RESUMO
UNLABELLED: Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease develops upon infiltration of HTLV-1-infected lymphocytes into the central nervous system, mostly the thoracic spinal cord. The central nervous system is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. In this study, we investigated the role of activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily, in the crossing of the BBB by HTLV-1-infected lymphocytes. We demonstrated that ALCAM is overexpressed on the surface of HTLV-1-infected lymphocytes, both in chronically infected cell lines and in primary infected CD4(+) T lymphocytes. ALCAM overexpression results from the activation of the canonical NF-κB pathway by the viral transactivator Tax. In contrast, staining of spinal cord sections of HAM/TSP patients showed that ALCAM expression is not altered on the BBB endothelium in the context of HTLV-1 infection. ALCAM blockade or downregulation of ALCAM levels significantly reduced the migration of HTLV-1-infected lymphocytes across a monolayer of human BBB endothelial cells. This study suggests a potential role for ALCAM in HAM/TSP pathogenesis. IMPORTANCE: Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease is the consequence of the infiltration of HTLV-1-infected lymphocytes into the central nervous system (CNS), mostly the thoracic spinal cord. The CNS is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. The mechanism of migration of lymphocytes into the CNS is unclear. Here, we show that the viral transactivator Tax increases activated leukocyte cell adhesion molecule (ALCAM/CD166) expression. This molecule facilitates the migration of lymphocytes across the BBB endothelium. Targeting this molecule could be of interest in preventing or reducing the development of HAM/TSP.
Assuntos
Antígenos CD/metabolismo , Barreira Hematoencefálica , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/virologia , Moléculas de Adesão Celular Neuronais/metabolismo , Movimento Celular , Proteínas Fetais/metabolismo , Interações Hospedeiro-Patógeno , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Linfócitos T CD4-Positivos/química , Linhagem Celular , Células Endoteliais/química , Produtos do Gene tax/metabolismo , Humanos , NF-kappa B/metabolismoRESUMO
Being capable of characterizing DNA local bending is essential to understand thoroughly many biological processes because they involve a local bending of the double helix axis, either intrinsic to the sequence or induced by the binding of proteins. Developing a method to measure DNA bend angles that does not perturb the conformation of the DNA itself or the DNA-protein complex is a challenging task. Here, we propose a joint theory-experiment high-throughput approach to rigorously measure such bend angles using the Tethered Particle Motion (TPM) technique. By carefully modeling the TPM geometry, we propose a simple formula based on a kinked Worm-Like Chain model to extract the bend angle from TPM measurements. Using constructs made of 575 base-pair DNAs with in-phase assemblies of one to seven 6A-tracts, we find that the sequence CA6CGG induces a bend angle of 19° ± 4°. Our method is successfully compared to more theoretically complex or experimentally invasive ones such as cyclization, NMR, FRET or AFM. We further apply our procedure to TPM measurements from the literature and demonstrate that the angles of bends induced by proteins, such as Integration Host Factor (IHF) can be reliably evaluated as well.
Assuntos
DNA/química , Sequência de Bases , DNA/metabolismo , Fatores Hospedeiros de Integração/metabolismo , Modelos Químicos , Movimento (Física) , Conformação de Ácido Nucleico , Física/métodosRESUMO
EVER1 and EVER2 are mutated in epidermodysplasia verruciformis patients, who are susceptible to human betapapillomavirus (HPV) infection. It is unknown whether their products control the infection of other viruses. Here, we show that the expression of both genes in B cells is activated immediately after Epstein-Barr virus (EBV) infection, whereas at later stages, it is strongly repressed via activation of the NF-κB signaling pathway by latent membrane protein 1 (LMP1). Ectopic expression of EVER1 impairs the ability of EBV to infect B cells.
