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1.
J Immunol ; 210(9): 1209-1221, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-36961448

RESUMO

Autosomal recessive PRKCD deficiency has previously been associated with the development of systemic lupus erythematosus in human patients, but the mechanisms underlying autoimmunity remain poorly understood. We introduced the Prkcd G510S mutation that we previously associated to a Mendelian cause of systemic lupus erythematosus in the mouse genome, using CRISPR-Cas9 gene editing. PrkcdG510S/G510S mice recapitulated the human phenotype and had reduced lifespan. We demonstrate that this phenotype is linked to a B cell-autonomous role of Prkcd. A detailed analysis of B cell activation in PrkcdG510S/G510S mice shows an upregulation of the PI3K/mTOR pathway after the engagement of the BCR in these cells, leading to lymphoproliferation. Treatment of mice with rapamycin, an mTORC1 inhibitor, significantly improves autoimmune symptoms, demonstrating in vivo the deleterious effect of mTOR pathway activation in PrkcdG510S/G510S mice. Additional defects in PrkcdG510S/G510S mice include a decrease in peripheral mature NK cells that might contribute to the known susceptibility to viral infections of patients with PRKCD mutations.


Assuntos
Autoimunidade , Lúpus Eritematoso Sistêmico , Humanos , Animais , Camundongos , Serina-Treonina Quinases TOR/metabolismo , Linfócitos B , Proliferação de Células
2.
PLoS Pathog ; 18(8): e1010798, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-36007070

RESUMO

Hepatitis E virus (HEV) infection is the most common cause of acute viral hepatitis worldwide. Hepatitis E is usually asymptomatic and self-limiting but it can become chronic in immunocompromised patients and is associated with increased fulminant hepatic failure and mortality rates in pregnant women. HEV genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Interestingly, HEV produces 3 isoforms of the ORF2 capsid protein which are partitioned in different subcellular compartments and perform distinct functions in the HEV lifecycle. Notably, the infectious ORF2 (ORF2i) protein is the structural component of virions, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. Here, by using a series of ORF2 capsid protein mutants expressed in the infectious genotype 3 p6 HEV strain as well as chimeras between ORF2 and the CD4 glycoprotein, we demonstrated how an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region controls the fate and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation likely to promote regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. It serves as maturation site of glycosylated ORF2 by furin, and promotes ORF2-host cell membrane interactions. The identification of ORF2 ARM as a unique central regulator of the HEV lifecycle uncovers how viruses settle strategies to condense their genetic information and hijack cellular processes.


Assuntos
Vírus da Hepatite E , Hepatite E , Motivos de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Feminino , Glicosilação , Hepatite E/genética , Hepatite E/metabolismo , Vírus da Hepatite E/crescimento & desenvolvimento , Humanos , Gravidez
3.
J Virol ; 95(3)2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33208442

RESUMO

Hepatitis C virus (HCV) infection triggers Golgi fragmentation through the Golgi-resident protein immunity-related GTPase M (IRGM). Here, we report the roles of NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) and ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain [CARD]), two inflammasome components, in the initial events leading to this fragmentation. We show that ASC resides at the Golgi with IRGM at homeostasis. Upon infection, ASC dissociates from both IRGM and the Golgi and associates with HCV-induced NLRP3. NLRP3 silencing inhibits Golgi fragmentation. ASC silencing disrupts the Golgi structure in both control and infected cells and reduces the localization of IRGM at the Golgi. IRGM depletion in the ASC-silenced cells cannot totally restore the Golgi structure. These data highlight a role for ASC, upstream of the formation of the inflammasome, in regulating IRGM through its control on the Golgi. A similar mechanism occurs in response to nigericin treatment, but not in cells infected with another member of the Flaviviridae family, Zika virus (ZIKV). We propose a model for a newly ascribed function of the inflammasome components in Golgi structural remodeling during certain stimuli.IMPORTANCE Numerous pathogens can affect cellular homeostasis and organelle dynamics. Hepatitis C virus (HCV) triggers Golgi fragmentation through the immunity-related GTPase M (IRGM), a resident Golgi protein, to enhance its lipid supply for replication. Here, we reveal the role of the inflammasome components NLRP3 and ASC in this process, thus uncovering a new interplay between effectors of inflammation and viral infection or stress. We show that the inflammasome component ASC resides at the Golgi under homeostasis and associates with IRGM. Upon HCV infection, ASC is recruited to NLRP3 and dissociates from IRGM, causing Golgi fragmentation. Our results uncover that aside from their known function in the inflammation response, these host defense regulators also ensure the maintenance of intact intracellular structure in homeostasis, while their activation relieves factors leading to Golgi remodeling.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Complexo de Golgi/fisiologia , Hepacivirus/isolamento & purificação , Hepatite C/virologia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Apoptose , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas de Ligação ao GTP/genética , Complexo de Golgi/virologia , Hepatite C/metabolismo , Hepatite C/patologia , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética
4.
PLoS Pathog ; 16(7): e1008737, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32726355

