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1.
Nucleic Acids Res ; 47(3): 1523-1531, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30481318

RESUMO

The HIV-1 trans-activator protein Tat binds the trans-activation response element (TAR) to facilitate recruitment of the super elongation complex (SEC) to enhance transcription of the integrated pro-viral genome. The Tat-TAR interaction is critical for viral replication and the emergence of the virus from the latent state, therefore, inhibiting this interaction has long been pursued to discover new anti-viral or latency reversal agents. However, discovering active compounds that directly target RNA with high affinity and selectivity remains a significant challenge; limiting pre-clinical development. Here, we report the rational design of a macrocyclic peptide mimic of the arginine rich motif of Tat, which binds to TAR with low pM affinity and 100-fold selectivity against closely homologous RNAs. Despite these unprecedented binding properties, the new ligand (JB181) only moderately inhibits Tat-dependent reactivation in cells and recruitment of positive transcription elongation factor (P-TEFb) to TAR. The NMR structure of the JB181-TAR complex revealed that the ligand induces a structure in the TAR loop that closely mimics the P-TEFb/Tat1:57/AFF4/TAR complex. These results strongly suggest that high-affinity ligands which bind the UCU bulge are not likely to inhibit recruitment of the SEC and suggest that targeting of the TAR loop will be an essential feature of effective Tat inhibitors.


Assuntos
Infecções por HIV/genética , Repetição Terminal Longa de HIV/genética , HIV-1/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Antivirais/química , Antivirais/farmacologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Repetição Terminal Longa de HIV/efeitos dos fármacos , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Humanos , Ligantes , Complexos Multiproteicos/efeitos dos fármacos , Complexos Multiproteicos/genética , Fator B de Elongação Transcricional Positiva/química , Fator B de Elongação Transcricional Positiva/genética , Ligação Proteica , RNA Viral/genética , Transcrição Gênica/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química
2.
mBio ; 4(3): e00332-13, 2013 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-23736286

RESUMO

ABSTRACT Human cytomegalovirus (HCMV) glycoproteins gB and gH/gL are both necessary and sufficient for cell-cell fusion. However, it is not clear what roles these glycoproteins play in virus entry, whether acting directly in membrane fusion or in binding receptors. With other herpesviruses, it appears that gB is the fusion protein and is triggered by gH/gL, which, in some cases, binds receptors. However, for HCMV, there is published evidence that gB binds cellular ligands necessary to promote virus entry into or signaling of cells. Most mechanistic information on herpesvirus fusion proteins involves cell-cell fusion assays, which do not allow a determination of whether gB or gH/gL in the virion envelope must be oriented toward cellular membranes that contain receptors. Here, we showed that HCMV virions lacking gB were unable to enter normal cells but entered cells that expressed gB. Analyses of gB mutants lacking the cytoplasmic domain or with substitutions in putative "fusion loops" provided evidence that gB fusion activity was required for this "entry in trans." In gB-mediated entry in trans, gB is oriented toward the virion envelope that apparently lacks receptors, arguing against an essential role for gB in binding receptors or signaling molecules. In contrast, particles lacking gH/gL did not enter cells expressing gH/gL, apparently because gH/gL must be oriented toward cellular membranes (which have receptors). Coupled with our previous interference studies, in which gH/gL expressed in cells blocked HCMV entry, our findings here support the hypothesis that HCMV gH/gL binds cellular receptors before triggering gB, which acts as the fusion protein. IMPORTANCE Human cytomegalovirus (HCMV) produces major disease in neonates and immunosuppressed transplant patients. As with other herpesviruses, HCMV requires two membrane glycoproteins, gB and gH/gL, to enter host cells. However, it has not been clear how gB and gH/gL function in two steps of the HCMV entry pathway, i.e., (i) binding of cellular receptors and (ii) fusion of the virion envelope with cellular membranes. There are studies that suggest that HCMV gB is required for receptor binding and other studies suggesting that gH/gL is the receptor binding protein and gB is the fusion protein. Here, we show that HCMV virions lacking gB can enter cells that express gB in cellular membranes. In contrast, virus particles lacking gH/gL could not enter cells expressing gH/gL. Our study supports the hypothesis that gB is the fusion protein and gH/gL acts upstream of gB to bind receptors and then activate gB for fusion.