Assuntos
Epidermodisplasia Verruciforme/patologia , Regulação da Expressão Gênica , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Proteínas de Membrana/biossíntese , Proteínas da Matriz Viral/metabolismo , Linfócitos B/virologia , Humanos , Proteínas de Membrana/genéticaRESUMO
Efficient HTLV-1 viral transmission occurs through cell-to-cell contacts. The Tax viral transcriptional activator protein facilitates this process. Using a comparative transcriptomic analysis, we recently identified a series of genes up-regulated in HTLV-1 Tax expressing T-lymphocytes. We focused our attention towards genes that are important for cytoskeleton dynamic and thus may possibly modulate cell-to-cell contacts. We first demonstrate that Gem, a member of the small GTP-binding proteins within the Ras superfamily, is expressed both at the RNA and protein levels in Tax-expressing cells and in HTLV-1-infected cell lines. Using a series of ChIP assays, we show that Tax recruits CREB and CREB Binding Protein (CBP) onto a c-AMP Responsive Element (CRE) present in the gem promoter. This CRE sequence is required to drive Tax-activated gem transcription. Since Gem is involved in cytoskeleton remodeling, we investigated its role in infected cells motility. We show that Gem co-localizes with F-actin and is involved both in T-cell spontaneous cell migration as well as chemotaxis in the presence of SDF-1/CXCL12. Importantly, gem knock-down in HTLV-1-infected cells decreases cell migration and conjugate formation. Finally, we demonstrate that Gem plays an important role in cell-to-cell viral transmission.
Assuntos
Citoesqueleto/metabolismo , Vírus Linfotrópico T Tipo 1 Humano , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Linfócitos T/metabolismo , Linfócitos T/virologia , Linhagem Celular , Quimiotaxia de Leucócito/fisiologia , Imunoprecipitação da Cromatina , Imunofluorescência , Regulação Viral da Expressão Gênica/fisiologia , Produtos do Gene tax/metabolismo , Immunoblotting , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/patologia , Ativação Transcricional/fisiologia , Transdução GenéticaRESUMO
The human T-cell leukemia/lymphoma virus type 1 (HTLV-1) p30 protein, essential for virus infectivity in vivo, is required for efficient infection of human dendritic cells (DCs) but not B and T cells in vitro. We used a human monocytic cell line, THP-1, and dendritic cells to study the mechanism of p30 and p12/p8 requirements in these cell types. p30 inhibited the expression of interferon (IFN)-responsive genes (ISG) following stimulation by lipopolysaccharide (LPS) of Toll-like receptor 4 (TLR4) and by poly(I·C) of TLR3 but not of TLR7/8 with imiquimod. Results with THP-1 mirrored those for ex vivo human primary monocytes and monocyte-derived dendritic cells (Mo-mDC). The effect of p30 on TLR signaling was also demonstrated by ablating its expression within a molecular clone of HTLV-1. HTLV-1 infection of monocytes inhibited TLR3- and TLR4-induced ISG expression by 50 to 90% depending on the genes, whereas the isogenic clone p30 knockout virus was less effective at inhibiting TLR3 and TRL4 signaling and displayed lower infectivity. Viral expression and inhibition of ISG transcription was, however, rescued by restoration of p30 expression. A chromatin immunoprecipitation assay demonstrated that p30 inhibits initiation and elongation of PU.1-dependent transcription of IFN-α1, IFN-ß, and TLR4 genes upon TLR stimulation. In contrast, experiments conducted with p12/p8 did not demonstrate an effect on ISG expression. These results provide a mechanistic explanation of the requirement of p30 for HTLV-1 infectivity in vivo, suggest that dampening interferon responses in monocytes and DCs is specific for p30, and represent an essential early step for permissive HTLV-1 infection and persistence.
