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1.
J Gen Virol ; 104(9)2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37668349

RESUMO

Human cytomegalovirus (HCMV) primary infections are typically asymptomatic in healthy individuals yet can cause increased morbidity and mortality in organ transplant recipients, AIDS patients, neonates, and the elderly. The successful, widespread dissemination of this virus among the population can be attributed in part to its wide cellular tropism and ability to establish life-long latency. HCMV infection is a multi-step process that requires numerous cellular and viral factors. The viral envelope consists of envelope protein complexes that interact with cellular factors; such interactions dictate virus recognition and attachment to different cell types, followed by fusion either at the cell membrane or within an endocytic vesicle. Several HCMV entry factors, including neuropilin-2 (Nrp2), THBD, CD147, OR14I1, and CD46, are characterized as participating in HCMV pentamer-specific entry of non-fibroblast cells such as epithelial, trophoblast, and endothelial cells, respectively. This study focuses on characterizing the structural elements of CD46 that impact HCMV infection. Infectivity studies of wild-type and CD46 knockout epithelial cells demonstrated that levels of CD46 expressed on the cell surface were directly related to HCMV infectivity. Overexpression of CD46 isomers BC1, BC2, and C2 enhanced infection. Further, CD46 knockout epithelial cells expressing CD46 deletion and chimeric molecules identified that the intact ectodomain was critical for rescue of HCMV infection in CD46 knockout cells. Collectively, these data support a model that the extracellular domain of CD46 participates in HCMV infection due to its surface expression.


Assuntos
Infecções por Citomegalovirus , Células Endoteliais , Proteína Cofatora de Membrana , Humanos , Membrana Celular , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Células Epiteliais , Proteína Cofatora de Membrana/genética
2.
Med ; 3(10): 705-721.e11, 2022 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-36044897

RESUMO

BACKGROUND: The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant and its BA.X lineages, has rendered ineffective a number of previously FDA emergency use authorized SARS-CoV-2 neutralizing antibody therapies. Furthermore, those approved antibodies with neutralizing activity against Omicron BA.1 are reportedly ineffective against the subset of Omicron subvariants that contain a R346K substitution, BA.1.1, and the more recently emergent BA.2, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern. METHODS: Following a campaign of antibody discovery based on the vaccination of Harbor H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167. FINDINGS: STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against each of the tested Omicron subvariants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice. CONCLUSIONS: With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for intravenous or intranasal use in human clinical trials. FUNDING: Funded by CRIPT (no. 75N93021R00014), DARPA (HR0011-19-2-0020), and NCI Seronet (U54CA260560).


Assuntos
Anticorpos Neutralizantes , Tratamento Farmacológico da COVID-19 , Administração Intranasal , Animais , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Humanos , Imunoglobulina G , Glicoproteínas de Membrana , Camundongos , Testes de Neutralização , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas do Envelope Viral
3.
Commun Biol ; 5(1): 387, 2022 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-35468974

RESUMO

Human cytomegalovirus (HCMV) is a ß-herpesvirus that increases morbidity and mortality in immunocompromised individuals including transplant recipients and newborns. New anti-HCMV therapies are an urgent medical need for diverse patient populations. HCMV infection of a broad range of host tissues is dependent on the gH/gL/gO trimer and gH/gL/UL28/UL130/UL131A pentamer complexes on the viral envelope. We sought to develop safe and effective therapeutics against HCMV by generating broadly-neutralizing, human monoclonal antibodies (mAbs) from VelocImmune® mice immunized with gH/gL cDNA. Following high-throughput binding and neutralization screening assays, 11 neutralizing antibodies were identified with unique CDR3 regions and a high-affinity (KD 1.4-65 nM) to the pentamer complex. The antibodies bound to distinct regions within Domains 1 and 2 of gH and effectively neutralized diverse clinical strains in physiologically relevant cell types including epithelial cells, trophoblasts, and monocytes. Importantly, combined adminstration of mAbs with ganciclovir, an FDA approved antiviral, greatly limited virus dissemination. Our work identifies several anti-gH/gL mAbs and sheds light on gH neutralizing epitopes that can guide future vaccine strategies.