Assuntos
Citomegalovirus/fisiologia , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Células Cultivadas , Citomegalovirus/genética , Análise Mutacional de DNA , Fibroblastos/virologia , Deleção de Genes , Humanos , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/genética
3.
Cell Host Microbe ; 13(2): 181-92, 2013 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-23414758

RESUMO

During retroviral RNA encapsidation, two full-length genomic (g) RNAs are selectively incorporated into assembling virions. Packaging involves a cis-acting packaging element (Ψ) within the 5' untranslated region of unspliced HIV-1 RNA genome. However, the mechanism(s) that selects and limits gRNAs for packaging remains uncertain. Using a dual complementation system involving bipartite HIV-1 gRNA, we observed that gRNA packaging is additionally dependent on a cis-acting RNA element, the genomic RNA packaging enhancer (GRPE), found within the gag p1-p6 domain and overlapping the Gag-Pol ribosomal frameshift signal. Deleting or disrupting the two conserved GRPE stem loops diminished gRNA packaging and infectivity >50-fold, while deleting gag sequences between Ψ and GRPE had no effect. Downregulating the translation termination factor eRF1 produces defective virus particles containing 20 times more gRNA. Thus, only the HIV-1 RNAs employed for Gag-Pol translation may be specifically selected for encapsidation, possibly explaining the limitation of two gRNAs per virion.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Proteínas de Fusão gag-pol/metabolismo , HIV-1/genética , RNA Viral/genética , Sequências Reguladoras de Ácido Ribonucleico , Montagem de Vírus , Proteínas de Fusão gag-pol/genética , Teste de Complementação Genética , Genoma Viral , Células HEK293 , HIV-1/fisiologia , Humanos , Sequências Repetidas Invertidas , Conformação de Ácido Nucleico , Fatores de Terminação de Peptídeos/genética , Fatores de Terminação de Peptídeos/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção
4.
J Virol ; 84(5): 2585-96, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20032184

RESUMO

Human cytomegalovirus (HCMV) depends upon a five-protein complex, gH/gL/UL128-131, to enter epithelial and endothelial cells. A separate HCMV gH/gL-containing complex, gH/gL/gO, has been described. Our prevailing model is that gH/gL/UL128-131 is required for entry into biologically important epithelial and endothelial cells and that gH/gL/gO is required for infection of fibroblasts. Genes encoding UL128-131 are rapidly mutated during laboratory propagation of HCMV on fibroblasts, apparently related to selective pressure for the fibroblast entry pathway. Arguing against this model in the accompanying paper by B. J. Ryckman et al. (J. Virol., 84:2597-2609, 2010), we describe evidence that clinical HCMV strain TR expresses a gO molecule that acts to promote endoplasmic reticulum (ER) export of gH/gL and that gO is not stably incorporated into the virus envelope. This was different from results involving fibroblast-adapted HCMV strain AD169, which incorporates gO into the virion envelope. Here, we constructed a TR gO-null mutant, TRDeltagO, that replicated to low titers, spread poorly among fibroblasts, but produced normal quantities of extracellular virus particles. TRDeltagO particles released from fibroblasts failed to infect fibroblasts and epithelial and endothelial cells, but the chemical fusogen polyethylene glycol (PEG) could partially overcome defects in infection. Therefore, TRDeltagO is defective for entry into all three cell types. Defects in entry were explained by observations showing that TRDeltagO incorporated about 5% of the quantities of gH/gL in extracellular virus particles compared with that in wild-type virions. Although TRDeltagO particles could not enter cells, cell-to-cell spread involving epithelial and endothelial cells was increased relative to TR, apparently resulting from increased quantities of gH/gL/UL128-131 in virions. Together, our data suggest that TR gO acts as a chaperone to promote ER export and the incorporation of gH/gL complexes into the HCMV envelope. Moreover, these data suggest that it is gH/gL, and not gH/gL/gO, that is present in virions and is required for infection of fibroblasts and epithelial and endothelial cells. Our observations that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial/endothelial cells differ from models for other beta- and gammaherpesviruses that use one of two different gH/gL complexes to enter different cells.