Assuntos
Células Dendríticas/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Monócitos/metabolismo , Transdução de Sinais , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas Virais/fisiologia , Sequência de Bases , Linhagem Celular , Imunoprecipitação da Cromatina , Primers do DNA , Humanos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: The role of innate immunity in general and of type I interferon (IFN-I) in particular in HTLV-1 pathogenesis is still a matter of debate. ADAR1-p150 is an Interferon Stimulated Gene (ISG) induced by IFN-I that can edit viral RNAs. We therefore investigated whether it could play the role of an anti-HTLV factor. RESULTS: We demonstrate here that ADAR1 is also expressed in the absence of IFN stimulation in activated primary T-lymphocytes that are the natural target of this virus and in HTLV-1 or HTLV-2 chronically infected T-cells. ADAR1 expression is also increased in primary lymphocytes obtained from HTLV-1 infected individuals. We show that ADAR1 enhances HTLV-1 and HTLV-2 infection in T-lymphocytes and that this proviral effect is independent from its editing activity. ADAR1 expression suppresses IFN-α inhibitory effect on HTLV-1 and HTLV-2 and acts through the repression of PKR phosphorylation. DISCUSSION: This study demonstrates that two interferon stimulated genes, i.e. PKR and ADAR1 have opposite effects on HTLV replication in vivo. The balanced expression of those proteins could determine the fate of the viral cycle in the course of infection.
Assuntos
Adenosina Desaminase/metabolismo , Interações Hospedeiro-Patógeno , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Vírus Linfotrópico T Tipo 2 Humano/fisiologia , Proteínas de Ligação a RNA/metabolismo , Replicação Viral , eIF-2 Quinase/antagonistas & inibidores , Células Cultivadas , Humanos , Inibição Psicológica , Dados de Sequência Molecular , Análise de Sequência de DNA , Linfócitos T/imunologia , Linfócitos T/virologiaRESUMO
Permanent activation of the NF-κB pathway by the human T cell leukemia virus type 1 (HTLV-1) Tax (Tax1) viral transactivator is a key event in the process of HTLV-1-induced T lymphocyte immortalization and leukemogenesis. Although encoding a Tax transactivator (Tax2) that activates the canonical NF-κB pathway, HTLV-2 does not cause leukemia. These distinct pathological outcomes might be related, at least in part, to distinct NF-κB activation mechanisms. Tax1 has been shown to be both ubiquitinated and SUMOylated, and these two modifications were originally proposed to be required for Tax1-mediated NF-κB activation. Tax1 ubiquitination allows recruitment of the IKK-γ/NEMO regulatory subunit of the IKK complex together with Tax1 into centrosome/Golgi-associated cytoplasmic structures, followed by activation of the IKK complex and RelA/p65 nuclear translocation. Herein, we compared the ubiquitination, SUMOylation, and acetylation patterns of Tax2 and Tax1. We show that, in contrast to Tax1, Tax2 conjugation to endogenous ubiquitin and SUMO is barely detectable while both proteins are acetylated. Importantly, Tax2 is neither polyubiquitinated on lysine residues nor ubiquitinated on its N-terminal residue. Consistent with these observations, Tax2 conjugation to ubiquitin and Tax2-mediated NF-κB activation is not affected by overexpression of the E2 conjugating enzyme Ubc13. We further demonstrate that a nonubiquitinable, non-SUMOylable, and nonacetylable Tax2 mutant retains a significant ability to activate transcription from a NF-κB-dependent promoter after partial activation of the IKK complex and induction of RelA/p65 nuclear translocation. Finally, we also show that Tax2 does not interact with TRAF6, a protein that was shown to positively regulate Tax1-mediated activation of the NF-κB pathway.
