and colocalization of Gag and Env toward CD63 and CD82 positive vesicle-like structures, including structures that seem to still be connected to the plasma membrane. This study identifies both a new negative regulator that targets the very late steps of the HIV-1 life cycle, and an assembly pathway that optimizes HIV-1 infectivity.

Low nuclear body formation and tax SUMOylation do not prevent NF-kappaB promoter activation

BackgroundThe Tax protein encoded by Human T-lymphotropic virus type 1 (HTLV-1) is a powerful activator of the NF-κB pathway, a property critical for HTLV-1-induced immortalization of CD4+ T lymphocytes. Tax permanently stimulates this pathway at a cytoplasmic level by activating the IκB kinase (IKK) complex and at a nuclear level by enhancing the binding of the NF-κB factor RelA to its cognate promoters and by forming nuclear bodies, believed to represent transcriptionally active structures. In previous studies, we reported that Tax ubiquitination and SUMOylation play a critical role in Tax localization and NF-κB activation. Indeed, analysis of lysine Tax mutants fused or not to ubiquitin or SUMO led us to propose a two-step model in which Tax ubiquitination first intervenes to activate IKK while Tax SUMOylation is subsequently required for promoter activation within Tax nuclear bodies. However, recent studies showing that ubiquitin or SUMO can modulate Tax activities in either the nucleus or the cytoplasm and that SUMOylated Tax can serve as substrate for ubiquitination suggested that Tax ubiquitination and SUMOylation may mediate redundant rather than successive functions.ResultsIn this study, we analyzed the properties of a new Tax mutant that is properly ubiquitinated, but defective for both nuclear body formation and SUMOylation. We report that reducing Tax SUMOylation and nuclear body formation do not alter the ability of Tax to activate IKK, induce RelA nuclear translocation, and trigger gene expression from a NF-κB promoter. Importantly, potent NF-κB promoter activation by Tax despite low SUMOylation and nuclear body formation is also observed in T cells, including CD4+ primary T lymphocytes. Moreover, we show that Tax nuclear bodies are hardly observed in HTLV-1-infected T cells. Finally, we provide direct evidence that the degree of NF-κB activation by Tax correlates with the level of Tax ubiquitination, but not SUMOylation.ConclusionsThese data reveal that the formation of Tax nuclear bodies, previously associated to transcriptional activities in Tax-transfected cells, is dispensable for NF-κB promoter activation, notably in CD4+ T cells. They also provide the first evidence that Tax SUMOylation is not a key determinant for Tax-induced NF-κB activation.

Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription

The viral Tax oncoprotein plays a key role in both Human T-cell lymphotropic virus type 1 (HTLV-1)-replication and HTLV-1-associated pathologies, notably adult T-cell leukemia. Tax governs the transcription from the viral 5’LTR, enhancing thereby its own expression, via the recruitment of dimers of phosphorylated CREB to cAMP-response elements located within the U3 region (vCRE). In addition to phosphorylation, CREB is also the target of O-GlcNAcylation, another reversible post-translational modification involved in a wide range of diseases, including cancers. O-GlcNAcylation consists in the addition of O-linked-N-acetylglucosamine (O-GlcNAc) on Serine or Threonine residues, a process controlled by two enzymes: O-GlcNAc transferase (OGT), which transfers O-GlcNAc on proteins, and O-GlcNAcase (OGA), which removes it. In this study, we investigated the status of O-GlcNAcylation enzymes in HTLV-1-transformed T cells. We found that OGA mRNA and protein expression levels are increased in HTLV-1-transformed T cells as compared to control T cell lines while OGT expression is unchanged. However, higher OGA production coincides with a reduction in OGA specific activity, showing that HTLV-1-transformed T cells produce high level of a less active form of OGA. Introducing Tax into HEK-293T cells or Tax-negative HTLV-1-transformed TL-om1 T cells is sufficient to inhibit OGA activity and increase total O-GlcNAcylation, without any change in OGT activity. Furthermore, Tax interacts with the OGT/OGA complex and inhibits the activity of OGT-bound OGA. Pharmacological inhibition of OGA increases CREB O-GlcNAcylation as well as HTLV-1-LTR transactivation by Tax and CREB recruitment to the LTR. Moreover, overexpression of wild-type CREB but not a CREB protein mutated on a previously described O-GlcNAcylation site enhances Tax-mediated LTR transactivation. Finally, both OGT and OGA are recruited to the LTR. These findings reveal the interplay between Tax and the O-GlcNAcylation pathway and identify new key molecular actors involved in the assembly of the Tax-dependent transactivation complex.

