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1.
Proc Natl Acad Sci U S A ; 121(12): e2312290121, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38483999

RESUMO

Human cytomegalovirus (HCMV) infection of monocytes is essential for viral dissemination and persistence. We previously identified that HCMV entry/internalization and subsequent productive infection of this clinically relevant cell type is distinct when compared to other infected cells. We showed that internalization and productive infection required activation of epidermal growth factor receptor (EGFR) and integrin/c-Src, via binding of viral glycoprotein B to EGFR, and the pentamer complex to ß1/ß3 integrins. To understand how virus attachment drives entry, we compared infection of monocytes with viruses containing the pentamer vs. those without the pentamer and then used a phosphoproteomic screen to identify potential phosphorylated proteins that influence HCMV entry and trafficking. The screen revealed that the most prominent pentamer-biased phosphorylated protein was the lipid- and protein-phosphatase phosphatase and tensin homolog (PTEN). PTEN knockdown with siRNA or PTEN inhibition with a PTEN inhibitor decreased pentamer-mediated HCMV entry, without affecting trimer-mediated entry. Inhibition of PTEN activity affected lipid metabolism and interfered with the onset of the endocytic processes required for HCMV entry. PTEN inactivation was sufficient to rescue pentamer-null HCMV from lysosomal degradation. We next examined dephosphorylation of a PTEN substrate Rab7, a regulator of endosomal maturation. Inhibition of PTEN activity prevented dephosphorylation of Rab7. Phosphorylated Rab7, in turn, blocked early endosome to late endosome maturation and promoted nuclear localization of the virus and productive infection.


Assuntos
Monócitos , Internalização do Vírus , Humanos , Células Cultivadas , Monócitos/metabolismo , Citomegalovirus/fisiologia , Receptores ErbB/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo
2.
J Virol ; 96(14): e0081922, 2022 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-35862696

RESUMO

Human cytomegalovirus (HCMV) exhibits a complex host-pathogen interaction with peripheral blood monocytes. We have identified a unique, cell-type specific retrograde-like intracellular trafficking pattern that HCMV utilizes to gain access to the monocyte nucleus and for productive infection. We show that infection of primary human monocytes, epithelial cells, and fibroblasts leads to an increase in the amount of the trafficking protein Syntaxin 6 (Stx6). However, only knockdown (KD) of Stx6 in monocytes inhibited viral trafficking to the trans-Golgi network (TGN), a requisite step for nuclear translocation in monocytes. Conversely, KD of Stx6 in epithelial cells and fibroblasts did not change the kinetics of nuclear translocation and productive infection. Stx6 predominantly functions at the level of the TGN where it facilitates retrograde transport, a trafficking pathway used by only a few cellular proteins and seldom by pathogens. We also newly identify that in monocytes, Stx6 exhibits an irregular vesicular localization rather than being concentrated at the TGN as seen in other cell-types. Lastly, we implicate that viral particles that associate with both Stx6 and EEA1 early in infection are the viral population that successfully traffics to the TGN at later time points and undergo nuclear translocation. Additionally, we show for the first time that HCMV enters the TGN, and that lack of Stx6 prevents viral trafficking to this organelle. We argue that we have identified an essential cell-type specific regulator that controls early steps in efficient productive infection of a cell-type required for viral persistence and disease. IMPORTANCE Human cytomegalovirus (HCMV) infection causes severe and often fatal disease in the immunocompromised. It is one of the leading infectious causes of birth defects and causes severe complications in transplant recipients. By uncovering the unique pathways used by the virus to infect key cells, such as monocytes, responsible for dissemination and persistence, we provide new potential targets for therapeutic intervention.