Assuntos
Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 2 Humano/patogenicidade , NF-kappa B/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Ubiquitina/metabolismo , Acetilação , Células HeLa , Humanos , Células Jurkat , Processamento de Proteína Pós-TraducionalRESUMO
Type I interferon (IFN-I) inhibits the replication of different viruses. However, the effect of IFN-I on the human T-lymphotropic virus type 1 (HTLV-1) viral cycle is controversial. Here, we investigated the consequences of IFN-α addition for different steps of HTLV-1 and HTLV-2 infection. We first show that alpha interferon (IFN-α) efficiently impairs HTLV-1 and HTLV-2 de novo infection in a T cell line and in primary lymphocytes. Using pseudotyped viruses expressing HTLV-1 envelope, we then show that cell-free infection is insensitive to IFN-α, demonstrating that the cytokine does not affect the early stages of the viral cycle. In contrast, intracellular levels of Gag, Env, or Tax protein are affected by IFN-α treatment in T cells, primary lymphocytes, or 293T cells transfected with HTLV-1 or HTLV-2 molecular clones, demonstrating that IFN-α acts during the late stages of infection. We show that IFN-α does not affect Tax-mediated transcription and acts at a posttranscriptional level. Using either small interfering RNA (siRNA) directed against PKR or a PKR inhibitor, we demonstrate that PKR, whose expression is induced by interferon, plays a major role in IFN-α-induced HTLV-1/2 inhibition. These results indicate that IFN-α has a strong repressive effect on the HTLV-1 and HTLV-2 viral cycle during de novo infection of cells that are natural targets of the viruses.
Assuntos
Infecções por HTLV-I/enzimologia , Infecções por HTLV-II/enzimologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Vírus Linfotrópico T Tipo 2 Humano/fisiologia , Interferon-alfa/metabolismo , eIF-2 Quinase/metabolismo , Linhagem Celular , Ativação Enzimática , Infecções por HTLV-I/genética , Infecções por HTLV-I/virologia , Infecções por HTLV-II/genética , Infecções por HTLV-II/virologia , Interações Hospedeiro-Patógeno , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 2 Humano/genética , Humanos , Interferon alfa-2 , Proteínas Recombinantes/metabolismo , eIF-2 Quinase/genéticaRESUMO
We present a simple and rapid procedure for producing polymer-coated substrates that can be easily functionalized by ion-chelating proteins. The procedure consists of depositing 18 nm metal-chelating cyclam-modified polymer nanoparticles (cyclam-nps) onto a conductive substrate (an Indium Tin Oxide (ITO) electrode) from an aqueous dispersion of Cu(2+)-loaded cyclam-nps while being subjected to a direct current (DC) field. The density of deposited nps as measured by AFM is shown to be in direct correlation to the concentration of nps in the dispersion with deposition of the particles taking less than 5 s. Because of the functionalization of the nps with cyclam groups, they can be used as anchoring sites for 6-Histidine (6-His) tagged proteins through complexation with divalent metal ions. In this work 6-His Green Fluorescent Protein (6-His GFP) is used as a model protein. The characterization by fluorescence microscopy clearly shows that the protein affinity was ion dependent and that the 6-His GFP density can be controlled by np density, which is itself easily tunable. AFM observations confirmed the immobilization of 6-His GFP onto cyclam-nps and its subsequent removal by treatment with ethylenediaminetetraacetic acid (EDTA).
Assuntos
Proteínas de Fluorescência Verde/química , Histidina/química , Nanotubos/química , Polímeros/química , Cobre/química , Eletrodos , Galvanoplastia , Microscopia de Força Atômica , Microscopia de Fluorescência , Compostos de Estanho/químicaRESUMO
MicroRNAs (miRNAs) are a class of small RNA molecules that function to control gene expression and restrict viral replication in host cells. The production of miRNAs is believed to be dependent upon the DICER enzyme. Available evidence suggests that in T lymphocytes, HIV-1 can both suppress and co-opt the host's miRNA pathway for its own benefit. In this study, we examined the state of miRNA production in monocytes and macrophages as well as the consequences of viral infection upon the production of miRNA. Monocytes in general express low amounts of miRNA-related proteins, and DICER in particular could not be detected until after monocytes were differentiated into macrophages. In the case where HIV-1 was present prior to differentiation, the expression of DICER was suppressed. MicroRNA chip results for RNA isolated from transfected and treated cells indicated that a drop in miRNA production coincided with DICER protein suppression in macrophages. We found that the expression of DICER in monocytes is restricted by miR-106a, but HIV-1 suppressed DICER expression via the viral gene Vpr. Additionally, analysis of miRNA expression in monocytes and macrophages revealed evidence that some miRNAs can be processed by both DICER and PIWIL4. Results presented here have implications for both the pathology of viral infections in macrophages and the biogenesis of miRNAs. First, HIV-1 suppresses the expression and function of DICER in macrophages via a previously unknown mechanism. Second, the presence of miRNAs in monocytes lacking DICER indicates that some miRNAs can be generated by proteins other than DICER.