Assuntos
Citomegalovirus , Proteínas do Envelope Viral , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes , Antígenos Virais , Anticorpos Amplamente Neutralizantes , Citomegalovirus/genética , Humanos , Recém-Nascido , Camundongos , Proteínas do Envelope Viral/genética
4.
Nat Immunol ; 22(4): 497-509, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33790474

RESUMO

Classic major histocompatibility complex class I (MHC-I) presentation relies on shuttling cytosolic peptides into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Viruses disable TAP to block MHC-I presentation and evade cytotoxic CD8+ T cells. Priming CD8+ T cells against these viruses is thought to rely solely on cross-presentation by uninfected TAP-functional dendritic cells. We found that protective CD8+ T cells could be mobilized during viral infection even when TAP was absent in all hematopoietic cells. TAP blockade depleted the endosomal recycling compartment of MHC-I molecules and, as such, impaired Toll-like receptor-regulated cross-presentation. Instead, MHC-I molecules accumulated in the ER-Golgi intermediate compartment (ERGIC), sequestered away from Toll-like receptor control, and coopted ER-SNARE Sec22b-mediated vesicular traffic to intersect with internalized antigen and rescue cross-presentation. Thus, when classic MHC-I presentation and endosomal recycling compartment-dependent cross-presentation are impaired in dendritic cells, cell-autonomous noncanonical cross-presentation relying on ERGIC-derived MHC-I counters TAP dysfunction to nevertheless mediate CD8+ T cell priming.


Assuntos
Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Modelos Animais de Doenças , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Feminino , Complexo de Golgi/imunologia , Complexo de Golgi/metabolismo , Complexo de Golgi/virologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/patogenicidade , Ativação Linfocitária , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/genética
5.
Nat Commun ; 10(1): 2699, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221976

RESUMO

Human cytomegalovirus (CMV) causes a wide array of disease to diverse populations of immune-compromised individuals. Thus, a more comprehensive understanding of how CMV enters numerous host cell types is necessary to further delineate the complex nature of CMV pathogenesis and to develop targeted therapeutics. To that end, we establish a vaccination strategy utilizing membrane vesicles derived from epithelial cells to generate a library of monoclonal antibodies (mAbs) targeting cell surface proteins in their native conformation. A high-throughput inhibition assay is employed to screen these antibodies for their ability to limit infection, and mAbs targeting CD46 are identified. In addition, a significant reduction of viral proliferation in CD46-KO epithelial cells confirms a role for CD46 function in viral dissemination. Further, we demonstrate a CD46-dependent entry pathway of virus infection in trophoblasts, but not in fibroblasts, highlighting the complexity of CMV entry and identifying CD46 as an entry factor in congenital infection.


Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Interações Hospedeiro-Patógeno/imunologia , Proteína Cofatora de Membrana/imunologia , Internalização do Vírus , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/imunologia , Linhagem Celular , Infecções por Citomegalovirus/prevenção & controle , Infecções por Citomegalovirus/virologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Fibroblastos/imunologia , Fibroblastos/virologia , Técnicas de Inativação de Genes , Humanos , Proteína Cofatora de Membrana/genética , RNA Interferente Pequeno/metabolismo , Trofoblastos/imunologia , Trofoblastos/virologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia
6.
Proc Natl Acad Sci U S A ; 115(5): 1069-1074, 2018 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-29339472

RESUMO

Human cytomegalovirus (HCMV) impacts more than one-half of the human population owing to its capacity to manipulate the cell and create latent reservoirs in the host. Despite an extensive understanding of HCMV biology during acute infection in fibroblasts, the molecular basis for latency in myeloid cells remains incomplete. This knowledge gap is due largely to the fact that the existing genetic systems require virus rescue in fibroblasts, precluding the study of genes that are essential during acute infection, yet likely play unique roles in myeloid cells or the establishment of latency. Here we present a solution to address this restriction. Through the exploitation of a hematopoietic-specific microRNA, we demonstrate a one-step recombineering approach that enables gene silencing only in cells associated with latency. As a proof of concept, here we describe a TB40/E variant that undergoes hematopoietic targeting of the Immediate Early-2 (IE2) gene to explore its function during infection of myeloid cells. While virus replication of the hematopoietic-targeted IE2 variant was unimpaired in fibroblasts, we observed a >100-fold increase in virus titers in myeloid cells. Virus replication in myeloid cells demonstrated that IE2 has a significant transcriptional footprint on both viral and host genes. These data implicate IE2 as an essential mediator of virus biology in myeloid cells and illustrate the utility of cell-specific microRNA-based targeting.