Assuntos
Citomegalovirus , Monócitos , Proteínas Qa-SNARE , Citomegalovirus/patogenicidade , Humanos , Monócitos/virologia , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Transdução de Sinais , Rede trans-Golgi/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(32): 19507-19516, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723814

RESUMO

Previous analysis of postentry events revealed that human cytomegalovirus (HCMV) displays a unique, extended nuclear translocation pattern in monocytes. We determined that c-Src signaling through pentamer engagement of integrins is required upon HCMV entry to avoid sorting of the virus into late endosomes and subsequent degradation. To follow up on this previous study, we designed experiments to investigate how HCMV-induced signaling through the other major axis-the epidermal growth factor receptor (EGFR) kinase-regulates viral postentry events. Here we show that HCMV induces chronic and functional EGFR signaling that is distinct to the virus as compared to the natural EGFR ligand: EGF. This chronic EGFR kinase activity in infected monocytes is required for the proper subcellular localization of the viral particle during trafficking events, as well as for promoting translocation of viral DNA into the host nucleus. Our data indicate that HCMV glycoprotein B (gB) binds to EGFR at the monocyte surface, the virus and EGFR are internalized together, and gB remains bound to EGFR throughout viral postentry events until de-envelopment to promote the chronic EGFR kinase activity required for viral trafficking and nuclear translocation. These data highlight how initial EGFR signaling via viral binding is necessary for entry, but not sufficient to promote each viral trafficking event. HCMV appears to manipulate the EGFR kinase postentry, via gB-EGFR interaction, to be active at the critical points throughout the trafficking process that leads to nuclear translocation and productive infection of peripheral blood monocytes.


Assuntos
Núcleo Celular/metabolismo , Citomegalovirus/fisiologia , Monócitos/virologia , Proteínas do Envelope Viral/metabolismo , Núcleo Celular/virologia , Células Cultivadas , DNA Viral/metabolismo , Endossomos/metabolismo , Endossomos/virologia , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Monócitos/metabolismo , Ligação Proteica , Transdução de Sinais , Rede trans-Golgi/metabolismo , Rede trans-Golgi/virologia
4.
Curr Top Microbiol Immunol ; 391: 221-35, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26428376

RESUMO

Epstein-Barr virus primarily, though not exclusively, infects B cells and epithelial cells. Many of the virus and cell proteins that are involved in entry into these two cell types in vitro have been identified, and their roles in attachment and fusion are being explored. This chapter discusses what is known about entry at the cellular level in vitro and describes what little is known about the process in vivo. It highlights some of the questions that still need to be addressed and considers some models that need further testing.


Assuntos
Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/fisiologia , Internalização do Vírus , Animais , Linfócitos B/virologia , Células Epiteliais/virologia , Herpesvirus Humano 4/genética , Humanos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
5.
J Virol ; 88(21): 12193-201, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25142593

RESUMO

UNLABELLED: Epstein-Barr virus (EBV) fusion with an epithelial cell requires virus glycoproteins gHgL and gB and is triggered by an interaction between gHgL and integrin αvß5, αvß6, or αvß8. Fusion with a B cell requires gHgL, gp42, and gB and is triggered by an interaction between gp42 and human leukocyte antigen class II. We report here that, like alpha- and betaherpesviruses, EBV, a gammaherpesvirus, can mediate cell fusion if gB and gHgL are expressed in trans. Entry of a gH-null virus into an epithelial cell is possible if the epithelial cell expresses gHgL, and entry of the same virus, which phenotypically lacks gHgL and gp42, into a B cell expressing gHgL is possible in the presence of a soluble integrin. Heat is capable of inducing the fusion of cells expressing only gB, and the proteolytic digestion pattern of gB in virions changes in the same way following the exposure of virus to heat or to soluble integrins. It is suggested that the Gibbs free energy released as a result of the high-affinity interaction of gHgL with an integrin contributes to the activation energy required to cause the refolding of gB from a prefusion to a postfusion conformation. IMPORTANCE: The core fusion machinery of herpesviruses consists of glycoproteins gB and gHgL. We demonstrate that as in alpha- and betaherpesvirus, gB and gHgL of the gammaherpesvirus EBV can mediate fusion and entry when expressed in trans in opposing membranes, implicating interactions between the ectodomains of the proteins in the activation of fusion. We further show that heat and exposure to a soluble integrin, both of which activate fusion, result in the same changes in the proteolytic digestion pattern of gB, possibly representing the refolding of gB from its prefusion to its postfusion conformation.