RESUMO

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. An unbalanced immune response, characterized by a weak production of type I interferons (IFN-Is) and an exacerbated release of proinflammatory cytokines, contributes to the severe forms of the disease. SARS-CoV-2 is genetically related to SARS-CoV and Middle East respiratory syndrome-related coronavirus (MERS-CoV), which caused outbreaks in 2003 and 2013, respectively. Although IFN treatment gave some encouraging results against SARS-CoV and MERS-CoV in animal models, its potential as a therapeutic against COVID-19 awaits validation. Here, we describe our current knowledge of the complex interplay between SARS-CoV-2 infection and the IFN system, highlighting some of the gaps that need to be filled for a better understanding of the underlying molecular mechanisms. In addition to the conserved IFN evasion strategies that are likely shared with SARS-CoV and MERS-CoV, novel counteraction mechanisms are being discovered in SARS-CoV-2-infected cells. Since the last coronavirus epidemic, we have made considerable progress in understanding the IFN-I response, including its spatiotemporal regulation and the prominent role of plasmacytoid dendritic cells (pDCs), which are the main IFN-I-producing cells. While awaiting the results of the many clinical trials that are evaluating the efficacy of IFN-I alone or in combination with antiviral molecules, we discuss the potential benefits of a well-timed IFN-I treatment and propose strategies to boost pDC-mediated IFN responses during the early stages of viral infection.


Assuntos
Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/imunologia , Infecções por Coronavirus/tratamento farmacológico , Células Dendríticas/imunologia , Imunidade Inata/imunologia , Interferon Tipo I/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Betacoronavirus/isolamento & purificação , COVID-19 , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Células Dendríticas/efeitos dos fármacos , Humanos , Imunidade Inata/efeitos dos fármacos , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Prognóstico , SARS-CoV-2 , Tratamento Farmacológico da COVID-19
5.
PLoS Pathog ; 15(2): e1007589, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30818370

RESUMO

Human T Lymphotropic virus (HTLV) infection can persist in individuals resulting, at least in part, from viral escape of the innate immunity, including inhibition of type I interferon response in infected T-cells. Plasmacytoid dendritic cells (pDCs) are known to bypass viral escape by their robust type I interferon production. Here, we demonstrated that pDCs produce type I interferons upon physical cell contact with HTLV-infected cells, yet pDC activation inversely correlates with the ability of the HTLV-producing cells to transmit infection. We show that pDCs sense surface associated-HTLV present with glycan-rich structure referred to as biofilm-like structure, which thus represents a newly described viral structure triggering the antiviral response by pDCs. Consistently, heparan sulfate proteoglycans and especially the cell surface pattern of terminal ß-galactoside glycosylation, modulate the transmission of the immunostimulatory RNA to pDCs. Altogether, our results uncover a function of virus-containing cell surface-associated glycosylated structures in the activation of innate immunity.