Assuntos
Citomegalovirus/genética , Proteínas Imediatamente Precoces/metabolismo , MicroRNAs/metabolismo , Transativadores/metabolismo , Biologia Computacional , Fibroblastos/metabolismo , Regulação Viral da Expressão Gênica , Inativação Gênica , Células-Tronco Hematopoéticas/citologia , Humanos , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Mutação , Células Mieloides/metabolismo , Ativação Transcricional , Transcriptoma , Proteínas do Envelope Viral/genética , Replicação Viral
7.
Nat Commun ; 7: 13627, 2016 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-27966523

RESUMO

The prototypic ß-herpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. The CMV envelope consists of various protein complexes that enable wide viral tropism. More specifically, the glycoprotein complex gH/gL/gO (gH-trimer) is required for infection of all cell types, while the gH/gL/UL128/130/131a (gH-pentamer) complex imparts specificity in infecting epithelial, endothelial and myeloid cells. Here we utilize state-of-the-art robotics and a high-throughput neutralization assay to screen and identify monoclonal antibodies (mAbs) targeting the gH glycoproteins that display broad-spectrum properties to inhibit virus infection and dissemination. Subsequent biochemical characterization reveals that the mAbs bind to gH-trimer and gH-pentamer complexes and identify the antibodies' epitope as an 'antigenic hot spot' critical for virus entry. The mAbs inhibit CMV infection at a post-attachment step by interacting with a highly conserved central alpha helix-rich domain. The platform described here provides the framework for development of effective CMV biologics and vaccine design strategies.


Assuntos
Anticorpos Neutralizantes/uso terapêutico , Infecções por Citomegalovirus/prevenção & controle , Citomegalovirus/patogenicidade , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/química , Linhagem Celular , Infecções por Citomegalovirus/imunologia , Humanos , Camundongos , Proteínas do Envelope Viral/química , Vacinas Virais , Internalização do Vírus
8.
mSphere ; 1(5)2016.
Artigo em Inglês | MEDLINE | ID: mdl-27704051

RESUMO

The recent Zika virus (ZIKV) outbreak has been linked to severe pathogenesis. Here, we report the construction of a plasmid carrying a cytomegalovirus (CMV) promoter-expressed prototype 1947 Uganda MR766 ZIKV cDNA that can initiate infection following direct plasmid DNA transfection of mammalian cells. Incorporation of a synthetic intron in the nonstructural protein 1 (NS1) region of the ZIKV polyprotein reduced viral cDNA-associated toxicity in bacteria. High levels of infectious virus were produced following transfection of the plasmid bearing the wild-type MR766 ZIKV genome, but not one with a disruption to the viral nonstructural protein 5 (NS5) polymerase active site. Multicycle growth curve and plaque assay experiments indicated that the MR766 virus resulting from plasmid transfection exhibited growth characteristics that were more similar to its parental isolate than previously published 2010 Cambodia and 2015 Brazil cDNA-rescued ZIKV. This ZIKV infectious clone will be useful for investigating the genetic determinants of ZIKV infection and pathogenesis and should be amenable to construction of diverse infectious clones expressing reporter proteins and representing a range of ZIKV isolates. IMPORTANCE The study of ZIKV, which has become increasingly important with the recent association of this virus with microcephaly and Guillain-Barré syndrome, would benefit from an efficient strategy to genetically manipulate the virus. This work describes a model system to produce infectious virus in cell culture. We created a plasmid carrying the prototype 1947 Uganda MR766 ZIKV genome that both was stable in bacteria and could produce high levels of infectious virus in mammalian cells through direct delivery of this DNA. Furthermore, growth properties of this rescued virus closely resembled those of the viral isolate from which it was derived. This model system will provide a simple and effective means to study how ZIKV genetics impact viral replication and pathogenesis.