Assuntos
Citomegalovirus/metabolismo , Células Endoteliais/virologia , Fibroblastos/virologia , Glicoproteínas de Membrana , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Vírion/metabolismo , Internalização do Vírus , Animais , Células Cultivadas , Citomegalovirus/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutação , Proteínas do Envelope Viral/genética , Proteínas Virais/genética , Vírion/genética , Vírion/ultraestrutura
5.
PLoS Pathog ; 4(2): e39, 2008 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-18282101

RESUMO

The control of Mycobacterium tuberculosis (Mtb) infection is heavily dependent on the adaptive Th1 cellular immune response. Paradoxically, optimal priming of the Th1 response requires activation of priming dendritic cells with Th1 cytokine IFN-gamma. At present, the innate cellular mechanisms required for the generation of an optimal Th1 T cell response remain poorly characterized. We hypothesized that innate Mtb-reactive T cells provide an early source of IFN-gamma to fully activate Mtb-exposed dendritic cells. Here, we report the identification of a novel population of Mtb-reactive CD4(-) alphabetaTCR(+) innate thymocytes. These cells are present at high frequencies, respond to Mtb-infected cells by producing IFN-gamma directly ex vivo, and display characteristics of effector memory T cells. This novel innate population of Mtb-reactive T cells will drive further investigation into the role of these cells in the containment of Mtb following infectious exposure. Furthermore, this is the first demonstration of a human innate pathogen-specific alphabetaTCR(+) T cell and is likely to inspire further investigation into innate T cells recognizing other important human pathogens.


Assuntos
Células Dendríticas/imunologia , Mycobacterium tuberculosis/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologia , Timo/imunologia , Tuberculose/imunologia , Contagem de Células , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Humanos , Imunidade Inata , Lactente , Recém-Nascido , Interferon Tipo I/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/microbiologia , Ativação Linfocitária , Subpopulações de Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/microbiologia , Timo/citologia , Timo/metabolismo
6.
J Clin Invest ; 116(1): 59-69, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16374516

RESUMO

The majority of acute clinical manifestations of atherosclerosis are due to the physical rupture of advanced atherosclerotic plaques. It has been hypothesized that macrophages play a key role in inducing plaque rupture by secreting proteases that destroy the extracellular matrix that provides physical strength to the fibrous cap. Despite reports detailing the expression of multiple proteases by macrophages in rupture-prone regions, there is no direct proof that macrophage-mediated matrix degradation can induce plaque rupture. We aimed to test this hypothesis by retrovirally overexpressing the candidate enzyme MMP-9 in macrophages of advanced atherosclerotic lesions of apoE-/- mice. Despite a greater than 10-fold increase in the expression of MMP-9 by macrophages, there was only a minor increase in the incidence of plaque fissuring. Subsequent analysis revealed that macrophages secrete MMP-9 predominantly as a proform, and this form is unable to degrade the matrix component elastin. Expression of an autoactivating form of MMP-9 in macrophages in vitro greatly enhances elastin degradation and induces significant plaque disruption when overexpressed by macrophages in advanced atherosclerotic lesions of apoE-/- mice in vivo. These data show that enhanced macrophage proteolytic activity can induce acute plaque disruption and highlight MMP-9 as a potential therapeutic target for stabilizing rupture-prone plaques.


Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/genética , Macrófagos/enzimologia , Metaloproteinase 9 da Matriz/genética , Animais , Apolipoproteínas E/genética , Aterosclerose/enzimologia , Aterosclerose/patologia , Transplante de Medula Óssea , Colagenases/metabolismo , Cruzamentos Genéticos , Feminino , Metaloproteinase 12 da Matriz , Metaloproteinase 13 da Matriz , Metaloproteinase 9 da Matriz/metabolismo , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
7.
J Biol Chem ; 280(3): 1826-37, 2005 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-15507448

RESUMO

Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26-28 kDa) and two minor (20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate (APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase Cdelta inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17- and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding.