Assuntos
Células Dendríticas/fisiologia , Infecções por HTLV-I/metabolismo , Citocinas , Galactosídeos/metabolismo , Glicosilação , Infecções por HTLV-I/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Vírus Linfotrópico T Tipo 2 Humano/imunologia , Vírus Linfotrópico T Tipo 2 Humano/patogenicidade , Humanos , Imunidade Inata/fisiologia , Interferon Tipo I/imunologia , Interferon-alfa/imunologia , Interferon-alfa/metabolismo , Células Jurkat , Linfócitos T/imunologia , Linfócitos T/fisiologia
6.
PLoS Pathog ; 13(9): e1006610, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28957419

RESUMO

IFITMs are broad antiviral factors that block incoming virions in endosomal vesicles, protecting target cells from infection. In the case of HIV-1, we and others reported the existence of an additional antiviral mechanism through which IFITMs lead to the production of virions of reduced infectivity. However, whether this second mechanism of inhibition is unique to HIV or extends to other viruses is currently unknown. To address this question, we have analyzed the susceptibility of a broad spectrum of viruses to the negative imprinting of the virion particles infectivity by IFITMs. The results we have gathered indicate that this second antiviral property of IFITMs extends well beyond HIV and we were able to identify viruses susceptible to the three IFITMs altogether (HIV-1, SIV, MLV, MPMV, VSV, MeV, EBOV, WNV), as well as viruses that displayed a member-specific susceptibility (EBV, DUGV), or were resistant to all IFITMs (HCV, RVFV, MOPV, AAV). The swapping of genetic elements between resistant and susceptible viruses allowed us to point to specificities in the viral mode of assembly, rather than glycoproteins as dominant factors of susceptibility. However, we also show that, contrarily to X4-, R5-tropic HIV-1 envelopes confer resistance against IFITM3, suggesting that viral receptors add an additional layer of complexity in the IFITMs-HIV interplay. Lastly, we show that the overall antiviral effects ascribed to IFITMs during spreading infections, are the result of a bimodal inhibition in which IFITMs act both by protecting target cells from incoming viruses and in driving the production of virions of reduced infectivity. Overall, our study reports for the first time that the negative imprinting of the virion particles infectivity is a conserved antiviral property of IFITMs and establishes IFITMs as a paradigm of restriction factor capable of interfering with two distinct phases of a virus life cycle.


Assuntos
Antígenos de Diferenciação/metabolismo , Vírion , Replicação Viral , Linhagem Celular , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Internalização do Vírus
7.
Gastroenterology ; 152(4): 895-907.e14, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27932311

RESUMO

BACKGROUND & AIMS: Daclatasvir is a direct-acting antiviral agent and potent inhibitor of NS5A, which is involved in replication of the hepatitis C virus (HCV) genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. Daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. We investigated the relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs, and inhibition of viral particle assembly. METHODS: Cell-culture-derived HCV particles were produced from Huh7.5 hepatocarcinoma cells in presence of daclatasvir for short time periods. Infectivity and production of physical particles were quantified and producer cells were subjected to subcellular fractionation. Intracellular colocalization between core, E2, NS5A, NS4B proteins, and viral RNAs was quantitatively analyzed by confocal microscopy and by structured illumination microscopy. RESULTS: Short exposure of HCV-infected cells to daclatasvir reduced viral assembly and induced clustering of structural proteins with non-structural HCV proteins, including core, E2, NS4B, and NS5A. These clustered structures appeared to be inactive assembly platforms, likely owing to loss of functional connection with replication complexes. Daclatasvir greatly reduced delivery of viral genomes to these core clusters without altering HCV RNA colocalization with NS5A. In contrast, daclatasvir neither induced clustered structures nor inhibited HCV assembly in cells infected with a daclatasvir-resistant mutant (NS5A-Y93H), indicating that daclatasvir targets a mutual, specific function of NS5A inhibiting both processes. CONCLUSIONS: In addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins because viral particles and replication complex vesicles cannot form or egress. This dual mode of action of daclatasvir could explain its efficacy in blocking HCV replication in cultured cells and in treatment of patients with HCV infection.