9.
Microbiol Mol Biol Rev ; 80(3): 663-77, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27307580

RESUMO

The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease.


Assuntos
Infecções por Citomegalovirus/transmissão , Citomegalovirus/imunologia , Glicoproteínas/imunologia , Evasão da Resposta Imune/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas Virais de Fusão/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Epitopos/imunologia , Glicoproteínas/genética , Humanos , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/genética , Ligação Viral , Internalização do Vírus
10.
Sci Rep ; 6: 23692, 2016 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-27026399

RESUMO

The prototypic betaherpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. While benign in healthy individuals, CMV poses a significant threat to the immune compromised, including transplant recipients and neonates. The CMV glycoprotein complex gH/gL/gO mediates infection of fibroblasts, and together with the gH/gL/UL128/130/131 a pentameric complex permits infection of epithelial, endothethial, and myeloid cells. Given the central role of the gH/gL complex during infection, we were interested in studying cellular trafficking of the gH/gL complex through generation of human cells that stably express gH and gL. When expressed alone, CMV gH and gL were degraded through the ER-associated degradation (ERAD) pathway. However, co-expression of these proteins stabilized the polypeptides and enhanced their cell-surface expression. To further define regulatory factors involved in gH/gL trafficking, a CMV gH chimera in which the gH transmembrane and cytoplasmic tail were replaced with that of human CD4 protein permitted cell surface gH expression in absence of gL. We thus demonstrate the ability of distinct cellular processes to regulate the trafficking of viral glycoproteins. Collectively, the data provide insight into the processing and trafficking requirements of CMV envelope protein complexes and provide an example of the co-opting of cellular processes by CMV.


Assuntos
Citomegalovirus/fisiologia , Degradação Associada com o Retículo Endoplasmático , Proteínas do Envelope Viral/metabolismo , Linhagem Celular , Expressão Gênica , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas do Envelope Viral/genética , Internalização do Vírus
11.
Immunity ; 44(1): 46-58, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26789921

RESUMO

Viruses are obligate parasites and thus require the machinery of the host cell to replicate. Inhibition of host factors co-opted during active infection is a strategy hosts use to suppress viral replication and a potential pan-antiviral therapy. To define the cellular proteins and processes required for a virus during infection is thus crucial to understanding the mechanisms of virally induced disease. In this report, we generated fully infectious tagged influenza viruses and used infection-based proteomics to identify pivotal arms of cellular signaling required for influenza virus growth and infectivity. Using mathematical modeling and genetic and pharmacologic approaches, we revealed that modulation of Sec61-mediated cotranslational translocation selectively impaired glycoprotein proteostasis of influenza as well as HIV and dengue viruses and led to inhibition of viral growth and infectivity. Thus, by studying virus-human protein-protein interactions in the context of active replication, we have identified targetable host factors for broad-spectrum antiviral therapies.


Assuntos
Interações Hospedeiro-Parasita/fisiologia , Vírus da Influenza A/fisiologia , Vírus da Influenza A/patogenicidade , Modelos Teóricos , Replicação Viral/fisiologia , Vírus da Dengue/patogenicidade , Vírus da Dengue/fisiologia , HIV/patogenicidade , HIV/fisiologia , Humanos , Imunoprecipitação , Espectrometria de Massas , Dobramento de Proteína , Proteômica
12.
J Virol ; 88(16): 9391-405, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24920803