Assuntos
Herpesvirus Humano 4/fisiologia , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus/efeitos da radiação , Animais , Linfócitos B/virologia , Linhagem Celular , Herpesvirus Humano 4/efeitos da radiação , Temperatura Alta , Humanos , Conformação Proteica
6.
PLoS Pathog ; 9(12): e1003806, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24367260

RESUMO

Herpes simplex virus (HSV)--and herpesviruses in general--encode for a multipartite entry/fusion apparatus. In HSV it consists of the HSV-specific glycoprotein D (gD), and three additional glycoproteins, gH/gL and gB, conserved across the Herpesviridae family and responsible for the execution of fusion. According to the current model, upon receptor binding, gD propagates the activation to gH/gL and to gB in a cascade fashion. Questions remain about how the cascade of activation is controlled and how it is synchronized with virion endocytosis, to avoid premature activation and exhaustion of the glycoproteins. We considered the possibility that such control might be carried out by as yet unknown receptors. Indeed, receptors for HSV gB, but not for gH/gL, have been described. In other members of the Herpesviridae family, such as Epstein-Barr virus, integrin receptors bind gH/gL and trigger conformational changes in the glycoproteins. We report that αvß6- and αvß8-integrins serve as receptors for HSV entry into experimental models of keratinocytes and other epithelial and neuronal cells. Evidence rests on loss of function experiments, in which integrins were blocked by antibodies or silenced, and gain of function experiments in which αvß6-integrin was expressed in integrin-negative cells. αvß6- and αvß8-integrins acted independently and are thus interchangeable. Both bind gH/gL with high affinity. The interaction profoundly affects the route of HSV entry and directs the virus to acidic endosomes. In the case of αvß8, but not αvß6-integrin, the portal of entry is located at lipid microdomains and requires dynamin 2. Thus, a major role of αvß6- or αvß8-integrin in HSV infection appears to be to function as gH/gL receptors and to promote virus endocytosis. We propose that placing the gH/gL activation under the integrin trigger point enables HSV to synchronize virion endocytosis with the cascade of glycoprotein activation that culminates in execution of fusion.


Assuntos
Antígenos de Neoplasias/fisiologia , Integrinas/fisiologia , Receptores Virais , Simplexvirus/fisiologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Animais , Células Cultivadas , Endocitose/fisiologia , Células HEK293 , Células HeLa , Humanos , Células K562 , Receptores Virais/metabolismo , Células Sf9 , Spodoptera
7.
Proc Natl Acad Sci U S A ; 109(48): 19792-7, 2012 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-23150579

RESUMO

Pathogens are sensed by Toll-like receptors (TLRs) and a growing number of non-TLR receptors. Integrins constitute a family of signaling receptors exploited by viruses and bacteria to access cells. By gain- and loss-of-function approaches we found that αvß3-integrin is a sensor of and plays a crucial role in the innate defense against herpes simplex virus (HSV). αvß3-integrin signaled through two pathways. One concurred with TLR2, affected activation/induction of interferons type 1 (IFNs-1), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), and a polarized set of cytokines and receptors. The virion glycoproteins gH/gL sufficed to induce IFN1 and NF-κB via this pathway. The other pathway was TLR2-independent, involved sarcoma (SRC)-spleen tyrosine kinase (SYK)-Caspase recruitment domain-containing protein 9 (CARD9)-TRIF (TIR-domain-containing adapter-inducing interferon-ß), and affected interferon regulatory factor 3 and 7 (IRF3-IRF7). The importance of αvß3-integrin-mediated defense is reflected in the observation that HSV evolved the immediate-early infected cellular protein 0 (ICP0) protein to counteract it. We propose that αvß3-integrin is considered a class of non-TLR pattern recognition receptors, a role likely exerted toward viruses and bacteria that interact with integrins and mount an innate response.