Assuntos
HIV-1/metabolismo , MicroRNAs/metabolismo , Monócitos/enzimologia , Ribonuclease III/metabolismo , Proteínas Argonautas , Diferenciação Celular/genética , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , MicroRNAs/genética , Monócitos/citologia , Monócitos/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas/genética , Proteínas/metabolismo , Proteínas de Ligação a RNA , Ribonuclease III/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Nuclear factor (NF)-kappaB is a major survival pathway engaged by the Human T-Lymphotropic Virus type 1 (HTLV-1) Tax protein. Tax1 activation of NF-kappaB occurs predominantly in the cytoplasm, where Tax1 binds NF-kappaB Essential Modulator (NEMO/IKKgamma) and triggers the activation of IkappaB kinases. Several independent studies have shown that Tax1-mediated NF-kappaB activation is dependent on Tax1 ubiquitination. Here, we identify by co-immunoprecipitation assays NEMO-Related Protein (NRP/Optineurin) as a binding partner for Tax1 in HTLV-1 infected and Tax1/NRP co-expressing cells. Immunofluorescence studies reveal that Tax1, NRP and NEMO colocalize in Golgi-associated structures. The interaction between Tax1 and NRP requires the ubiquitin-binding activity of NRP and the ubiquitination sites of Tax1. In addition, we observe that NRP increases the ubiquitination of Tax1 along with Tax1-dependent NF-kappaB signaling. Surprisingly, we find that in addition to Tax1, NRP interacts cooperatively with the Tax1 binding protein TAX1BP1, and that NRP and TAX1BP1 cooperate to modulate Tax1 ubiquitination and NF-kappaB activation. Our data strongly suggest for the first time that NRP is a critical adaptor that regulates the assembly of TAX1BP1 and post-translationally modified forms of Tax1, leading to sustained NF-kappaB activation.
Assuntos
Produtos do Gene tax/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Fator de Transcrição TFIIIA/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Produtos do Gene tax/genética , Complexo de Golgi , Células HeLa , Humanos , Imunoprecipitação , Espaço Intracelular/metabolismo , Proteínas de Membrana Transportadoras , Domínios e Motivos de Interação entre Proteínas , Técnicas do Sistema de Duplo-Híbrido , UbiquitinaçãoRESUMO
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 naive 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.
Assuntos
Antígenos de Deltaretrovirus/metabolismo , Deltaretrovirus/metabolismo , Produtos do Gene env/metabolismo , Receptores Virais/metabolismo , Sequência de Aminoácidos , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Linhagem Celular , Deltaretrovirus/genética , Deltaretrovirus/fisiologia , Transportador de Glucose Tipo 1/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Alinhamento de Sequência , Transdução Genética , Ligação ViralRESUMO
This manuscript describes a novel method for the biofunctionalization of glass surfaces with polyhistidine-tagged proteins. The main innovation of this methodology consists of the covalent binding between the nitrilotriacetic acid (NTA) moiety and the proteins, ensuring not only orientation, but also stability of the recombinant proteins on NTA-covered surfaces. In this work, as C-terminal polyhistidine tagged cadherin extracellular fragments have been used, this methodology guarantees the proper orientation of these proteins, by mimicking their insertion into cell plasma membranes. These biofunctionalized surfaces have been characterized by confocal microscopy, X-ray photoelectron spectroscopy, contact angle, and atomic force microscopy, showing a high density of cadherins on the glass surfaces and the stability of the linkage. The prepared materials exhibited a high tendency to promote cell spreading, demonstrating the functionality of the protein and the high utility of these biomaterials to promote cell adhesion events. Interestingly, differences in the cytoskeleton organization have been observed in cells adhering to surfaces with no cadherins or with nonoriented cadherins, in comparison to surfaces functionalized with well-oriented cadherins. This method, which allows the robust immobilization of polyhistidine tagged proteins due to their covalent binding and with a defined orientation, may also find particular usefulness in the making of protein biochips, for analysis of protein-protein interactions, as well as structural and single-molecule studies.