Recurrent TET2 mutations in adult T cell leukemia

Deregulation of DNA methylation has been recently identified in malignant hematologic diseases such as inactivation of the Ten-ElevenTranslocation 2 (TET2) gene by haplo-insufficiency. Inactivating mutations of TET2 were first described in myeloid disorders and more recently in peripheral T-cell lymphomas especially those that are harboring T follicular helper features like angio-immunoblastic T cell lymphoma. In order to determine new oncogenic pathways in Adult T cell leukemia/lymphoma (ATLL) that could cooperate with viral oncogenic proteins, we investigated the presence of TET2 coding sequence mutations and their clinical relevance in a retrospective cohort of 49 ATL patients. We identify inactivating mutations of TET2 gene in 10 patients of 49 analyzed (20%). Of the 37 patients with aggressive forms (acute and lymphoma), 9 (24%) had TET2 mutations as only one (8%) of the 12 with indolent forms had a TET2 mutation. This last patient had nevertheless a poor outcome and died four years from the diagnosis from relapse. In addition, five patients show the same recurrent point mutation, which conducts to the loss of coding sequence in one allele and lead to haplo-insufficiency. Analysis of different clinical cases suggests that TET2 mutation could be acquired at different steps of the T cell oncogenesis and could even in some cases precede HTLV-1 infection. Analysis of viral integration is still ongoing. Here, we show that loss of TET2 is frequently associated with ATLL and seems to more frequent in aggressive forms.

Predominant role of Tax sumoylation in Tax-induced NF-kB activation in T cells

Tax is a powerful activator of the NF-kB pathway, a property that is required for HTLV-1-induced T cell immortalization. Tax activates the NF-kB pathway by acting both at cytoplasmic and nuclear levels. In the cytoplasm, Tax binds to and activates the IkB Kinase (IKK) complex while in the nucleus, Tax assembles transcriptional active nuclear bodies. Others and we have previously demonstrated that the cytoplasmic/nuclear partition and NF-kB activity of Tax critically depend on its post-translational modifications. NEMO binding and IKK activation in the cytoplasm depends on Tax ubiquitination while Tax SUMOylation facilitates Tax nuclear body formation. Based on these findings, the current view is thatTax ubiquitination and SUMOylation cooperate to ensure optimal NF-kB activation by successively regulating the cytoplasmic and nuclear events. However, many questions remain regarding the individual properties of ubiquitinated or SUMOylated Tax and how the intracellular trafficking of Tax is coordinated to NF-kB activation. To explore these issues, we took advantage of the isolation of new Tax mutants that are ubiquitinated but poorly SUMOylated. We found that lack of SUMOylation modifies neither Tax stability nor Tax ubiquitination. In addition, while absence of SUMOylation prevents Tax nuclear body formation, this does not preclude Tax import into the nucleus. Finally, absence of SUMOylation reduces the NF-kB activity of Tax by around 70% in T cells. We are currently investigating the effect of fusion to SUMO isoforms on the activity of the mutants. Based on these new findings, we will propose a refined model for Tax-induced NF-kB activation in T cells.

Exclusion from the Golgi and very low levels of HTLV-2 Tax ubiquitination do not prevent IKK-gamma/NEMO relocalization and NF-κB activation

Permanent activation of the NF-κB pathway by the HTLV-1 Tax (Tax1) viral transactivator is a key event in the induction of T-cell immortalization and participates in HTLV-1-induced leukemogenesis. Tax1-induced NF-κB activation occurs through the ubiquitin-dependent recruitment of the IKK-gamma/NEMO regulatory subunit in centrosome/Golgi-associated cytoplasmic structures, which allows RelA nuclear translocation and transcription from NF-κB-dependent promoters. Although encoding a Tax protein (Tax2) that is also able to activate NF-κB, HTLV-2 does not cause leukemia. It was hence proposed that distinct Tax localizations and transactivation mechanisms could account for these differences in pathogenesis. We therefore compared the ubiquitination status of Tax2 and Tax1 as well as their ability to induce IKK-gamma/NEMO relocalization. Surprisingly, while endogenous ubiquitination of Tax1 was easily detected, endogenous Tax2 ubiquitination was barely detectable. Indeed, Tax2 ubiquitination was only seen upon ectopic expression of ubiquitin. In addition and contrary to Tax1, Tax2 was excluded from GM130-positive Golgi structures but colocalized with calreticulin, suggesting a link between Tax ubiquitination and association with the Golgi. We further showed that a non-ubiquitinable lysine-less Tax2 mutant retained the ability to induce IKK-gamma/NEMO relocalization, RelA nuclear translocation and transcription from a NF-κB-dependent promoter. Our data indicate that contrary to Tax1, Tax2 ubiquitination is dispensable for NF-κB activation. Altogether, our results reveal new significant differences between Tax1 and Tax2 and suggest an unexpected ubiquitin-independent mechanism for Tax2-induced NF-κB activation.