Assuntos
Herpesvirus Humano 1/imunologia , Imunidade Inata/fisiologia , Integrina alfaVbeta3/fisiologia , Linhagem Celular , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Inativação Gênica , Herpesvirus Humano 1/fisiologia , Humanos , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase , Replicação Viral
8.
Chin J Cancer ; 33(11): 545-8, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25322867

RESUMO

Epstein-Barr virus (EBV) infection occurs by distinct mechanisms across different cell types. EBV infection of B cells in vitro minimally requires 5 viral glycoproteins and 2 cellular proteins. By contrast, infection of epithelial cells requires a minimum of 3 viral glycoproteins, which are capable of interacting with one or more of 3 different cellular proteins. The full complement of proteins involved in entry into all cell types capable of being infected in vivo is unknown. This review discusses the events that occur when the virus is delivered into the cytoplasm of a cell, the players known to be involved in these events, and the ways in which these players are thought to function.


Assuntos
Herpesvirus Humano 4 , Proteínas Virais , Fenômenos Fisiológicos Virais , Linfócitos B , Células Epiteliais , Infecções por Vírus Epstein-Barr
9.
J Virol ; 85(24): 13214-23, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21957301

RESUMO

Fusion of herpesviruses with their target cells requires a minimum of three glycoproteins, namely, gB and a complex of gH and gL. Epstein-Barr virus (EBV) fusion with an epithelial cell requires no additional virus glycoproteins, and we have shown previously that it can be initiated by an interaction between integrin αvß6 or αvß8 and gHgL. We now report that integrin αvß5 can also bind to gHgL and trigger fusion. Binding of gHgL to integrins is a two-step reaction. The first step, analyzed by surface plasmon resonance, was fast, with high association and low dissociation rate constants. The second step, detected by fluorescence spectroscopy of gHgL labeled at cysteine 153 at the domain I-domain II interface with the environmentally sensitive probes acrylodan and IANBD, involved a slower conformational change. Interaction of gHgL with neutralizing monoclonal antibodies or Fab' fragments was also consistent with a two-step reaction involving fast high-affinity binding and a subsequent slower conformational change. None of the antibodies bound to the same epitope, and none completely inhibited integrin binding. However, binding of each decreased the rate of conformational change induced by integrin binding, suggesting that neutralization might involve a conformational change that precludes fusion. Overall, the data are consistent with the interaction of gHgL with an integrin inducing a functionally important rearrangement at the domain I-domain II interface.


Assuntos
Células Epiteliais/virologia , Herpesvirus Humano 4/patogenicidade , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Receptores de Vitronectina/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus , Antígenos de Neoplasias/metabolismo , Humanos , Integrinas/metabolismo , Glicoproteínas de Membrana/química , Modelos Moleculares , Chaperonas Moleculares/química , Ligação Proteica , Conformação Proteica , Análise Espectral , Ressonância de Plasmônio de Superfície , Proteínas do Envelope Viral/química , Proteínas Virais/química
10.
Proc Natl Acad Sci U S A ; 106(48): 20464-9, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19920174

RESUMO

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that is causally implicated in the development of lymphoid and epithelial tumors. Entry of virus requires fusion of virus envelopes and cell membranes. Fusion with B lymphocytes requires virus glycoprotein gB and a 3-part complex of glycoproteins, gHgLgp42. It is triggered by interactions between glycoprotein 42 (gp42) and HLA class II. However, fusion with epithelial cells is impeded by gp42 and instead is triggered by interactions between an unknown epithelial protein and a 2-part complex of gHgL. We report here that gHgL binds with high affinity to epithelial cells and that affinity of binding is increased by 3 orders of magnitude in the presence of Mn(2+). Binding and infection can be reduced by fibronectin and vitronectin, by down-regulation of integrin alphav, or by a peptide corresponding to 13 aa of gH which include a KGDE motif. Fusion of cells expressing gB and gHgL can be blocked by vitronectin or triggered by addition of soluble truncated integrins alphavbeta6 and alphavbeta8. We conclude that the direct interaction between EBV gHgL and integrins alphavbeta6 and alphavbeta8 can provide the trigger for fusion of EBV with an epithelial cell.