RESUMO

UNLABELLED: The ability of human cytomegalovirus (HCMV) to establish lifelong persistence and reactivate from latency is critical to its success as a pathogen. Here we describe a short-term in vitro model representing the events surrounding HCMV latency and reactivation in circulating peripheral blood monocytes that was developed in order to study the immunological consequence of latent virus carriage. Infection of human CD14(+) monocytes by HCMV resulted in the immediate establishment of latency, as evidenced by the absence of particular lytic gene expression, the transcription of latency-associated mRNAs, and the maintenance of viral genomes. Latent HCMV induced cellular differentiation to a macrophage lineage, causing production of selective proinflammatory cytokines and myeloid-cell chemoattractants that most likely play a role in virus dissemination in the host. Analysis of global cellular gene expression revealed activation of innate immune responses and the modulation of protein and lipid synthesis to accommodate latent HCMV infection. Remarkably, monocytes harboring latent virus exhibited selective responses to secondary stimuli known to induce an antiviral state. Furthermore, when challenged with type I and II interferon, latently infected cells demonstrated a blockade of signaling at the level of STAT1 phosphorylation. The data demonstrate that HCMV reprograms specific cellular pathways in monocytes, most notably innate immune responses, which may play a role in the establishment of, maintenance of, and reactivation from latency. The modulation of innate immune responses is likely a viral evasion strategy contributing to viral dissemination and pathogenesis in the host. IMPORTANCE: HCMV has the ability to establish a lifelong infection within the host, a phenomenon termed latency. We have established a short-term model system in human peripheral blood monocytes to study the immunological relevance of latent virus carriage. Infection of CD14(+) monocytes by HCMV results in the generation of latency-specific transcripts, maintenance of viral genomes, and the capacity to reenter the lytic cycle. During short-term latency in monocytes the virus initiates a program of differentiation to inflammatory macrophages that coincides with the modulation of cytokine secretion and specific cellular processes. HCMV-infected monocytes are hindered in their capacity to exert normal immunoprotective mechanisms. Additionally, latent virus disrupts type I and II interferon signaling at the level of STAT1 phosphorylation. This in vitro model system can significantly contribute to our understanding of the molecular and inflammatory factors that initiate HCMV reactivation in the host and allow the development of strategies to eradicate virus persistence.


Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Imunidade Inata/imunologia , Monócitos/imunologia , Latência Viral/imunologia , Linhagem Celular , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Citocinas/genética , Citocinas/imunologia , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Expressão Gênica/genética , Expressão Gênica/imunologia , Humanos , Imunidade Inata/genética , Inflamação/genética , Inflamação/imunologia , Inflamação/virologia , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/virologia , Monócitos/virologia , Células Mieloides/imunologia , Células Mieloides/virologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Transcrição Gênica/imunologia , Latência Viral/genética
13.
Viruses ; 6(3): 1202-18, 2014 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-24625810

RESUMO

Human cytomegalovirus (HCMV) encodes a number of viral proteins with homology to cellular G protein-coupled receptors (GPCRs). These viral GPCRs, including US27, US28, UL33, and UL78, have been ascribed numerous functions during infection, including activating diverse cellular pathways, binding to immunomodulatory chemokines, and impacting virus dissemination. To investigate the role of US28 during virus infection, two variants of the clinical isolate TB40/E were generated: TB40/E-US28(YFP) expressing a C-terminal yellow fluorescent protein tag, and TB40/E-FLAG(YFP) in which a FLAG-YFP cassette replaces the US28 coding region. The TB40/E-US28(YFP) protein localized as large perinuclear fluorescent structures at late times post-infection in fibroblasts, endothelial, and epithelial cells. Interestingly, US28(YFP) is a non-glycosylated membrane protein throughout the course of infection. US28 appears to impact cell-to-cell spread of virus, as the DUS28 virus (TB40/E-FLAG(YFP)) generated a log-greater yield of extracellular progeny whose spread could be significantly neutralized in fibroblasts. Most strikingly, in epithelial cells, where dissemination of virus occurs exclusively by the cell-to-cell route, TB40/E-FLAG(YFP) (DUS28) displayed a significant growth defect. The data demonstrates that HCMV US28 may contribute at a late stage of the viral life cycle to cell-to-cell dissemination of virus.