Assuntos
Antivirais/farmacologia , Genoma Viral , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Imidazóis/farmacologia , Transporte de RNA/efeitos dos fármacos , RNA Viral/metabolismo , Carbamatos , Linhagem Celular Tumoral , Hepacivirus/fisiologia , Humanos , Transporte Proteico/efeitos dos fármacos , Pirrolidinas , Valina/análogos & derivados , Proteínas do Core Viral/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus/efeitos dos fármacos
8.
J Virol ; 90(22): 10050-10053, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27605675

RESUMO

All cells possess signaling pathways designed to trigger antiviral responses, notably characterized by type I interferon (IFN) production, upon recognition of invading viruses. Especially, host sensors recognize viral nucleic acids. Nonetheless, virtually all viruses have evolved potent strategies that preclude host responses within the infected cells. The plasmacytoid dendritic cell (pDC) is an immune cell type known as a robust type I IFN producer in response to viral infection. Evidence suggests that such functionality of the pDCs participates in viral clearance. Nonetheless, their contribution, which is likely complex and varies depending on the pathogen, is still enigmatic for many viruses. pDCs are not permissive to most viral infections, and consistently, recent examples suggest that pDCs respond to immunostimulatory viral RNA transferred via noninfectious and/or noncanonical viral/cellular carriers. Therefore, the pDC response likely bypasses innate signaling blockages induced by virus within infected cells. Importantly, the requirement for cell-cell contact is increasingly recognized as a hallmark of the pDC-mediated antiviral state, triggered by evolutionarily divergent RNA viruses.


Assuntos
Células Dendríticas/imunologia , Viroses/imunologia , Animais , Citocinas/imunologia , Humanos , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Transdução de Sinais/imunologia
9.
Nature ; 474(7352): 467-71, 2011 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-21666665

RESUMO

Polyhydroxylated steroids are regulators of body shape and size in higher organisms. In metazoans, intracellular receptors recognize these molecules. Plants, however, perceive steroids at membranes, using the membrane-integral receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1). Here we report the structure of the Arabidopsis thaliana BRI1 ligand-binding domain, determined by X-ray diffraction at 2.5 Å resolution. We find a superhelix of 25 twisted leucine-rich repeats (LRRs), an architecture that is strikingly different from the assembly of LRRs in animal Toll-like receptors. A 70-amino-acid island domain between LRRs 21 and 22 folds back into the interior of the superhelix to create a surface pocket for binding the plant hormone brassinolide. Known loss- and gain-of-function mutations map closely to the hormone-binding site. We propose that steroid binding to BRI1 generates a docking platform for a co-receptor that is required for receptor activation. Our findings provide insight into the activation mechanism of this highly expanded family of plant receptors that have essential roles in hormone, developmental and innate immunity signalling.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/química , Colestanóis/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Esteroides Heterocíclicos/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Sítios de Ligação , Brassinosteroides , Colestanóis/química , Cristalografia por Raios X , Ativação Enzimática , Modelos Moleculares , Dados de Sequência Molecular , Reguladores de Crescimento de Plantas/química , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Esteroides Heterocíclicos/química , Relação Estrutura-Atividade
10.
J Biol Chem ; 290(38): 23173-87, 2015 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-26224633