Assuntos
Células Epiteliais/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Integrinas/metabolismo , Manganês/farmacologia , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/virologia , Western Blotting , Células Epiteliais/virologia , Fibronectinas/farmacologia , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunoprecipitação , Integrinas/genética , Interferência de RNA , Vitronectina/farmacologia
11.
Methods Mol Biol ; 2244: 233-246, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555590

RESUMO

During the binding and infection of monocytes, HCMV binds to at least two major cell surface receptors/receptor families: the epidermal growth factor receptor (EGFR) to initiate downstream signaling through the EGFR-PI3K pathway, and to ß1- and ß3-integrins to initiate downstream signaling through the integrin-c-Src pathway (Nogalski et al. PLoS Pathog 9:e1003463, 2013; Chan et al. Proc Natl Acad Sci U S A 106:22369-22374, 2009; Kim et al. Proc Natl Acad Sci U S A 113:8819-8824, 2016; Wang et al. Nature 424:456-461, 2003; Wang et al. Nat Med 11:515-521, 2005; Yurochko et al. Proc Natl Acad Sci U S A 89:9034-9038, 1992). Signaling through these receptors can occur rapidly with phosphorylation observed as early as 15 s after EGF-EGFR interaction, for example (Alvarez-Salamero et al. Front Immunol 8:938, 2017). The ability to detect signaling and the consequences of that signaling are critical for our understanding of how HCMV-receptor engagement promotes infection and modulates the biology of different target cells. In this chapter we describe how we used an ELISA-based antibody platform to perform an assessment of the rapid phosphorylation events that occur in monocytes following infection. This assay can be adapted to other infection systems, time points and cell types as needed. Together, we examined via an ELISA-based antibody array a phosphoproteomic screen to search for potential phosphorylated proteins that might influence HCMV infection.


Assuntos
Infecções por Citomegalovirus/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Monócitos/virologia , Fosfoproteínas/análise , Células Cultivadas , Citomegalovirus/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Integrinas/metabolismo , Monócitos/metabolismo , Monócitos/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Transdução de Sinais , Internalização do Vírus
12.
J Mol Biol ; 351(5): 1081-100, 2005 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-16051265

RESUMO

We show that human wild-type alpha synuclein (WT alpha-syn), and the inherited mutants A53T or A30P, when expressed in the yeast Saccharomyces cerevisiae triggers events that are diagnostic of apoptosis: loss of membrane asymmetry due to the externalization of phosphatidylserine, accumulation of reactive oxygen species (ROS), and the release of cytochrome c from mitochondria. A brief heat shock was strikingly protective in that alpha-syn-expressing cells receiving a heat shock exhibited none of these apoptotic markers. Because the heat shock did not decrease the expression level of alpha-syn, a protective protein or proteins, induced by the heat shock, must be responsible for inhibition of alpha-syn-induced apoptosis. Using ROS accumulation as a marker of apoptosis, the role of various genes and various drugs in controlling alpha-syn-induced apoptosis was investigated. Treatment with geldanamycin or glutathione, overexpression of Ssa3 (Hsp70), or deletion of the yeast metacaspase gene YCA1 abolishes the ability of alpha-syn to induce ROS accumulation. Deletion of YCA1 also promotes vigorous growth of alpha-syn-expressing cells compared to cells that contain a functional copy of YCA1. These findings indicate that alpha-syn-induced ROS generation is mediated by the caspase, according to alpha-syn-->caspase-->ROS-->apoptosis. It is shown by co-immunoprecipitation that Ssa3 binds to alpha-syn in a nucleotide-dependent manner. Thus, we propose that Hsp70 chaperones inhibit this sequence of events by binding and sequestering alpha-syn.