Assuntos
Citomegalovirus/fisiologia , Receptores de Quimiocinas/metabolismo , Proteínas Virais/metabolismo , Fusão Gênica Artificial , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Células Cultivadas , Citomegalovirus/genética , Citomegalovirus/crescimento & desenvolvimento , Citomegalovirus/isolamento & purificação , Citoplasma/química , Células Endoteliais/virologia , Células Epiteliais/virologia , Fibroblastos/virologia , Deleção de Genes , Genes Reporter , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Receptores de Quimiocinas/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Coloração e Rotulagem , Proteínas Virais/genética
14.
Toxins (Basel) ; 6(1): 33-53, 2013 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-24366208

RESUMO

Ricin toxin, an A-B toxin from Ricinus communis, induces cell death through the inhibition of protein synthesis. The toxin binds to the cell surface via its B chain (RTB) followed by its retrograde trafficking through intracellular compartments to the ER where the A chain (RTA) is transported across the membrane and into the cytosol. Ricin A chain is transported across the ER membrane utilizing cellular proteins involved in the disposal of aberrant ER proteins by a process referred to as retrograde translocation. Given the current lack of therapeutics against ricin intoxication, we developed a high-content screen using an enzymatically attenuated RTA chimera engineered with a carboxy-terminal enhanced green fluorescent protein (RTA(E177Q)egfp) to identify compounds that target RTA retrograde translocation. Stabilizing RTA(E177Q)egfp through the inclusion of proteasome inhibitor produced fluorescent peri-nuclear granules. Quantitative analysis of the fluorescent granules provided the basis to discover compounds from a small chemical library (2080 compounds) with known bioactive properties. Strikingly, the screen found compounds that stabilized RTA molecules within the cell and several compounds limited the ability of wild type RTA to suppress protein synthesis. Collectively, a robust high-content screen was developed to discover novel compounds that stabilize intracellular ricin and limit ricin intoxication.


Assuntos
Ricina/antagonistas & inibidores , Ricina/química , Bibliotecas de Moléculas Pequenas/química , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Ricinus/química , Toxinas Biológicas/antagonistas & inibidores , Toxinas Biológicas/química
15.
J Biol Chem ; 286(24): 21231-8, 2011 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-21527639

RESUMO

Ricin is a potent A-B toxin that is transported from the cell surface to the cytosol, where it inactivates ribosomes, leading to cell death. Ricin enters cells via endocytosis, where only a minute number of ricin molecules reach the endoplasmic reticulum (ER) lumen. Subsequently, the ricin A chain traverses the ER bilayer by a process referred to as dislocation or retrograde translocation to gain access to the cytosol. To study the molecular processes of ricin A chain dislocation, we have established, for the first time, a human cell system in which enzymatically attenuated ricin toxin A chains (RTA(E177D) and RTA(Δ177-181)) are expressed in the cell and directed to the ER. Using this human cell-based system, we found that ricin A chains underwent a rapid dislocation event that was quite distinct from the dislocation of a canonical ER soluble misfolded protein, null Hong Kong variant of α(1)-antitrypsin. Remarkably, ricin A chain dislocation occurred via a membrane-integrated intermediate and utilized the ER protein SEL1L for transport across the ER bilayer to inhibit protein synthesis. The data support a model in which ricin A chain dislocation occurs via a novel strategy of utilizing the hydrophobic nature of the ER membrane and selective ER components to gain access to the cytosol.


Assuntos
Retículo Endoplasmático/metabolismo , Bicamadas Lipídicas/química , Ricina/química , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Citosol/metabolismo , Epitopos/química , Glicosídeo Hidrolases/química , Humanos , Focalização Isoelétrica , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Dobramento de Proteína , alfa 1-Antitripsina/química
16.
J Clin Invest ; 120(11): 3969-78, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20978345