RESUMO

Lipoprotein components are crucial factors for hepatitis C virus (HCV) assembly and entry. As hepatoma cells producing cell culture-derived HCV (HCVcc) particles are impaired in some aspects of lipoprotein metabolism, it is of upmost interest to biochemically and functionally characterize the in vivo produced viral particles, particularly regarding how lipoprotein components modulate HCV entry by lipid transfer receptors such as scavenger receptor BI (SR-BI). Sera from HCVcc-infected liver humanized FRG mice were separated by density gradients. Viral subpopulations, termed HCVfrg particles, were characterized for their physical properties, apolipoprotein association, and infectivity. We demonstrate that, in contrast to the widely spread distribution of apolipoproteins across the different HCVcc subpopulations, the most infectious HCVfrg particles are highly enriched in apoE, suggesting that such apolipoprotein enrichment plays a role for entry of in vivo derived infectious particles likely via usage of apolipoprotein receptors. Consistent with this salient feature, we further reveal previously undefined functionalities of SR-BI in promoting entry of in vivo produced HCV. First, unlike HCVcc, SR-BI is a particularly limiting factor for entry of HCVfrg subpopulations of very low density. Second, HCVfrg entry involves SR-BI lipid transfer activity but not its capacity to bind to the viral glycoprotein E2. In conclusion, we demonstrate that composition and biophysical properties of the different subpopulations of in vivo produced HCVfrg particles modulate their levels of infectivity and receptor usage, hereby featuring divergences with in vitro produced HCVcc particles and highlighting the powerfulness of this in vivo model for the functional study of the interplay between HCV and liver components.


Assuntos
Hepacivirus/metabolismo , Hepatite C/metabolismo , Fígado/virologia , Internalização do Vírus , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Hepacivirus/genética , Hepatite C/genética , Hepatite C/patologia , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
11.
PLoS Pathog ; 10(10): e1004434, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25340500

RESUMO

Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. Like many viruses, DENV has evolved potent mechanisms that abolish the antiviral response within infected cells. Nevertheless, several in vivo studies have demonstrated a key role of the innate immune response in controlling DENV infection and disease progression. Here, we report that sensing of DENV infected cells by plasmacytoid dendritic cells (pDCs) triggers a robust TLR7-dependent production of IFNα, concomitant with additional antiviral responses, including inflammatory cytokine secretion and pDC maturation. We demonstrate that unlike the efficient cell-free transmission of viral infectivity, pDC activation depends on cell-to-cell contact, a feature observed for various cell types and primary cells infected by DENV, as well as West Nile virus, another member of the Flavivirus genus. We show that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response.


Assuntos
Células Dendríticas/virologia , Vírus da Dengue , Proteínas do Envelope Viral/metabolismo , Citoesqueleto de Actina/metabolismo , Evolução Biológica , Linhagem Celular , Humanos , Imunidade Inata/imunologia , Fusão de Membrana/fisiologia
12.
Plant J ; 77(2): 322-37, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24147788

RESUMO

Phosphatidylinositolphosphates (PIPs) are phospholipids that contain a phosphorylated inositol head group. PIPs represent a minor fraction of total phospholipids, but are involved in many regulatory processes, such as cell signalling and intracellular trafficking. Membrane compartments are enriched or depleted in specific PIPs, providing a unique composition for these compartments and contributing to their identity. The precise subcellular localization and dynamics of most PIP species is not fully understood in plants. Here, we designed genetically encoded biosensors with distinct relative affinities and expressed them stably in Arabidopsis thaliana. Analysis of this multi-affinity 'PIPline' marker set revealed previously unrecognized localization of various PIPs in root epidermis. Notably, we found that PI(4,5)P2 is able to localize PIP2 -interacting protein domains to the plasma membrane in non-stressed root epidermal cells. Our analysis further revealed that there is a gradient of PI4P, with the highest concentration at the plasma membrane, intermediate concentration in post-Golgi/endosomal compartments, and the lowest concentration in the Golgi. Finally, we also found a similar gradient of PI3P from high in late endosomes to low in the tonoplast. Our library extends the range of available PIP biosensors, and will allow rapid progress in our understanding of PIP dynamics in plants.


