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1.
Sci Rep ; 11(1): 3266, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547379

RESUMO

West Nile virus (WNV) is a Flavivirus, which can cause febrile illness in humans that may progress to encephalitis. Like any other obligate intracellular pathogens, Flaviviruses hijack cellular protein functions as a strategy for sustaining their life cycle. Many cellular proteins display globular domain known as PDZ domain that interacts with PDZ-Binding Motifs (PBM) identified in many viral proteins. Thus, cellular PDZ-containing proteins are common targets during viral infection. The non-structural protein 5 (NS5) from WNV provides both RNA cap methyltransferase and RNA polymerase activities and is involved in viral replication but its interactions with host proteins remain poorly known. In this study, we demonstrate that the C-terminal PBM of WNV NS5 recognizes several human PDZ-containing proteins using both in vitro and in cellulo high-throughput methods. Furthermore, we constructed and assayed in cell culture WNV replicons where the PBM within NS5 was mutated. Our results demonstrate that the PBM of WNV NS5 is important in WNV replication. Moreover, we show that knockdown of the PDZ-containing proteins TJP1, PARD3, ARHGAP21 or SHANK2 results in the decrease of WNV replication in cells. Altogether, our data reveal that interactions between the PBM of NS5 and PDZ-containing proteins affect West Nile virus replication.


Assuntos
Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/fisiologia , Animais , Sítios de Ligação , Linhagem Celular , Células HEK293 , Humanos , Domínios PDZ , Proteínas não Estruturais Virais/química , Febre do Nilo Ocidental/metabolismo
2.
Mol Cell Proteomics ; 20: 100049, 2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33515806

RESUMO

Viruses manipulate the central machineries of host cells to their advantage. They prevent host cell antiviral responses to create a favorable environment for their survival and propagation. Measles virus (MV) encodes two nonstructural proteins MV-V and MV-C known to counteract the host interferon response and to regulate cell death pathways. Several molecular mechanisms underlining MV-V regulation of innate immunity and cell death pathways have been proposed, whereas MV-C host-interacting proteins are less studied. We suggest that some cellular factors that are controlled by MV-C protein during viral replication could be components of innate immunity and the cell death pathways. To determine which host factors are targeted by MV-C, we captured both direct and indirect host-interacting proteins of MV-C protein. For this, we used a strategy based on recombinant viruses expressing tagged viral proteins followed by affinity purification and a bottom-up mass spectrometry analysis. From the list of host proteins specifically interacting with MV-C protein in different cell lines, we selected the host targets that belong to immunity and cell death pathways for further validation. Direct protein interaction partners of MV-C were determined by applying protein complementation assay and the bioluminescence resonance energy transfer approach. As a result, we found that MV-C protein specifically interacts with p65-iASPP protein complex that controls both cell death and innate immunity pathways and evaluated the significance of these host factors on virus replication.

3.
Proc Natl Acad Sci U S A ; 118(2)2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33402530

RESUMO

The recent emergence and reemergence of viruses in the human population has highlighted the need to develop broader panels of therapeutic molecules. High-throughput screening assays opening access to untargeted steps of the viral replication cycle will provide powerful leverage to identify innovative antiviral molecules. We report here the development of an innovative protein complementation assay, termed αCentauri, to measure viral translocation between subcellular compartments. As a proof of concept, the Centauri fragment was either tethered to the nuclear pore complex or sequestered in the nucleus, while the complementary α fragment (<16 amino acids) was attached to the integrase proteins of infectious HIV-1. The translocation of viral ribonucleoproteins from the cytoplasm to the nuclear envelope or to the nucleoplasm efficiently reconstituted superfolder green fluorescent protein or NanoLuc αCentauri reporters. These fluorescence- or bioluminescence-based assays offer a robust readout of specific steps of viral infection in a multiwell format that is compatible for high-throughput screening and is validated by a short hairpin RNA-based prototype screen.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Viroses/metabolismo , Replicação Viral/fisiologia , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Infecções por HIV/metabolismo , Células HeLa , Humanos , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Ribonucleoproteínas/metabolismo , Replicação Viral/efeitos dos fármacos
4.
Chemistry ; 27(6): 2112-2123, 2021 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-33137225