RESUMO

US28 is a constitutively active chemokine receptor encoded by CMV (also referred to as human herpesvirus 5), a highly prevalent human virus that infects a broad spectrum of cells, including intestinal epithelial cells (IECs). To study the role of US28 in vivo, we created transgenic mice (VS28 mice) in which US28 expression was targeted to IECs. Expression of US28 was detected in all IECs of the small and large intestine, including in cells expressing leucine rich repeat containing GPCR5 (Lgr5), a marker gene of intestinal epithelial stem cells. US28 expression in IECs inhibited glycogen synthase 3ß (GSK-3ß) function, promoted accumulation of ß-catenin protein, and increased expression of Wnt target genes involved in the control of the cell proliferation. VS28 mice showed a hyperplastic intestinal epithelium and, strikingly, developed adenomas and adenocarcinomas by 40 weeks of age. When exposed to an inflammation-driven tumor model (azoxymethane/dextran sodium sulfate), VS28 mice developed a significantly higher tumor burden than control littermates. Transgenic coexpression of the US28 ligand CCL2 (an inflammatory chemokine) increased IEC proliferation as well as tumor burden, suggesting that the oncogenic activity of US28 can be modulated by inflammatory factors. Together, these results indicate that expression of US28 promotes development of intestinal dysplasia and cancer in transgenic mice and suggest that CMV infection may facilitate development of intestinal neoplasia in humans.


Assuntos
Mucosa Intestinal/patologia , Neoplasias Intestinais/imunologia , Neoplasias Intestinais/patologia , Camundongos Transgênicos , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL
17.
J Biol Chem ; 284(9): 5905-14, 2009 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-19121997

RESUMO

The human cytomegalovirus proteins US2 and US11 have co-opted endoplasmic reticulum (ER) quality control to facilitate the destruction of major histocompatibility complex class I heavy chains. The class I heavy chains are dislocated from the ER to the cytosol, where they are deglycosylated and subsequently degraded by the proteasome. We examined the role of TRAM1 (translocating chain-associated membrane protein-1) in the dislocation of class I molecules using US2- and US11-expressing cells. TRAM1 is an ER protein initially characterized for its role in processing nascent polypeptides. Co-immunoprecipitation studies demonstrated that TRAM1 can complex with the wild type US2 and US11 proteins as well as deglycosylated and polyubiquitinated class I degradation intermediates. In studies using US2- and US11-TRAM1 knockdown cells, we observed an increase in levels of class I heavy chains. Strikingly, increased levels of glycosylated heavy chains were observed in TRAM1 knockdown cells when compared with control cells in a pulse-chase experiment. In fact, US11-mediated class I dislocation was more sensitive to the lack of TRAM1 than US2. These results provide further evidence that these viral proteins may utilize distinct complexes to facilitate class I dislocation. For example, US11-mediated class I heavy chain degradation requires Derlin-1 and SEL1L, whereas signal peptide peptidase is critical for US2-induced class I destabilization. In addition, TRAM1 can complex with the dislocation factors Derlin-1 and signal peptide peptidase. Collectively, the data support a model in which TRAM1 functions as a cofactor to promote efficient US2- and US11-dependent dislocation of major histocompatibility complex class I heavy chains.


Assuntos
Astrocitoma/metabolismo , Retículo Endoplasmático/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Glicosilação , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoprecipitação , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Transporte Proteico , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares , Células Tumorais Cultivadas , Ubiquitina/metabolismo , Proteínas do Envelope Viral/genética , Proteínas Virais/genética
18.
J Virol ; 83(3): 1359-67, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19004944

RESUMO

Throughout the course of natural evolution with its host, the human cytomegalovirus (HCMV) has developed a variety of strategies to avoid immune recognition and clearance. The major histocompatibility complex (MHC) class I antigen presentation pathway is a major target of the virus. HCMV encodes at least six gene products that modulate the processing of endoplasmic reticulum (ER)-resident MHC class I molecules. Here, we show that two virus-encoded proteins, US2 and US3, coordinate their functions toward the common goal of attenuating class I protein surface expression. In cells stably expressing both US2 and US3, class I molecules were almost completely downregulated from the cell surface. In addition, pulse-chase analysis revealed that the proteasome-dependent turnover of class I molecules occurs more rapidly in cells expressing both US2 and US3 than either US2 or US3 alone. The ability of US3 to retain class I molecules in the ER produces a target-rich environment for US2 to mediate the destruction of class I heavy chains. In fact, expression of US3 enhanced the association between US2 and class I molecules, thus encouraging their dislocation and degradation. This immune evasion strategy ensures that viral antigens are not presented on the cell surface during the early phase of HCMV infection, a critical time of replication and viral proliferation.