Assuntos
Biomimética/métodos , Caderinas/química , Animais , Caderinas/metabolismo , Adesão Celular , Linhagem Celular , Quelantes/química , Vidro/química , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Metais/química , Camundongos , Ácido Nitrilotriacético/química , Oligopeptídeos/metabolismo , Concentração Osmolar , Ligação Proteica , Silanos/química , Propriedades de SuperfícieRESUMO
Cells in normal tissues or in tumors have extensive opportunities for adhesion to their neighbors and the importance of cell to cell contact in the study of fundamental cellular processes is beginning to emerge. In this review, we discuss recent evidence of dramatic changes in the activity of an important signal transducer found to be profoundly affected by cell to cell adhesion, the signal transducer and activator of transcription-3 (Stat3). Direct cadherin engagement, growth of cells to postconfluence, or formation of multicellular aggregates were found to induce a striking increase in the levels of Stat3 activity, Rac1/Cdc42, and members of the IL6 receptor family in different settings. This activation was specific to Stat3, in that the levels of the extracellular signal regulated kinase (Erk1/2), a signal transducer often coordinately activated with Stat3 by a number of growth factors or oncogenes, remained unaffected by cell density. Density-dependent Stat3 activation may play a key role in survival, and could contribute to the establishment of cell polarity. It is clear that at any given time the total Stat3 activity levels in a cell are the sum of the effects of cell to cell adhesion plus the conventional Stat3 activating factors present.
Assuntos
Caderinas/metabolismo , Sobrevivência Celular/fisiologia , Fator de Transcrição STAT3/metabolismo , Animais , Comunicação Autócrina/fisiologia , Caderinas/genética , Linhagem Celular , Interleucina-6/metabolismo , Janus Quinase 1/metabolismo , Fator de Transcrição STAT3/genética , Transdução de Sinais/fisiologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
We and others have uncovered the existence of human T-cell lymphotropic virus type 3 (HTLV-3). We have now generated an HTLV-3 proviral clone. We established that gag, env, pol, pro, and tax/rex as well as minus-strand mRNAs are present in cells transfected with the HTLV-3 clone. HTLV-3 p24(gag) protein is detected in the cell culture supernatant. Transfection of 293T-long terminal repeat (LTR)-green fluorescent protein (GFP) cells with the HTLV-3 clone promotes formation of syncytia, a hallmark of Env expression, together with the appearance of fluorescent cells, demonstrating that Tax is expressed. Viral particles are visible by electron microscopy. These particles are infectious, as demonstrated by infection experiments with purified virions.