RESUMO

In this work on the design and studies of luciferins related to the blue-hued coelenterazine, the synthesis of heterocyclic analogues susceptible to produce a photon, possibly at a different wavelength, is undertaken. Here, the synthesis of O-acetylated derivatives of imidazo[1,2-b]pyridazin-3(5 H)-one, imidazo[2,1-f][1,2,4]triazin-7(1 H)-one, imidazo[1,2-a]pyridin-3-ol, imidazo[1,2-a]quinoxalin-1(5 H)-one, benzo[f]imidazo[1,2-a]quinoxalin-3(11 H)-one, imidazo[1',2':1,6]pyrazino[2,3-c]quinolin-3(11 H)-one, and 5,11-dihydro-3 H-chromeno[4,3-e]imidazo[1,2-a]pyrazin-3-one is described thanks to extensive use of the Buchwald-Hartwig N-arylation reaction. The acidic hydrolysis of these derivatives then gave solutions of the corresponding luciferin analogues, which were studied. Not too unexpectedly, even if these were "dressed" with substituents found in actual substrates of the nanoKAZ/NanoLuc luciferase, no bioluminescence was observed with these compounds. However, in a phosphate buffer, all produced a light signal, by chemiluminescence, with extensive variations in their respective intensity and this could be increased by adding a quaternary ammonium salt in the buffer. This aspect was actually instrumental to determine the emission spectra of many of these luciferin analogues.

5.
Nucleic Acids Res ; 48(18): 10428-10440, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32960265

RESUMO

Cellular exonucleases involved in the processes that regulate RNA stability and quality control have been shown to restrict or to promote the multiplication cycle of numerous RNA viruses. Influenza A viruses are major human pathogens that are responsible for seasonal epidemics, but the interplay between viral proteins and cellular exonucleases has never been specifically studied. Here, using a stringent interactomics screening strategy and an siRNA-silencing approach, we identified eight cellular factors among a set of 75 cellular proteins carrying exo(ribo)nuclease activities or involved in RNA decay processes that support influenza A virus multiplication. We show that the exoribonuclease ERI1 interacts with the PB2, PB1 and NP components of the viral ribonucleoproteins and is required for viral mRNA transcription. More specifically, we demonstrate that the protein-protein interaction is RNA dependent and that both the RNA binding and exonuclease activities of ERI1 are required to promote influenza A virus transcription. Finally, we provide evidence that during infection, the SLBP protein and histone mRNAs co-purify with vRNPs alongside ERI1, indicating that ERI1 is most probably recruited when it is present in the histone pre-mRNA processing complex in the nucleus.


Assuntos
Exorribonucleases/genética , Vírus da Influenza A/genética , Influenza Humana/genética , Proteínas Nucleares/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Linhagem Celular , Histonas/genética , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/patogenicidade , Influenza Humana/virologia , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Interferente Pequeno , RNA Viral/genética , Ribonucleoproteínas/genética , Transcrição Genética/genética , Proteínas Virais/genética , Replicação Viral/genética
6.
G3 (Bethesda) ; 10(9): 3399-3402, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32763951

RESUMO

The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.


Assuntos
Betacoronavirus/genética , Fases de Leitura Aberta/genética , Betacoronavirus/isolamento & purificação , COVID-19 , Clonagem Molecular , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Escherichia coli/metabolismo , Humanos , Pandemias , Plasmídeos/genética , Plasmídeos/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Potyvirus/genética , SARS-CoV-2
7.
mBio ; 11(2)2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32265326

