Daniel C. Bertolette

Cell-free HTLV-1 infects dendritic cells leading to transmission and transformation of CD4 + T cells

Cell-free human T-lymphotropic virus type 1 (HTLV-1) virions are poorly infectious in vitro for their primary target cells, CD4+ T cells. Here, we show that HTLV-1 can efficiently infect myeloid and plasmacytoid dendritic cells (DCs). Moreover, DCs exposed to HTLV-1, both before and after being productively infected, can rapidly, efficiently and reproducibly transfer virus to autologous primary CD4+ T cells. This DC-mediated transfer of HTLV-1 involves heparan sulfate proteoglycans and neuropilin-1 and results in long-term productive infection and interleukin-2–independent transformation of the CD4+ T cells. These studies, along with observations of HTLV-1–infected DCs in the peripheral blood of infected individuals, indicate that DCs have a central role in HTLV-1 transmission, dissemination and persistence in vivo. In addition to altering the current paradigm concerning how HTLV-1 transmission occurs, these studies suggest that impairment of DC function after HTLV-1 infection plays a part in pathogenesis.

Heparan sulfate proteoglycans mediate attachment and entry of human T-cell leukemia virus type 1 virions into CD4+ T cells.

ABSTRACT Heparan sulfate proteoglycans (HSPGs) are used by a number of viruses to facilitate entry into host cells. For the retrovirus human T-cell leukemia virus type 1 (HTLV-1), it has recently been reported that HSPGs are critical for efficient binding of soluble HTLV-1 SU and the entry of HTLV pseudotyped viruses into non-T cells. However, the primary in vivo targets of HTLV-1, CD4+ T cells, have been reported to express low or undetectable levels of HSPGs. For this study, we reexamined the expression of HSPGs in CD4+ T cells and examined their role in HTLV-1 attachment and entry. We observed that while quiescent primary CD4+ T cells do not express detectable levels of HSPGs, HSPGs are expressed on primary CD4+ T cells following immune activation. Enzymatic modification of HSPGs on the surfaces of either established CD4+ T-cell lines or primary CD4+ T cells dramatically reduced the binding of both soluble HTLV-1 SU and HTLV-1 virions. HSPGs also affected the efficiency of HTLV-1 entry, since blocking the interaction with HSPGs markedly reduced both the internalization of HTLV-1 virions and the titer of HTLV-1 pseudotyped viral infection in CD4+ T cells. Thus, HSPGs play a critical role in the binding and entry of HTLV-1 into CD4+ T cells.

Human T-Cell Leukemia Virus Type 1 (HTLV-1) and HTLV-2 Use Different Receptor Complexes To Enter T Cells

ABSTRACT Studies using adherent cell lines have shown that glucose transporter-1 (GLUT-1) can function as a receptor for human T-cell leukemia virus type 1 (HTLV). In primary CD4+ T cells, heparan sulfate proteoglycans (HSPGs) are required for efficient entry of HTLV-1. Here, the roles of HSPGs and GLUT-1 in HTLV-1 and HTLV-2 Env-mediated binding and entry into primary T cells were studied. Examination of the cell surface of activated primary T cells revealed that CD4+ T cells, the primary target of HTLV-1, expressed significantly higher levels of HSPGs than CD8+ T cells. Conversely, CD8+ T cells, the primary target of HTLV-2, expressed GLUT-1 at dramatically higher levels than CD4+ T cells. Under these conditions, the HTLV-2 surface glycoprotein (SU) binding and viral entry were markedly higher on CD8+ T cells while HTLV-1 SU binding and viral entry were higher on CD4+ T cells. Binding studies with HTLV-1/HTLV-2 SU recombinants showed that preferential binding to CD4+ T cells expressing high levels of HSPGs mapped to the C-terminal portion of SU. Transfection studies revealed that overexpression of GLUT-1 in CD4+ T cells increased HTLV-2 entry, while expression of HSPGs on CD8+ T cells increased entry of HTLV-1. These studies demonstrate that HTLV-1 and HTLV-2 differ in their T-cell entry requirements and suggest that the differences in the in vitro cellular tropism for transformation and in vivo pathobiology of these viruses reflect different interactions between their Env proteins and molecules on CD4+ and CD8+ T cells involved in entry.