Assuntos
Temperatura Alta , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Animais , Anexina A5/química , Apoptose , Benzoquinonas , Western Blotting , Caspases/genética , Membrana Celular/metabolismo , Separação Celular , Inibidores de Cisteína Proteinase/farmacologia , Citocromos c/metabolismo , Citometria de Fluxo , Deleção de Genes , Genótipo , Glutationa/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Lactamas Macrocíclicas , Mitocôndrias/metabolismo , Mutação , Plasmídeos/metabolismo , Quinonas/farmacologia , Espécies Reativas de Oxigênio , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sinucleínas , Fatores de Tempo , alfa-Sinucleína
13.
Curr Protoc Protein Sci ; 83: 28.10.1-28.10.18, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26836408

RESUMO

Protein aggregation is a common problem in protein biochemistry and is linked to many cellular pathologies and human diseases. The molecular chaperone ClpB can resolubilize and reactivate aggregated proteins. This unit describes the procedure for following reactivation of an aggregated enzyme glucose-6-phosphate dehydrogenase mediated by ClpB from Escherichia coli in cooperation with another molecular chaperone, DnaK. The procedures for purification of these chaperones are also described.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Glucosefosfato Desidrogenase/química , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico/química , Agregados Proteicos , Endopeptidase Clp , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Solubilidade
14.
Virology ; 494: 23-8, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27061054

RESUMO

The cell surface molecules used by Epstein-Barr virus (EBV) to attach to epithelial cells are not well-defined, although when CD21, the B cell receptor for EBV is expressed epithelial cell infection increases disproportionately to the increase in virus bound. Many herpesviruses use low affinity charge interactions with molecules such as heparan sulfate to attach to cells. We report here that the EBV glycoprotein gp150 binds to heparan sulfate proteoglycans, but that attachment via this glycoprotein is not productive of infection. We also report that only the aminoterminal two short consensus repeats of CD21 are required for efficient infection, This supports the hypothesis that, when expressed on an epithelial cell CD21 serves primarily to cluster the major attachment protein gp350 in the virus membrane and enhance access of other important glycoproteins to the epithelial cell surface.


Assuntos
Células Epiteliais/virologia , Heparitina Sulfato/metabolismo , Herpesvirus Humano 4/fisiologia , Glicoproteínas de Membrana/metabolismo , Receptores de Complemento 3d/metabolismo , Proteínas Virais/metabolismo , Ligação Viral , Animais , Células CHO , Linhagem Celular , Células Cultivadas , Cricetulus , Humanos , Glicoproteínas de Membrana/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Virais/química
15.
FEBS Lett ; 565(1-3): 65-9, 2004 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-15135054

RESUMO

Recent reports have indicated that insect antimicrobial peptides kill bacteria by inhibiting the molecular chaperone DnaK. It was proposed that the antimicrobial peptide, all-L-pyrrhocoricin (L-PYR), binds to two sites on DnaK, the conventional substrate-binding site and the multi-helical C-terminal lid, and that inhibition of DnaK comes about from the lid mode of binding. In this report, we show using two different assays that L-PYR binds to and stimulates the ATPase activity of both wild-type and a lidless variant of DnaK. Our study shows that L-PYR interacts with DnaK much like the all-L NR (NRLLLTG) peptide, which is known to bind in the conventional substrate-binding site of DnaK. L-PYR antimicrobial activity is thus a consequence of the competitive inhibition of bacterial DnaK.