RESUMO

The multifunctional nature of viral proteins is essentially driven by posttranslational modifications (PTMs) and is key for the successful outcome of infection. For influenza A viruses (IAVs), a composite pattern of PTMs regulates the activity of viral proteins. However, almost none are known that target the PB2 replication protein, except for inducing its degradation. We show here that PB2 undergoes a nonproteolytic ubiquitination during infection. We identified E3 ubiquitin ligases catalyzing this ubiquitination as two multicomponent RING-E3 ligases based on cullin 4 (CRL4s), which are both contributing to the levels of ubiquitinated forms of PB2 in infected cells. The CRL4 E3 ligase activity is required for the normal progression of the viral cycle and for maximal virion production, indicating that the CRL4s mediate a ubiquitin signaling that promotes infection. The CRL4s are recruiting PB2 through an unconventional bimodal interaction with both the DDB1 adaptor and DCAF substrate receptors. While able to bind to PB2 when engaged in the viral polymerase complex, the CRL4 factors do not alter transcription and replication of the viral segments during infection. CRL4 ligases catalyze different patterns of lysine ubiquitination on PB2. Recombinant viruses mutated in the targeted lysines showed attenuated viral production, suggesting that CRL4-mediated ubiquitination of PB2 contributes to IAV infection. We identified K29-linked ubiquitin chains as main components of the nonproteolytic PB2 ubiquitination mediated by the CRL4s, providing the first example of the role of this atypical ubiquitin linkage in the regulation of a viral infection.IMPORTANCE Successful infection by influenza A virus, a pathogen of major public health importance, involves fine regulation of the multiple functions of the viral proteins, which often relies on post-translational modifications (PTMs). The PB2 protein of influenza A viruses is essential for viral replication and a key determinant of host range. While PTMs of PB2 inducing its degradation have been identified, here we show that PB2 undergoes a regulating PTM signaling detected during infection, based on an atypical K29-linked ubiquitination and mediated by two multicomponent E3 ubiquitin ligases. Recombinant viruses impaired for CRL4-mediated ubiquitination are attenuated, indicating that ubiquitination of PB2 is necessary for an optimal influenza A virus infection. The CRL4 E3 ligases are required for normal viral cycle progression and for maximal virion production. Consequently, they represent potential candidate host factors for antiviral targets.


Assuntos
Proteínas Culina/metabolismo , Vírus da Influenza A/química , Provírus/enzimologia , RNA Polimerase Dependente de RNA/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Virais/química , Replicação Viral , Células A549 , Proteínas Culina/genética , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/fisiologia , Processamento de Proteína Pós-Traducional
8.
Nature ; 580(7803): 402-408, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32296183

RESUMO

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships1,2. Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome3, transcriptome4 and proteome5 data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes.


Assuntos
Proteoma/metabolismo , Espaço Extracelular/metabolismo , Humanos , Especificidade de Órgãos , Mapeamento de Interação de Proteínas
9.
Chemistry ; 26(4): 948-958, 2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31765054

RESUMO

We describe here an extensive structure-bioluminescence relationship study of a chemical library of analogues of coelenterazine, using nanoKAZ/NanoLuc, a mutated luciferase originated from the catalytic subunit of the deep-sea shrimp Oplophorus gracilirostris. Out of the 135 O-acetylated precursors that were prepared by using our recently reported synthesis and following their hydrolysis to give solutions of the corresponding luciferins, notable bioluminescence improvements were achieved in comparison with furimazine, which is currently amongst the best substrates of nanoKAZ/NanoLuc. For instance, the rather more lipophilic analogue 8-(2,3-difluorobenzyl)-2-((5-methylfuran-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one provided a 1.5-fold improvement of the total light output over a 2 h period, a close to threefold increase of the initial signal intensity and a signal-to-background ratio five times greater than furimazine. The kinetic parameters for the enzymatic reaction were obtained for a selection of luciferin analogues and provided unexpected insights into the luciferase activity. Most prominently, along with a general substrate-dependent and irreversible inactivation of this enzyme, in the case of the optimized luciferin mentioned above, the consumption of 2664 molecules was found to be required for the detection of a single Relative Light Unit (RLU; a luminometer-dependent fraction of a photon).