Induction of Human T Cell Leukemia Virus Type I Receptors on Quiescent Naive T Lymphocytes by TGF-β

The retrovirus human T cell leukemia virus (HTLV) type I (HTLV-I) is primarily transmitted by breast-feeding or sexual contact, by cell-to-cell contact between T cells. TGF-β, which has been shown to enhance transmission of HTLV-I in vitro, is found at high levels in breast milk and semen. In this study, the ability of TGF-β to regulate expression of molecules involved in HTLV-I binding and entry was examined. Previous studies using a soluble form of the HTLV-I envelope protein SU have shown that quiescent human T cells do not express cell surface molecules that specifically bind SU. After T cell activation, HTLV SU binding proteins are rapidly induced. In this study, we report that TGF-β induces expression of proteins that bind soluble HTLV SU and HTLV virions on naive CD4+ T lymphocytes. The induction of these proteins occurred without cell cycle entry or expression of activation markers, involved TGF-β-induced intracellular signaling, and required de novo transcription and translation. Treatment of naive CD4+ T lymphocytes with TGF-β induced expression of GLUT-1, which has recently been reported to function as a receptor for HTLV. Treatment of a TGF-β-sensitive human myeloid cell line increased the titer of both HTLV-I- and HTLV-II-pseudotyped viruses. Although earlier studies suggested that HTLV SU binding proteins might be an early marker of T cell activation and/or cell proliferation, we report in this study that TGF-β induces binding of HTLV virions and expression of glucose transporter type 1 in primary CD4+ T lymphocytes that remain quiescent.

Nuclear localization of v-Abl leads to complex formation with cyclic AMP response element (CRE)-binding protein and transactivation through CRE motifs.

Deregulated expression of v-abl and BCR/abl genes has been associated with myeloproliferative syndromes and myelodysplasia, both of which can progress to acute leukemia. These studies identify the localization of the oncogenic form of the abl gene product encoded by the Abelson murine leukemia virus in the nuclei of myeloid cells and the association of the v-Abl protein with the transcriptional regulator cyclic AMP response element-binding protein (CREB). We have mapped the specific domains within each of the proteins responsible for this interaction. We have shown that complex formation is a prerequisite for transcriptional potentiation of CREB. Transient overexpression of the homologous cellular protein c-Abl also results in the activation of promoters containing an intact CRE. These observations identify a novel function for v-Abl, that of a transcriptional activator that physically interacts with a transcription factor.

The Receptor Complex Associated with Human T-Cell Lymphotropic Virus Type 3 (HTLV-3) Env-Mediated Binding and Entry Is Distinct from, but Overlaps with, the Receptor Complexes of HTLV-1 and HTLV-2

ABSTRACT Little is known about the transmission or tropism of the newly discovered human retrovirus, human T-cell lymphotropic virus type 3 (HTLV-3). Here, we examine the entry requirements of HTLV-3 using independently expressed Env proteins. We observed that HTLV-3 surface glycoprotein (SU) binds efficiently to both activated CD4+ and CD8+ T cells. This contrasts with both HTLV-1 SU, which primarily binds to activated CD4+ T cells, and HTLV-2 SU, which primarily binds to activated CD8+ T cells. Binding studies with heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), two molecules important for HTLV-1 entry, revealed that these molecules also enhance HTLV-3 SU binding. However, unlike HTLV-1 SU, HTLV-3 SU can bind efficiently in the absence of both HSPGs and NRP-1. Studies of entry performed with HTLV-3 Env-pseudotyped viruses together with SU binding studies revealed that, for HTLV-1, glucose transporter 1 (GLUT-1) functions at a postbinding step during HTLV-3 Env-mediated entry. Further studies revealed that HTLV-3 SU binds efficiently to naïve CD4+ T cells, which do not bind either HTLV-1 or HTLV-2 SU and do not express detectable levels of HSPGs, NRP-1, and GLUT-1. These results indicate that the complex of receptor molecules used by HTLV-3 to bind to primary T lymphocytes differs from that of both HTLV-1 and HTLV-2.

Levels of Smad7 regulate Smad and mitogen activated kinases (MAPKs) signaling and controls erythroid and megakaryocytic differentiation of erythroleukemia cells

Smad and MAPK signaling cascades are involved in erythroid and megakaryocytic differentiation. The inhibitory Smad for TGF-β/activin signaling, Smad7, may directly or indirectly affect these signaling pathways. By modulating Smad7 expression, we attempted to delineate the relevance of Smad7 during erythro-megakaryocytic (E/M) differentiation of human erythroleukemia cells. Smad7 transcripts were detected at low levels in different erythroleukemia cell lines (TF-1, HEL and K562). Reduction of expression of endogenous Smad7 by RNA interference enhanced erythroid differentiation of K562 cells in response to physiological doses of activin-A/TGF-β1. Stable over-expression of Smad7 in K562 cells (K562/7) prevented activation of Smad2/3 and MAPK (ERK1/2, p38 and JNK1/2) proteins by activin-A/TGF-β1and subsequent induction of erythroid differentiation. High levels of Smad7 also interfered with hydroxyurea- and butyrate-, but not hemin-induced erythroid differentiation. Interestingly, K562/7 cells were found to harbor a significant proportion (about 35%) of large ploy nucleated cells compared to fewer than 12% in control cells. K562/7 cells treated with phorbol 12-myristate 13-acetate (PMA), showed a great shift in ploidy towards high ploidy classes (≥8N) accompanied with an increase in the expression of the maturation marker CD42b. We showed here that: (a) low levels of endogenous Smad7 in erythroleukemia cells are physiologically relevant, and (b) high levels of Smad7 interferes with TGF-β/activin-induced Smad/MAPK signaling and erythro-differentiation and promotes megakaryocytic differentiation, possibly by blocking autocrine TGF-β.