Assuntos
Arabidopsis/metabolismo , Biomarcadores/metabolismo , Fosfatidilinositóis/metabolismo , Técnicas Biossensoriais , Compartimento Celular
13.
Proc Natl Acad Sci U S A ; 109(16): 6205-10, 2012 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-22492964

RESUMO

Hepatitis C virus (HCV) infects ∼2% of the world's population. It is estimated that there are more than 500,000 new infections annually in Egypt, the country with the highest HCV prevalence. An effective vaccine would help control this expanding global health burden. HCV is highly variable, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human mAbs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies to HCV.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Hepacivirus/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos/imunologia , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/imunologia , Genótipo , Células HEK293 , Hepacivirus/genética , Hepatite C/imunologia , Hepatite C/prevenção & controle , Hepatite C/virologia , Anticorpos Anti-Hepatite C/imunologia , Humanos , Immunoblotting , Camundongos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Tetraspanina 28/imunologia , Proteínas do Envelope Viral/genética , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/imunologia
14.
Hepatology ; 57(2): 492-504, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23081796

RESUMO

UNLABELLED: Scavenger receptor class B type I (SR-BI) is a high-density lipoprotein (HDL) receptor highly expressed in the liver and modulating HDL metabolism. Hepatitis C virus (HCV) is able to directly interact with SR-BI and requires this receptor to efficiently enter into hepatocytes to establish productive infection. A complex interplay between lipoproteins, SR-BI and HCV envelope glycoproteins has been reported to take place during this process. SR-BI has been demonstrated to act during binding and postbinding steps of HCV entry. Although the SR-BI determinants involved in HCV binding have been partially characterized, the postbinding function of SR-BI remains largely unknown. To uncover the mechanistic role of SR-BI in viral initiation and dissemination, we generated a novel class of anti-SR-BI monoclonal antibodies that interfere with postbinding steps during the HCV entry process without interfering with HCV particle binding to the target cell surface. Using the novel class of antibodies and cell lines expressing murine and human SR-BI, we demonstrate that the postbinding function of SR-BI is of key impact for both initiation of HCV infection and viral dissemination. Interestingly, this postbinding function of SR-BI appears to be unrelated to HDL interaction but to be directly linked to its lipid transfer function. CONCLUSION: Taken together, our results uncover a crucial role of the SR-BI postbinding function for initiation and maintenance of viral HCV infection that does not require receptor-E2/HDL interactions. The dissection of the molecular mechanisms of SR-BI-mediated HCV entry opens a novel perspective for the design of entry inhibitors interfering specifically with the proviral function of SR-BI.


Assuntos
Antígenos CD36/metabolismo , Hepacivirus/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos CD36/imunologia , Linhagem Celular , HDL-Colesterol/antagonistas & inibidores , HDL-Colesterol/metabolismo , Hepacivirus/imunologia , Hepacivirus/patogenicidade , Hepatite C/prevenção & controle , Humanos , Lipoproteínas HDL/metabolismo , Camundongos , Ratos , Receptores de Lipoproteínas/metabolismo
15.
J Biol Chem ; 287(37): 31242-57, 2012 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-22767607

RESUMO

Hepatitis C virus (HCV) particles assemble along the very low density lipoprotein pathway and are released from hepatocytes as entities varying in their degree of lipid and apolipoprotein (apo) association as well as buoyant densities. Little is known about the cell entry pathway of these different HCV particle subpopulations, which likely occurs by regulated spatiotemporal processes involving several cell surface molecules. One of these molecules is the scavenger receptor BI (SR-BI), a receptor for high density lipoprotein that can bind to the HCV glycoprotein E2. By studying the entry properties of infectious virus subpopulations differing in their buoyant densities, we show that these HCV particles utilize SR-BI in a manifold manner. First, SR-BI mediates primary attachment of HCV particles of intermediate density to cells. These initial interactions involve apolipoproteins, such as apolipoprotein E, present on the surface of HCV particles, but not the E2 glycoprotein, suggesting that lipoprotein components in the virion act as host-derived ligands for important entry factors such as SR-BI. Second, we found that in contrast to this initial attachment, SR-BI mediates entry of HCV particles independent of their buoyant density. This function of SR-BI does not depend on E2/SR-BI interaction but relies on the lipid transfer activity of SR-BI, probably by facilitating entry steps along with other HCV entry co-factors. Finally, our results underscore a third function of SR-BI governed by specific residues in hypervariable region 1 of E2 leading to enhanced cell entry and depending on SR-BI ability to bind to E2.