Assuntos
Cálcio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/fisiologia , Metaloendopeptidases/fisiologia , Acetato de Fenilmercúrio/análogos & derivados , Acetato de Fenilmercúrio/farmacologia , Proteínas ADAM , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide , Sequência de Bases , Betacelulina , Linhagem Celular Transformada , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Humanos , Transporte de Íons
8.
J Immunol ; 172(6): 3678-85, 2004 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-15004171

RESUMO

CXC chemokine ligand (CXCL)16 and scavenger receptor for phosphatidylserine and oxidized low-density lipoprotein were independently identified as a chemokine and a scavenger receptor, respectively, but have since been shown to be identical. CXCL16 is synthesized as a transmembrane protein with its chemokine domain at the end of a mucin-rich stalk. When expressed at the cell surface, CXCL16 functions as a scavenger receptor, binding and internalizing oxidized low-density lipoprotein and bacteria. As a soluble form, CXCL16 is a chemoattractant for activated CD4+ and CD8+ T cells through binding its receptor, CXCR6. In this study, we examined the mechanisms that regulate the conversion between these two functionally distinct forms of CXCL16. We demonstrate that murine CXCL16 is synthesized as an intracellular precursor that is rapidly transported to the cell surface where it undergoes metalloproteinase-dependent cleavage, causing the release of a fragment that constitutes the majority of the CXCL16 extracellular domain. Using a novel retroviral system for the generation of short interfering RNAs, we show that knockdown of a disintegrin and metalloproteinase (ADAM) family protease ADAM10 decreases this constitutive shedding of CXCL16. Furthermore, we show that overexpression of ADAM10 increases CXCL16 shedding, whereas overexpression of a dominant-negative form of ADAM10 lowers shedding of CXCL16 in a similar manner to short interfering RNAs. Through the modulation of ADAM10 function, we demonstrate that ADAM10-mediated constitutive shedding is a key regulator of CXCL16 cell surface expression. The identification of ADAM10 as a major protease responsible for the conversion of CXCL16 from a membrane-bound scavenger receptor to a soluble chemoattractant will provide new information for understanding the physiological function of this molecule.


Assuntos
Quimiocinas CXC/biossíntese , Quimiocinas CXC/metabolismo , Desintegrinas/fisiologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Metaloendopeptidases/fisiologia , Receptores Imunológicos/biossíntese , Receptores Imunológicos/metabolismo , Proteínas ADAM , Proteína ADAM10 , Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide , Animais , Catálise , Linhagem Celular , Membrana Celular/genética , Membrana Celular/imunologia , Membrana Celular/metabolismo , Quimiocina CXCL16 , Quimiocina CXCL6 , Quimiocinas CXC/antagonistas & inibidores , Quimiocinas CXC/genética , Vetores Genéticos , Hidrólise , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-Traducional/imunologia , Estrutura Terciária de Proteína/genética , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/genética , Receptores Depuradores
9.
J Biol Chem ; 278(39): 37459-64, 2003 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-12878595

RESUMO

A variety of cell surface adhesion molecules can exist as both transmembrane proteins and soluble circulating forms. Increases in the levels of soluble adhesion molecules have been correlated with a variety of inflammatory diseases, suggesting a pathological role. Although soluble forms are thought to result from proteolytic cleavage from the cell surface, relatively little is known about the proteases responsible for their release. In this report we demonstrate that under normal culture conditions, cells expressing vascular cell adhesion molecule 1 (VCAM-1) release a soluble form of the extracellular domain that is generated by metalloproteinase-mediated cleavage. VCAM-1 release can be rapidly simulated by phorbol 12-myristate 13-acetate (PMA), and this induced VCAM-1 shedding is mediated by metalloproteinase cleavage of VCAM-1 near the transmembrane domain. PMA-induced VCAM-1 shedding occurs as the result of activation of a specific pathway, as the generation of soluble forms of three other adhesion molecules, E-selectin, platelet-endothelial cell adhesion molecule 1, and intercellular adhesion molecule 1, are not altered by PMA stimulation. Using cells derived from genetically deficient mice, we identify tumor necrosis factor-alpha-converting enzyme (TACE or ADAM 17) as the protease responsible for PMA-induced VCAM-1 release, including shedding of endogenously expressed VCAM-1 by murine endothelial cells. Therefore, TACE-mediated shedding of VCAM-1 may be important for the regulation of VCAM-1 function at the cell surface.


Assuntos
Metaloendopeptidases/fisiologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Proteínas ADAM , Proteína ADAM17 , Animais , Linhagem Celular , Feminino , Metaloproteases/fisiologia , Camundongos , Acetato de Tetradecanoilforbol/farmacologia
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