Assuntos
Citomegalovirus/fisiologia , Regulação para Baixo , Antígenos de Histocompatibilidade Classe I/imunologia , Astrocitoma/virologia , Linhagem Celular Tumoral , Citometria de Fluxo , Glicoproteínas/metabolismo , Glicoproteínas/fisiologia , Humanos , Hidrólise , Proteínas Imediatamente Precoces/metabolismo , Proteínas Imediatamente Precoces/fisiologia , Imunoprecipitação , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/fisiologia
19.
Biosci Rep ; 29(3): 173-81, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18811591

RESUMO

NCLs (neuronal ceroid lipofuscinoses), a group of inherited neurodegenerative lysosomal storage diseases that predominantly affect children, are the result of autosomal recessive mutations within one of the nine cln genes. The wild-type cln gene products are composed of membrane and soluble proteins that localize to the lysosome or the ER (endoplasmic reticulum). However, the destiny of the Cln variants has not been fully characterized. To explore a possible link between ER quality control and processing of Cln mutants, we investigated the fate of two NCL-related Cln6 mutants found in patient samples (Cln6(G123D) and Cln6(M241T)) in neuronal-derived human cells. The point mutations are predicted to be in the putative transmembrane domains and most probably generate misfolded membrane proteins that are subjected to ER quality control. Consistent with this paradigm, both mutants underwent rapid proteasome-mediated degradation and complexed with components of the ER extraction apparatus, Derlin-1 and p97. In addition, knockdown of SEL1L [sel-1 suppressor of lin-12-like (Caenorhabditis elegans)], a member of an E3 ubiquitin ligase complex involved in ER protein extraction, rescued significant amounts of Cln6(G123D) and Cln6(M241T) polypeptides. The results implicate ER quality control in the instability of the Cln variants that probably contributes to the development of NCL.


Assuntos
Proteínas de Membrana/metabolismo , Mutação , Lipofuscinoses Ceroides Neuronais/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Substituição de Aminoácidos , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/genética , Lipofuscinoses Ceroides Neuronais/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteínas/genética , Ubiquitina-Proteína Ligases/genética
20.
J Gen Virol ; 89(Pt 5): 1122-1130, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18420789

RESUMO

Inhibition of cell-surface expression of major histocompatibility complex class I molecules by human cytomegalovirus (HCMV, a beta-herpesvirus) promotes escape from recognition by CD8+ cytotoxic T cells. The HCMV US2 and US11 gene products induce class I downregulation during the early phase of HCMV infection by facilitating the degradation of class I heavy chains. The HCMV proteins promote the transport of the class I heavy chains across the endoplasmic reticulum (ER) membrane into the cytosol by a process referred to as 'dislocation', which is then followed by proteasome degradation. This process has striking similarities to the degradation of misfolded ER proteins mediated by ER quality control. Even though the major steps of the dislocation reaction have been characterized, the cellular proteins, specifically the ER chaperones involved in targeting class I for dislocation, have not been fully delineated. To elucidate the chaperones involved in HCMV-mediated class I dislocation, we utilized a chimeric class I heavy chain with an affinity tag at its carboxy terminus. Interestingly, US2 but not US11 continued to target the class I chimera for destruction, suggesting a structural limitation for US11-mediated degradation. Association studies in US2 cells and in cells that express a US2 mutant, US2-186HA, revealed that class I specifically interacts with calnexin, BiP and calreticulin. These findings demonstrate that US2-mediated class I destruction utilizes specific chaperones to facilitate class I dislocation. The data suggest a more general model in which the chaperones that mediate protein folding may also function during ER quality control to eliminate aberrant ER proteins.


Assuntos
Citomegalovirus/fisiologia , Retículo Endoplasmático/enzimologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Envelope Viral/metabolismo , Calnexina/metabolismo , Calreticulina/metabolismo , Linhagem Celular , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Humanos , Imunoprecipitação , Ligação Proteica , Mapeamento de Interação de Proteínas
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