10.
mBio ; 10(6)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848284

RESUMO

Listeria monocytogenes is a pathogenic bacterium causing potentially fatal foodborne infections in humans and animals. While the mechanisms used by Listeria to manipulate its host have been thoroughly characterized, how the host controls bacterial virulence factors remains to be extensively deciphered. Here, we found that the secreted Listeria virulence protein InlC is monoubiquitinated by the host cell machinery on K224, restricting infection. We show that the ubiquitinated form of InlC interacts with the intracellular alarmin S100A9, resulting in its stabilization and in increased reactive oxygen species production by neutrophils in infected mice. Collectively, our results suggest that posttranslational modification of InlC exacerbates the host response upon Listeria infection.IMPORTANCE The pathogenic potential of Listeria monocytogenes relies on the production of an arsenal of virulence determinants that have been extensively characterized, including surface and secreted proteins of the internalin family. We have previously shown that the Listeria secreted internalin InlC interacts with IκB kinase α to interfere with the host immune response (E. Gouin, M. Adib-Conquy, D. Balestrino, M.-A. Nahori, et al., Proc Natl Acad Sci USA, 107:17333-17338, 2010, https://doi.org/10.1073/pnas.1007765107). In the present work, we report that InlC is monoubiquitinated on K224 upon infection of cells and provide evidence that ubiquitinated InlC interacts with and stabilizes the alarmin S100A9, which is a critical regulator of the immune response and inflammatory processes. Additionally, we show that ubiquitination of InlC causes an increase in reactive oxygen species production by neutrophils in mice and restricts Listeria infection. These findings are the first to identify a posttranscriptional modification of an internalin contributing to host defense.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Listeria/fisiologia , Listeriose/metabolismo , Listeriose/microbiologia , Calgranulina B/metabolismo , Suscetibilidade a Doenças , Células Epiteliais , Humanos , Ubiquitinação
11.
Sci Signal ; 12(601)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575732

RESUMO

The retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) RIG-I, MDA5, and LGP2 stimulate inflammatory and antiviral responses by sensing nonself RNA molecules produced during viral replication. Here, we investigated how LGP2 regulates the RIG-I- and MDA5-dependent induction of type I interferon (IFN) signaling and showed that LGP2 interacted with different components of the RNA-silencing machinery. We identified a direct protein-protein interaction between LGP2 and the IFN-inducible, double-stranded RNA binding protein PACT. The LGP2-PACT interaction was mediated by the regulatory C-terminal domain of LGP2 and was necessary for inhibiting RIG-I-dependent responses and for amplifying MDA5-dependent responses. We described a point mutation within LGP2 that disrupted the LGP2-PACT interaction and led to the loss of LGP2-mediated regulation of RIG-I and MDA5 signaling. These results suggest a model in which the LGP2-PACT interaction regulates the inflammatory responses mediated by RIG-I and MDA5 and enables the cellular RNA-silencing machinery to coordinate with the innate immune response.


Assuntos
Antivirais/metabolismo , Proteína DEAD-box 58/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Chlorocebus aethiops , Proteína DEAD-box 58/genética , Enterovirus Humano B/genética , Enterovirus Humano B/fisiologia , Células HEK293 , Células HeLa , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Mengovirus/genética , Mengovirus/fisiologia , Ligação Proteica , RNA Helicases/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Transdução de Sinais/genética , Células Vero
12.
Nat Commun ; 10(1): 3907, 2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31467278

RESUMO

Complementary assays are required to comprehensively map complex biological entities such as genomes, proteomes and interactome networks. However, how various assays can be optimally combined to approach completeness while maintaining high precision often remains unclear. Here, we propose a framework for binary protein-protein interaction (PPI) mapping based on optimally combining assays and/or assay versions to maximize detection of true positive interactions, while avoiding detection of random protein pairs. We have engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions, differing by protein expression systems and tagging configurations. The resulting union of N2H versions recovers as many PPIs as 10 distinct assays combined. Thus, to further improve PPI mapping, developing alternative versions of existing assays might be as productive as designing completely new assays. Our findings should be applicable to systematic mapping of other biological landscapes.


Assuntos
Bioensaio/métodos , Mapeamento de Interação de Proteínas/métodos , Proteoma/análise , Bases de Dados de Proteínas , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala/métodos , Humanos , Mapas de Interação de Proteínas , Proteínas/metabolismo , Proteômica/métodos , Técnicas do Sistema de Duplo-Híbrido
13.
Methods Mol Biol ; 1998: 291-304, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250310

RESUMO

The endosomal sorting complex required for transport (ESCRT) machinery comprises five complexes that act sequentially to recruit and cluster transmembrane cargo proteins (ESCRT-0), drive membrane curving (ESCRT-I and II), catalyze fission of cargo-containing vesicles (ESCRT-III and VPS/VTA1), and disassemble and recycle the ESCRT-III complex (VPS/VTA1). Since interactions between ESCRT components and cellular or microbial proteins are typically weak, transient, and involve membrane proteins, they are often difficult to study by standard technologies. Here, we describe the utility of high-throughput protein-fragment complementation assays based on the reconstitution of a split luciferase reporter to screen for interactions between any protein and a library of ESCRT proteins in mammalian cells and provide a detailed protocol for these assays.