Assuntos
Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 3 de Primatas/genética , RNA Mensageiro/metabolismo , Linhagem Celular , Clonagem Molecular , Primers do DNA/genética , Células Gigantes/virologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Vírus Linfotrópico T Tipo 3 de Primatas/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Human T-cell leukemia virus type 1 and type 2 are related human retroviruses. HTLV-1 is the etiological agent of the Adult T-cell Leukemia/Lymphoma and of the Tropical Spastic Paraparesis/HTLV-1 Associated Myelopathy, whereas, HTLV-2 infection has not been formally associated with any T-cell malignancy. HTLV-1 and 2 genomes encode, respectively, the Tax1 and Tax2 proteins whose role is to transactivate the viral promoter. HTLV-1 and HTLV-2 Tax sequences display 28% divergence at the amino acid level. Tax1 is a shuttling protein that possesses both a non canonical nuclear import (NLS) and a nuclear export (NES) signal. We have recently demonstrated that Tax1 and Tax2 display different subcellular localization and that residues 90-100 are critical for this process. We investigate in the present report, whether Tax2 also possesses a functional NES. RESULTS: We first used a NES prediction method to determine whether the Tax2 protein might contain a NES and the results do suggest the presence of a NES sequence in Tax2. Using Green Fluorescent Protein-NES (GFP-NES) fusion proteins, we demonstrate that the Tax2 sequence encompasses a functional NES (NES2). As shown by microscope imaging, NES2 is able to mediate translocation of GFP from the nucleus, without the context of a full length Tax protein. Furthermore, point mutations or leptomycin B treatment abrogate NES2 function. However, within the context of full length Tax2, similar point mutations in the NES2 leucine rich stretch do not modify Tax2 localization. Finally, we also show that Tax1 NES function is dependent upon the positioning of the nuclear export signal "vis-à-vis" GFP. CONCLUSION: HTLV-2 Tax NES is functional but dispensable for the protein localization in vitro.
Assuntos
Produtos do Gene tax/química , Produtos do Gene tax/fisiologia , Sinais de Exportação Nuclear , Células HeLa , Humanos , Carioferinas/fisiologia , Leucina , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/fisiologia , Proteína Exportina 1RESUMO
Human T-cell Leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are pathogenic retroviruses that infect humans and cause severe hematological and neurological diseases. Both viruses have simian counterparts (STLV-1 and STLV-2). STLV-3 belongs to a third group of lymphotropic viruses which infect numerous African monkeys species. Among 240 Cameroonian plasma tested for the presence of HTLV-1 and/or HTLV-2 antibodies, 48 scored positive by immunofluorescence. Among those, 27 had indeterminate western-blot pattern. PCR amplification of pol and tax regions, using HTLV-1, -2 and STLV-3 highly conserved primers, demonstrated the presence of a new human retrovirus in one DNA sample. tax (180 bp) and pol (318 bp) phylogenetic analyses demonstrated the strong relationships between the novel human strain (Pyl43) and STLV-3 isolates from Cameroon. The virus, that we tentatively named HTLV-3, originated from a 62 years old Bakola Pygmy living in a remote settlement in the rain forest of Southern Cameroon. The plasma was reactive on MT2 cells but was negative on C19 cells. The HTLV 2.4 western-blot exhibited a strong reactivity to p19 and a faint one to MTA-1. On the INNO-LIA strip, it reacted faintly with the generic p19 (I/II), but strongly to the generic gp46 (I/II) and to the specific HTLV-2 gp46. The molecular relationships between Pyl43 and STLV-3 are thus not paralleled by the serological results, as most of the STLV-3 infected monkeys have an "HTLV-2 like" WB pattern. In the context of the multiple interspecies transmissions which occurred in the past, and led to the present-day distribution of the PTLV-1, it is thus very tempting to speculate that this newly discovered human retrovirus HTLV-3 might be widespread, at least in the African continent.
Assuntos
Deltaretrovirus/classificação , Deltaretrovirus/isolamento & purificação , Animais , Western Blotting , Camarões , Linhagem Celular , DNA Viral/sangue , Deltaretrovirus/genética , Infecções por Deltaretrovirus/virologia , Produtos do Gene pol/genética , Produtos do Gene tax/genética , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Vírus Linfotrópico T Tipo 3 de Primatas , Análise de Sequência de DNARESUMO
We detail herein readily accessible processes to control previously unobserved robust self-assemblies of nanodiamonds (diamondoids) in micro- and nanocrystals from their mild vapor deposition. The chemical functionalization of uniform and discernible nanodiamonds was found to be a key parameter, and depending on the type of functional group (hydroxy, fluorine, etc.) and its position on the diamondoid, the structure of the discrete deposits can vary dramatically. Thus, well-defined anisotropic structures such as rod, needle, triangle or truncated octahedron shapes can be obtained, and self-assembled edifices of sizes ranging from 20 nm to several hundred micrometers formed with conservation of a similar structure for a given diamondoid. Key thermodynamic data including sublimation enthalpy of diamondoid derivatives are reported, and the SEM of the self-assemblies coupled with EDX analyses and XRD attest the nature and purity of nanodiamond crystal deposits. This attractive method is simple and outperforms in terms of deposit quality dip-coating methods we used. This vapor phase deposition approach is expected to allow for an easy formation of diamondoid nanoobjects on different types of substrates.