Assuntos
Adenosina Trifosfatases/química , Antibacterianos/química , Anti-Infecciosos/química , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Insetos , Animais , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sítios de Ligação , Relação Dose-Resposta a Droga , Proteínas de Choque Térmico HSP70/metabolismo , Insetos , Cinética , Modelos Químicos , Modelos Moleculares , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Temperatura , Fatores de Tempo
16.
Virulence ; 1(5): 395-8, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21178476

RESUMO

Epstein-Barr virus is a ubiquitous orally-transmitted human herpesvirus that is carried by most of the adult population. It establishes latent infections in B lymphocytes, reactivates periodically from latency and can be amplified in epithelial cells where it is thought more commonly to undergo lytic replication. Entry into either cell involves fusion of the virus envelope with a cell membrane. Fusion with a B cell requires four envelope glycoproteins, gB and a ternary complex of gHgLgp42. Fusion is triggered by an interaction between gp42 and HLA class II. Fusion with an epithelial cell requires three envelope glycoproteins, gB and a binary complex of gHgL. The presence of gp42 blocks infection and blocks the interaction of gHgL with a specific receptor on the epithelial cell surface. We recently demonstrated that both integrins αvß6 and αvß8 can serve as specific receptors for gHgL and that on binding to gHgL, even in a soluble form, can provide the trigger for direct virus fusion with the epithelial cell plasma membrane. It reveals yet another way in which an integrin can be used by a pathogen to invade a cell.


Assuntos
Antígenos de Neoplasias/metabolismo , Células Epiteliais/virologia , Herpesvirus Humano 4/fisiologia , Integrinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Receptores Virais/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Humanos , Ligação Proteica , Internalização do Vírus
17.
J Virol ; 80(19): 9628-33, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16973566

RESUMO

Epstein-Barr virus (EBV) is a persistent, orally transmitted herpesvirus that replicates in B cells and epithelial cells and is associated with lymphoid and epithelial malignancies. The virus binds to CD21 on B cells via glycoprotein gp350/220 and infects efficiently. Infection of cultured epithelial cells has not typically been efficient but can occur in the absence of gp350/220 and CD21 and in vivo is thought to be important to the development of nasopharyngeal carcinoma. We report here that antibodies to gp350/220, which inhibit EBV infection of B cells, enhance infection of epithelial cells. The effect is not mediated by Fc receptor binding but is further enhanced by antibody cross-linking, which may patch gp350/220 in the virus envelope. Saliva from EBV-seropositive individuals has similar effects that can be reversed by depletion of antibody. The results are consistent with a model in which gp350/220 interferes with the access of other important players to the epithelial cell surface. The results may have implications for the development of nasopharyngeal carcinoma in high-risk populations in which elevated titers of antibody to EBV lytic cycle proteins are prognostic.


Assuntos
Anticorpos/imunologia , Células Epiteliais/imunologia , Herpesvirus Humano 4/imunologia , Proteínas da Matriz Viral/imunologia , Linfócitos B/imunologia , Linhagem Celular , Células Epiteliais/metabolismo , Humanos , Receptores de Complemento 3d/metabolismo , Saliva/imunologia , Solubilidade
18.
Biochemistry ; 44(33): 11224-33, 2005 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-16101306

RESUMO

Hsp70 chaperones are heterotropic allosteric systems in which ATP and misfolded or aggregated polypeptides are the activating ligands. To gain insight into the mechanism by which ATP and polypeptides regulate Hsp70 chaperone activity, the effect of a short peptide on the K(M) for ATP was analyzed using the Escherichia coli Hsp70 called DnaK. In the absence of peptide, the K(-P)(M) for ATP is 52 +/- 11 nM, whereas this value jumps to 14.6 +/- 1.6 microM in the presence of saturating peptide. This finding supports a mechanism in which ATP binding drives the chaperone in one direction and peptide binding pushes the chaperone back in the opposite direction (and thus increases K(M)), according to ATP + DnaK.P <==> ATP.DnaK.P <==> ATP.DnaK* + P, where ATP.DnaK.P is an intermediate from which competing ATP hydrolysis occurs (ATP.DnaK.P --> ADP.DnaK.P). We show that this branched mechanism can even explain how DnaK hydrolyzes ATP in the absence of peptide and that the true rate constant for DnaK-mediated ATP hydrolysis (k(hy)) in the absence of peptide may be as high as 0.5 s(-)(1) (rather than 5 x 10(-)(4) s(-)(1) as often stated in the literature). What happens is that a conformational equilibrium outcompetes ATP hydrolysis and effectively reduces the concentration of the intermediate by a factor of a thousand, resulting in the following relation: k(cat) = k(hy)/1000 = 5 x 10(-)(4) s(-)(1). How polypeptide substrates and the co-chaperone DnaJ modulate DnaK to achieve its theoretical maximal rate of ATP hydrolysis, which we suggest is 0.5 s(-)(1), is discussed.