Assuntos
Bioensaio/métodos , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Mapeamento de Interação de Proteínas/métodos , Animais , Copépodes , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Luciferases/química , Luciferases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção
14.
Proc Natl Acad Sci U S A ; 116(22): 10968-10977, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31076555

RESUMO

New therapeutic strategies targeting influenza are actively sought due to limitations in current drugs available. Host-directed therapy is an emerging concept to target host functions involved in pathogen life cycles and/or pathogenesis, rather than pathogen components themselves. From this perspective, we focused on an essential host partner of influenza viruses, the RED-SMU1 splicing complex. Here, we identified two synthetic molecules targeting an α-helix/groove interface essential for RED-SMU1 complex assembly. We solved the structure of the SMU1 N-terminal domain in complex with RED or bound to one of the molecules identified to disrupt this complex. We show that these compounds inhibiting RED-SMU1 interaction also decrease endogenous RED-SMU1 levels and inhibit viral mRNA splicing and viral multiplication, while preserving cell viability. Overall, our data demonstrate the potential of RED-SMU1 destabilizing molecules as an antiviral therapy that could be active against a wide range of influenza viruses and be less prone to drug resistance.


Assuntos
Antivirais/farmacologia , Proteínas Cromossômicas não Histona/metabolismo , Citocinas/metabolismo , Orthomyxoviridae/efeitos dos fármacos , Fatores de Processamento de RNA/metabolismo , Células A549 , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Citocinas/química , Citocinas/genética , Células HEK293 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Orthomyxoviridae/patogenicidade , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Splicing de RNA , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/genética , Spliceossomos/efeitos dos fármacos
15.
Org Biomol Chem ; 17(15): 3709-3713, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30882838

RESUMO

An original gram-scale synthesis of O-acetylated forms of coelenterazine, furimazine or hydroxy-bearing analogues of luciferins is described. The comparison over two hours of their bioluminescence, using the nanoKAZ/NanoLuc luciferase, provides remarkable insights useful for the selection of a substrate adapted for a given application.


Assuntos
Luciferina de Vaga-Lumes/síntese química , Imidazóis/síntese química , Pirazinas/síntese química , Acetilação , Animais , Vaga-Lumes , Luciferina de Vaga-Lumes/análogos & derivados , Luciferina de Vaga-Lumes/química , Imidazóis/química , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes , Estrutura Molecular , Pirazinas/química
16.
Cell Rep ; 26(7): 1800-1814.e5, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30759391

RESUMO

The mechanisms that regulate envelopment of HCV and other viruses that bud intracellularly and/or lack late-domain motifs are largely unknown. We reported that K63 polyubiquitination of the HCV nonstructural (NS) 2 protein mediates HRS (ESCRT-0 component) binding and envelopment. Nevertheless, the ubiquitin signaling that governs NS2 ubiquitination remained unknown. Here, we map the NS2 interactome with the ubiquitin proteasome system (UPS) via mammalian cell-based screens. NS2 interacts with E3 ligases, deubiquitinases, and ligase regulators, some of which are candidate proviral or antiviral factors. MARCH8, a RING-finger E3 ligase, catalyzes K63-linked NS2 polyubiquitination in vitro and in HCV-infected cells. MARCH8 is required for infection with HCV, dengue, and Zika viruses and specifically mediates HCV envelopment. Our data reveal regulation of HCV envelopment via ubiquitin signaling and both a viral protein substrate and a ubiquitin K63-linkage of the understudied MARCH8, with potential implications for cell biology, virology, and host-targeted antiviral design.


