Cari Petrow-Sadowski

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.

HTLV-1 uses HSPG and neuropilin-1 for entry by molecular mimicry of VEGF165

Human T-cell lymphotropic virus type 1 (HTLV-1) entry involves the interaction between the surface (SU) subunit of the Env proteins and cellular receptor(s). Previously, our laboratories demonstrated that heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), a receptor of VEGF(165), are essential for HTLV-1 entry. Here we investigated whether, as when binding VEGF(165), HSPGs and NRP-1 work in concert during HTLV-1 entry. VEGF(165) binds to the b domain of NRP-1 through both HSPG-dependent and -independent interactions, the latter involving its exon 8. We show that VEGF(165) is a selective competitor of HTLV-1 entry and that HTLV-1 mimics VEGF(165) to recruit HSPGs and NRP-1: (1) the NRP-1 b domain is required for HTLV-1 binding; (2) SU binding to target cells is blocked by the HSPG-binding domain of VEGF(165); (3) the formation of Env/NRP-1 complexes is enhanced by HSPGs; and (4) the HTLV SU contains a motif homologous to VEGF(165) exon 8. This motif directly binds to NRP-1 and is essential for HTLV-1 binding to, internalization into, and infection of CD4(+) T cells and dendritic cells. These findings demonstrate that HSPGs and NRP-1 function as HTLV-1 receptors in a cooperative manner and reveal an unexpected mimicry mechanism that may have major implications in vivo.

Infection of Lymphoid Cells by Integration-Defective Human Immunodeficiency Virus Type 1 Increases De Novo Methylation

ABSTRACT DNA methylation, by regulating the transcription of genes, is a major modifier of the eukaryotic genome. DNA methyltransferases (DNMTs) are responsible for both maintenance and de novo methylation. We have reported that human immunodeficiency virus type 1 (HIV-1) infection increases DNMT1 expression and de novo methylation of genes such as the gamma interferon gene in CD4+ cells. Here, we examined the mechanism(s) by which HIV-1 infection increases the cellular capacity to methylate genes. While the RNAs and proteins of all three DNMTs (1, 3a, and 3b) were detected in Hut 78 lymphoid cells, only the expression of DNMT1 was significantly increased 3 to 5 days postinfection. This increase was observed with either wild-type HIV-1 or an integrase (IN) mutant, which renders HIV replication defective, due to the inability of the provirus to integrate into the host genome. Unintegrated viral DNA is a common feature of many retroviral infections and is thought to play a role in pathogenesis. These results indicate another mechanism by which unintegrated viral DNA affects the host. In addition to the increase in overall genomic methylation, hypermethylation and reduced expression of thep16 INK4A gene, one of the most commonly altered genes in human cancer, were seen in cells infected with both wild-type and IN-defective HIV-1. Thus, infection of lymphoid cells with integration-defective HIV-1 can increase the methylation of CpG islands in the promoters of genes such as thep16 INK4A gene, silencing their expression.

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.

Neutralization of Autocrine Transforming Growth Factor‐β in Human Cord Blood CD34+CD38−Lin− Cells Promotes Stem‐Cell‐Factor‐Mediated Erythropoietin‐Independent Early Erythroid Progenitor Development and Reduces Terminal Differentiation

Transforming growth factor (TGF)‐β1 exerts autocrine and paracrine effects on hematopoiesis. Here, we have attempted to evaluate the effect of endogenous TGF‐β1 on early erythroid development from primitive human hematopoietic stem cells (HSCs) and to assess the effects of TGF‐β1 on different phases of erythropoiesis. Cord blood CD34+CD38− lineage‐marker‐negative (Lin−) cells were cultured in serum‐free conditions using various combinations of stem cell factor (SCF), erythropoietin (Epo), and TGF‐β‐neutralizing antibody. Generation of erythroid progenitors was assessed using colony assay and flow cytometry. Terminal erythroid differentiation was examined when SCF/Epo‐stimulated cells were recultured in the presence of Epo with and without TGF‐β1. Anti‐TGF‐β augmented the proliferation of CD34+CD38−Lin− cells (day 21) in SCF‐stimulated (6.4‐fold ± 1.5‐fold) and SCF/Epo‐stimulated (2.9‐fold ± 1.2‐fold) cultures. Cells stimulated by SCF/Epo underwent similar levels of erythroid differentiation with and without anti‐TGF‐β. While SCF alone stimulated the production of tryptase‐positive mast cells, cells stimulated by SCF/anti‐TGF‐β were predominantly erythroid (CD36+CD14− and glycophorin A positive). A distinct expansion of erythroid progenitors (CD34+CD36+CD14−) with the potential to form erythroid colonies was seen, revealing early Epo‐independent erythroid development. In contrast, the kinetics of erythroid progenitor generation from primitive HSCs indicate that TGF‐β1 is not inhibitory in late erythropoiesis, but it accelerated the conversion of large BFU‐E into colony‐forming units‐erythroid. Finally, TGF‐β1 accelerated Epo‐induced terminal erythroid differentiation and resulted in a greater level of enucleation (22% ± 6% versus 7% ± 3%) in serum‐free conditions. Serum addition stimulated enucleation (54% ± 18%), which was lower (26% ± 14%) with anti‐TGF‐β, suggesting that optimal erythroid enucleation is Epo dependent, requiring serum factors including TGF‐β1.

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.

Similar Regulation of Cell Surface Human T-Cell Leukemia Virus Type 1 (HTLV-1) Surface Binding Proteins in Cells Highly and Poorly Transduced by HTLV-1-Pseudotyped Virions

ABSTRACT Little is known about the requirements for human T-cell leukemia virus type 1 (HTLV-1) entry, including the identity of the cellular receptor(s). Previous studies have shown that although the HTLV receptor(s) are widely expressed on cell lines of various cell types from different species, cell lines differ dramatically in their susceptibility to HTLV-Env-mediated fusion. Human cells (293, HeLa, and primary CD4+ T cells) showed higher levels of binding at saturation than rodent (NIH 3T3 and NRK) cells to an HTLV-1 SU immunoadhesin. A direct comparison of the binding of the HTLV-1 surface glycoprotein (SU) immunoadhesin and transduction by HTLV-1 pseudotyped virus revealed parallels between the level of binding and the titer for various cell lines. When cells were treated with phorbol myristate acetate (PMA), which down-modulates a number of cell surface molecules, the level of SU binding was markedly reduced. However, PMA treatment only slightly reduced the titer of murine leukemia virus(HTLV-1) on both highly susceptible and poorly susceptible cells. Treatment of target cells with trypsin greatly reduced binding, indicating that the majority of HTLV SU binding is to proteins. Polycations, which enhance the infectivity of several other retroviruses, inhibited HTLV-1 Env-mediated binding and entry on both human and rodent cells. These results suggest that factors other than the number of primary binding receptors are responsible for the differences in the titers of HTLV-1 pseudotypes between highly susceptible cells and poorly susceptible cells.

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.

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.