Differential inhibitory effects of cyanovirin-N, griffithsin, and scytovirin on entry mediated by envelopes of gammaretroviruses and deltaretroviruses.

ABSTRACT The antiviral lectins griffithsin (GRFT), cyanovirin-N (CV-N), and scytovirin (SVN), which inhibit several enveloped viruses, including lentiviruses, were examined for their ability to inhibit entry mediated by Env proteins of delta- and gammaretroviruses. The glycoproteins from human T-cell leukemia virus type 1 (HTLV-1) were resistant to the antiviral effects of all three lectins. For gammaretroviruses, CV-N inhibited entry mediated by some but not all of the envelopes examined, whereas GRFT and SVN displayed only little or no effect.

Crosstalk between the Smad and the Mitogen-Activated Protein Kinase Pathways is Essential for Erythroid Differentiation of Erythroleukemia Cells Induced by TGF-β, Activin, Hydroxyurea and Butyrate

The role of crosstalk between the Smad and the MAPK signaling pathways in activin-, transforming growth factor-β (TGF-β)-, hydroxyurea (HU) - and butyrate-dependent erythroid differentiation of K562 leukemic cells was studied. Treatment with all four inducers caused transient phosphorylation of Smad2/3 and MAPK proteins including ERK, p38 and JNK. Use of specific inhibitors of p38, ERK and JNK MAPK proteins, and TGF-β type I receptor indicated that differentiation induced by each of these agents involves activation of Smad2/3 and p38 MAPK, and inhibition of ERK MAPK. Also, treatment of cells with an inhibitor of protein serine/threonine phosphatase, okadaic acid (OA), induced phosphorylation of Smad2/3, and p38 MAPK, coincident with its induction of erythroid differentiation. Specific inhibition of TGF-β type I receptor kinase activity not only abolished TGF-β/activin effects but also prevented Smad2/3 activation and erythroid differentiation induced by OA, HU and butyrate. The TGF-β type I receptor kinase inhibitor blocked OA-induced differentiation but not p38 MAPK phosphorylation demonstrating that signals from both pathways are needed. As previously observed, addition of ERK1/2 MAPK inhibitors upregulated Smad2/3 phosphorylation and enhanced differentiation, but these effects were dependent on signals from the TGF-β type I receptor. These data indicate that activation of both Smad2/3 and p38 MAPK signaling pathways is a prerequisite to induce erythroid differentiation of erythroleukemia cells by activin, TGF-β, HU, OA and butyrate.

Different routes of entry of HTLV-1 during infection of primary dendritic cells and CD4+ T cells

Although HTLV-1 is primarily found in T cells in infected individuals, ex vivo cultures of T cells are not readily infected by cell-free HTLV-1. In contrast, cell-free HTLV-1 efficiently infects cultures of primary dendritic cells. Little is known about the productive route of infection of dendritic cells, or the stage at which infection of T cells is blocked. Comparison of primary CD4+ T cells and monocyte-derived dendritic cells (MDDC) exposed to HTLV-1 revealed that, for both cell types, HTLV-1 virions bound to and entered the cells. Entry was dependent on interactions with HSPGs and neuropilin-1, molecules known to be involved in HTLV-1 entry. The observation that cell-free HTLV-1 can enter both types of cells but only efficiently infect DCs suggests that the virus can enter cells by both productive and non-productive pathways. Dendritic cells use a type of constitutive, actin-dependent endocytosis called macropinocytosis to capture antigens, and several viruses use this route to infect host cells. In T cells, macropinocytosis is not constitutive, but can be induced. Studies with inhibitors revealed that HTLV-1 infection of MDDC is markedly decreased when the cells are treated with an actin-depolymerizing agent (cytochalasin D) or a specific inhibitor of macropinocytosis (EIPA). Strikingly, treatment of primary CD4+ T cells with a peptide that induces macropinocytosis dramatically increases infection following exposure to cell-free HTLV-1. These results suggest that HTLV-1 can enter cells by both productive and non-productive pathways, and that altering the route of entry can alter the susceptibility of a given cell type to HTLV-1 infection.