Assuntos
Hepacivirus/metabolismo , Hepatite C/virologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Células HEK293 , Hepacivirus/patogenicidade , Hepatite C/genética , Hepatite C/metabolismo , Humanos , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
17.
SLAS Discov ; 24(1): 25-37, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30184441

RESUMO

Natural killer (NK) cells are essential players of the innate immune response that secrete cytolytic factors and cytokines such as IFN-γ when contacting virus-infected or tumor cells. They represent prime targets in immunotherapy as defects in NK cell functions are hallmarks of many pathological conditions, such as cancer and chronic infections. The functional screening of chemical libraries or biologics would greatly help identify new modulators of NK cell activity, but commonly used methods such as flow cytometry are not easily scalable to high-throughput settings. Here we describe an efficient assay to measure the natural cytotoxicity of primary NK cells where the bioluminescent enzyme NanoLuc is constitutively expressed in the cytoplasm of target cells and is released in co-culture supernatants when lysis occurs. We fully characterized this assay using either purified NK cells or total peripheral blood mononuclear cells (PBMCs), including some patient samples, as effector cells. A pilot screen was also performed on a library of 782 metabolites, xenobiotics, and common drugs, which identified dextrometorphan and diphenhydramine as novel NK cell inhibitors. Finally, this assay was further improved by developing a dual-reporter cell line to simultaneously measure NK cell cytotoxicity and IFN-γ secretion in a single well, extending the potential of this system.


Assuntos
Células Matadoras Naturais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , Citometria de Fluxo/métodos , Células HEK293 , Humanos , Interferon gama/metabolismo , Células K562 , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Luciferases/metabolismo , Projetos Piloto
18.
Oncotarget ; 9(17): 13102-13115, 2018 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-29568343

RESUMO

The SRC Kinase Adaptor Phosphoprotein 2 (SKAP2) is a broadly expressed adaptor associated with the control of actin-polymerization, cell migration, and oncogenesis. After activation of different receptors at the cell surface, this dimeric protein serves as a platform for assembling other adaptors such as FYB and some SRC family kinase members, although these mechanisms are still poorly understood. The goal of this study is to map the SKAP2 interactome and characterize which domains or binding motifs are involved in these interactions. This is a prerequisite to finely analyze how these pathways are integrated in the cell machinery and to study their role in cancer and other human diseases when this network of interactions is perturbed. In this work, the domain and the binding motif of fourteen proteins interacting with SKAP2 were precisely defined and a new interactor, FAM102A was discovered. Herein, a fine-tuning between the binding of SRC kinases and their activation was identified. This last process, which depends on SKAP2 dimerization, indirectly affects the binding of FYB protein. Analysis of conformational changes associated with activation/inhibition of SRC family members, presently limited to their effect on kinase activity, is extended to their interactive network, which paves the way for therapeutic development.

20.
mSphere ; 2(6)2017.
Artigo em Inglês | MEDLINE | ID: mdl-29202037

RESUMO

The optimized exploitation of cell resources is one cornerstone of a successful infection. Differential mapping of host-pathogen protein-protein interactions (PPIs) on the basis of comparative interactomics of multiple strains is an effective strategy to highlight correlations between host proteome hijacking and biological or pathogenic traits. Here, we developed an interactomic pipeline to deliver high-confidence comparative maps of PPIs between a given pathogen and the human ubiquitin proteasome system (UPS). This subarray of the human proteome represents a range of essential cellular functions and promiscuous targets for many viruses. The screening pipeline was applied to the influenza A virus (IAV) PB2 polymerase proteins of five strains representing different levels of virulence in humans. An extensive PB2-UPS interplay has been detected that recapitulates the evolution of IAVs in humans. Functional validation with several IAV strains, including the seasonal H1N1pdm09 and H3N2 viruses, confirmed the biological relevance of most identified UPS factors and revealed strain-independent and strain-specific effects of UPS factor invalidation on IAV infection. This strategy is applicable to proteins from any other virus or pathogen, providing a valuable resource with which to explore the UPS-pathogen interplay and its relationship with pathogenicity. IMPORTANCE Influenza A viruses (IAVs) are responsible for mild-to-severe seasonal respiratory illness of public health concern worldwide, and the risk of avian strain outbreaks in humans is a constant threat. Elucidating the requisites of IAV adaptation to humans is thus of prime importance. In this study, we explored how PB2 replication proteins of IAV strains with different levels of virulence in humans hijack a major protein modification pathway of the human host cell, the ubiquitin proteasome system (UPS). We found that the PB2 protein engages in an extended interplay with the UPS that evolved along with the virus's adaptation to humans. This suggests that UPS hijacking underlies the efficient infection of humans and can be used as an indicator for evaluation of the potential of avian IAVs to infect humans. Several UPS factors were found to be necessary for infection with circulating IAV strains, pointing to potential targets for therapeutic approaches.

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