Assuntos
Trifosfato de Adenosina/química , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico HSP70/química , Peptídeos/química , Trifosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Hidrólise , Cinética , Peptídeos/metabolismo , Ligação Proteica/fisiologia
19.
Arch Biochem Biophys ; 444(1): 61-5, 2005 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-16289019

RESUMO

ClpB and DnaK form a bi-chaperone system that reactivates strongly aggregated proteins in vivo and in vitro. Previously observed interaction between purified ClpB and DnaK suggested that one of the chaperones might recruit its partner during substrate reactivation. We show that ClpB from Escherichia coli binds at the substrate binding site of DnaK and the interaction is supported by the N-terminal domain and the middle domain of ClpB. Moreover, the interaction between ClpB and DnaK depends on the nucleotide-state of DnaK: it is stimulated by ADP and inhibited by ATP. These observations indicate that DnaK recognizes selected structural motifs in ClpB as "pseudo-substrates" and that ClpB may compete with bona fide substrates of DnaK. We conclude that direct interaction between ClpB and DnaK does not mediate a substrate transfer between the chaperones, it may, however, play a role in the recruitment of the bi-chaperone system to specific recognition sites in aggregated particles.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico/química , Chaperonas Moleculares/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Cromatografia em Gel , Endopeptidase Clp , Ligação Proteica , Estrutura Terciária de Proteína
20.
Biochemistry ; 42(30): 9028-40, 2003 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-12885236

RESUMO

In this study, we have used surface plasmon resonance (SPR) and isothermal microtitration calorimetry (ITC) to study the mechanism of complex formation between the Hsp70 molecular chaperone, DnaK, and its cochaperone, GrpE, which is a nucleotide exchange factor. Experiments were geared toward understanding the influence of DnaK's three domains, the ATPase (residues 1-388), substrate-binding (residues 393-507), and lid (residues 508-638) domains, on complex formation with GrpE. We show that the equilibrium dissociation constants for the interaction of GrpE with wtDnaK, lidless DnaK(2-517), the ATPase domain (2-388), and the substrate-binding fragment (393-507) are 64 (+/-16) nM, 4.0 (+/-1.5) nM, 35 (+/-10) nM, and 67 (+/-11) microM, respectively, and that the on-rate constant for the different reactions varies by over 4 orders of magnitude. SPR experiments revealed that GrpE-DnaK(393-507) complex formation is inhibited by added peptide and abolished when the 33-residue flexible "tail" of GrpE is deleted. Such results strongly suggest that the 33-residue flexible N-terminal tail of GrpE binds in the substrate-binding pocket of DnaK. This unique mode of binding between GrpE's tail and DnaK contributes to, but does not fully explain, the decrease in K(d) from 64 to 4 nM upon deletion of DnaK's lid. The possibility that deletion of DnaK's lid creates a more symmetrically shaped molecule, with enhanced affinity to GrpE, is also discussed. Our results reveal a complex set of molecular interactions between DnaK and its cochaperone GrpE. We discuss the impact of each domain on complex formation and dissociation.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico/metabolismo , Deleção de Sequência , Termodinâmica , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Catálise , Colorimetria/métodos , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Cinética , Substâncias Macromoleculares , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Especificidade por Substrato/genética , Ressonância de Plasmônio de Superfície/métodos
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