Assuntos
Apolipoproteínas E/metabolismo , Hepacivirus/fisiologia , Receptores Depuradores Classe B/metabolismo , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Animais , Apolipoproteínas E/genética , Linhagem Celular Tumoral , Humanos , Camundongos , Estrutura Terciária de Proteína , Ratos , Receptores Depuradores Classe B/genética , Proteínas do Envelope Viral/genética
16.
Methods Mol Biol ; 2618: 289-315, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36905525

RESUMO

Dendritic cells (DCs) are key regulators of both innate and adaptive immunity via varied functions, including cytokine production and antigen presentation. Plasmacytoid DC (pDC) is a DC subset specialized in the production of type I and III interferons (IFNs). They are thus pivotal players of the host antiviral response during the acute phase of infection by genetically distant viruses. The pDC response is primarily triggered by the endolysosomal sensors Toll-like receptors, which recognize nucleic acids from pathogens. In some pathologic contexts, pDC response can also be triggered by host nucleic acids, hereby contributing to the pathogenesis of autoimmune diseases, such as, e.g., systemic lupus erythematosus. Importantly, recent in vitro studies from our laboratory and others uncovered that pDCs sense viral infections when a physical contact is established with infected cells. This specialized synapse-like feature enables a robust type I and III IFN secretion at the infected site. Therefore, this concentrated and confined response likely limits the correlated deleterious impacts of excessive cytokine production to the host, notably due to tissue damages. Here we provide a pipeline of methods for ex vivo studies of pDC antiviral functions, designed to address how pDC activation is regulated by cell-cell contact with virally infected cells and the current approaches enabling to decipher the underlying molecular events leading to an efficient antiviral response.


Assuntos
Interferon Tipo I , Ácidos Nucleicos , Imunidade Inata , Antivirais , Interferons , Células Dendríticas , Interferon Tipo I/metabolismo
17.
Nat Commun ; 14(1): 694, 2023 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-36755036

RESUMO

Type I and III interferons (IFN-I/λ) are important antiviral mediators against SARS-CoV-2 infection. Here, we demonstrate that plasmacytoid dendritic cells (pDC) are the predominant IFN-I/λ source following their sensing of SARS-CoV-2-infected cells. Mechanistically, this short-range sensing by pDCs requires sustained integrin-mediated cell adhesion with infected cells. In turn, pDCs restrict viral spread by an IFN-I/λ response directed toward SARS-CoV-2-infected cells. This specialized function enables pDCs to efficiently turn-off viral replication, likely via a local response at the contact site with infected cells. By exploring the pDC response in SARS-CoV-2 patients, we further demonstrate that pDC responsiveness inversely correlates with the severity of the disease. The pDC response is particularly impaired in severe COVID-19 patients. Overall, we propose that pDC activation is essential to control SARS-CoV-2-infection. Failure to develop this response could be important to understand severe cases of COVID-19.


Assuntos
COVID-19 , Interferon Tipo I , Humanos , SARS-CoV-2/metabolismo , Antivirais/metabolismo , Células Dendríticas/metabolismo , Interferon lambda
18.
J Biol Chem ; 286(35): 30361-30376, 2011 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-21737455

RESUMO

Hepatitis C virus (HCV) entry into permissive cells is a complex process that involves interactions with at least four co-factors followed by endocytosis and low pH-dependent fusion with endosomes. The precise sequence of receptor engagement and their roles in promoting HCV E1E2 glycoprotein-mediated fusion are poorly characterized. Because cell-free HCV tolerates an acidic environment, we hypothesized that binding to one or more receptors on the cell surface renders E1E2 competent to undergo low pH-induced conformational changes and promote fusion with endosomes. To test this hypothesis, we examined the effects of low pH and of the second extracellular loop (ECL2) of CD81, one of the four entry factors, on HCV infectivity. Pretreatment with an acidic buffer or with ECL2 enhanced infection through changing the E1E2 conformation, as evidenced by the altered reactivity of these proteins with conformation-specific antibodies and stable association with liposomes. However, neither of the two treatments alone permitted direct fusion with the cell plasma membrane. Sequential HCV preincubation with ECL2 and acidic buffer in the absence of target cells resulted in a marked loss of infectivity, implying that the receptor-bound HCV is primed for low pH-dependent conformational changes. Indeed, soluble receptor-pretreated HCV fused with the cell plasma membrane at low pH under conditions blocking an endocytic entry pathway. These findings suggest that CD81 primes HCV for low pH-dependent fusion early in the entry process. The simple triggering paradigm and intermediate conformations of E1E2 identified in this study could help guide future vaccine and therapeutic efforts to block HCV infection.