RESUMO
AIMS: We aimed to study the interactions between human mesenchymal stem cells and the bone integration of nanostructured titanium implants. MATERIALS & METHODS: Nanopores of 20, 30 and 50 nm were prepared by anodization of titanium at 5, 10 and 20 V in a mixture of fluorhydric and acetic acid. Ti 30 and 50 nanostructures promoted early osteoblastic gene differentiation of the human mesenchymal stem cells without osteogenic supplements. The osseointegration of nanostructured and control titanium implants was compared by implantation in rat tibias for 1 and 3 weeks. RESULTS: The nanostructures significantly accelerated bone apposition and bone bonding strength in vivo in correlation with in vitro results. CONCLUSION: These findings demonstrate that specific nanostructures controlled the differentiation of cells and, thus, the integration of implants in tissues. These nanoporous titanium surfaces may be of considerable interest for dental and orthopedic implants.
Assuntos
Substitutos Ósseos/química , Células-Tronco Mesenquimais/citologia , Nanoestruturas/química , Osseointegração , Alicerces Teciduais/química , Titânio/química , Animais , Substitutos Ósseos/metabolismo , Diferenciação Celular , Células Cultivadas , Eletrodos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Mesenquimais/metabolismo , Nanoporos/ultraestrutura , Nanotecnologia , Osteoblastos/citologia , Ratos , Ratos Wistar , Propriedades de Superfície , Tíbia/cirurgia , Titânio/metabolismoRESUMO
Human T-cell Lymphotropic Viruses type 1 (HTLV-1) is the etiological agent of Adult T-cell Leukemia/Lymphoma. Although associated with lymphocytosis, HTLV-2 infection is not associated with any malignant hematological disease. Similarly, no infection-related symptom has been detected in HTLV-3-infected individuals studied so far. Differences in individual Tax transcriptional activity might account for these distinct physiopathological outcomes. Tax-1 and Tax-3 possess a PDZ binding motif in their sequence. Interestingly, this motif, which is critical for Tax-1 transforming activity, is absent from Tax-2. We used the DNA microarray technology to analyze and compare the global gene expression profiles of different T- and non T-cell types expressing Tax-1, Tax-2 or Tax-3 viral transactivators. In a T-cell line, this analysis allowed us to identify 48 genes whose expression is commonly affected by all Tax proteins and are hence characteristic of the HTLV infection, independently of the virus type. Importantly, we also identified a subset of genes (nâ=â70) which are specifically up-regulated by Tax-1 and Tax-3, while Tax-1 and Tax-2 shared only 1 gene and Tax-2 and Tax-3 shared 8 genes. These results demonstrate that Tax-3 and Tax-1 are closely related in terms of cellular gene deregulation. Analysis of the molecular interactions existing between those Tax-1/Tax-3 deregulated genes then allowed us to highlight biological networks of genes characteristic of HTLV-1 and HTLV-3 infection. The majority of those up-regulated genes are functionally linked in biological processes characteristic of HTLV-1-infected T-cells expressing Tax such as regulation of transcription and apoptosis, activation of the NF-κB cascade, T-cell mediated immunity and induction of cell proliferation and differentiation. In conclusion, our results demonstrate for the first time that, in T- and non T-cells types, Tax-3 is a functional analogue of Tax-1 in terms of transcriptional activation and suggest that HTLV-3 might share pathogenic features with HTLV-1 in vivo.