Assuntos
Antígenos CD/metabolismo , Hepacivirus/metabolismo , Anticorpos Monoclonais/química , Soluções Tampão , Linhagem Celular , Membrana Celular/metabolismo , Sistema Livre de Células , Endocitose , Humanos , Concentração de Íons de Hidrogênio , Cinética , Lipossomos/química , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Tetraspanina 28 , Proteínas Virais/química
19.
J Virol ; 85(11): 5513-23, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21430055

RESUMO

The recent development of a cell culture model of hepatitis C virus (HCV) infection based on the JFH-1 molecular clone has enabled discovery of new antiviral agents. Using a cell-based colorimetric screening assay to interrogate a 1,200-compound chemical library for anti-HCV activity, we identified a family of 1,2-diamines derived from trans-stilbene oxide that prevent HCV infection at nontoxic, low micromolar concentrations in cell culture. Structure-activity relationship analysis of ~ 300 derivatives synthesized using click chemistry yielded compounds with greatly enhanced low nanomolar potency and a > 1,000:1 therapeutic ratio. Using surrogate models of HCV infection, we showed that the compounds selectively block the initiation of replication of incoming HCV RNA but have no impact on viral entry, primary translation, or ongoing HCV RNA replication, nor do they suppress persistent HCV infection. Selection of an escape variant revealed that NS5A is directly or indirectly targeted by this compound. In summary, we have identified a family of HCV inhibitors that target a critical step in the establishment of HCV infection in which NS5A translated de novo from an incoming genomic HCV RNA template is required to initiate the replication of this important human pathogen.


Assuntos
Antivirais/farmacologia , Diaminas/farmacologia , Hepacivirus/efeitos dos fármacos , Estilbenos/farmacologia , Replicação Viral/efeitos dos fármacos , Antivirais/química , Antivirais/isolamento & purificação , Antivirais/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Diaminas/química , Diaminas/isolamento & purificação , Diaminas/toxicidade , Avaliação Pré-Clínica de Medicamentos/métodos , Farmacorresistência Viral , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Testes de Sensibilidade Microbiana , RNA Viral/metabolismo , Estilbenos/química , Estilbenos/isolamento & purificação , Estilbenos/toxicidade , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
20.
Proc Natl Acad Sci U S A ; 106(33): 14046-51, 2009 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-19666601

RESUMO

In addition to its cellular homeostasis function, autophagy is emerging as a central component of antimicrobial host defense against diverse infections. To counteract this mechanism, many pathogens have evolved to evade, subvert, or exploit autophagy. Here, we report that autophagy proteins (i.e., Beclin-1, Atg4B, Atg5, and Atg12) are proviral factors required for translation of incoming hepatitis C virus (HCV) RNA and, thereby, for initiation of HCV replication, but they are not required once infection is established. These results illustrate a previously unappreciated role for autophagy in the establishment of a viral infection and they suggest that different host factors regulate the translation of incoming viral genome and translation of progeny HCV RNA once replication is established.


Assuntos
Autofagia , Hepacivirus/genética , Hepacivirus/metabolismo , Replicação Viral , Linhagem Celular , Regulação Viral da Expressão Gênica , Genoma Viral , Glicoproteínas/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lentivirus/genética , Microscopia de Fluorescência , Fagossomos/metabolismo , RNA Viral/metabolismo , Proteínas Virais/metabolismo
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