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1.
Virology ; 589: 109921, 2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-37939648

RESUMEN

Human norovirus is the leading cause of acute gastroenteritis worldwide, however despite the significance of this pathogen, we have a limited understanding of how noroviruses cause disease, and modulate the innate immune response. Programmed cell death (PCD) is an important part of the innate response to invading pathogens, but little is known about how specific PCD pathways contribute to norovirus replication. Here, we reveal that murine norovirus (MNV) virus-induced PCD in macrophages correlates with the release of infectious virus. We subsequently show, genetically and chemically, that MNV-induced cell death and viral replication occurs independent of the activity of inflammatory mediators. Further analysis revealed that MNV infection promotes the cleavage of apoptotic caspase-3 and PARP. Correspondingly, pan-caspase inhibition, or BAX and BAK deficiency, perturbed viral replication rates and delayed virus release and cell death. These results provide new insights into how MNV harnesses cell death to increase viral burden.


Asunto(s)
Infecciones por Caliciviridae , Norovirus , Ratones , Humanos , Animales , Macrófagos , Apoptosis , Inmunidad Innata , Norovirus/fisiología , Replicación Viral
2.
EMBO Rep ; 24(11): e56865, 2023 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-37846472

RESUMEN

Programmed cell death pathways play an important role in innate immune responses to infection. Activation of intrinsic apoptosis promotes infected cell clearance; however, comparatively little is known about how this mode of cell death is regulated during infections and whether it can induce inflammation. Here, we identify that the pro-survival BCL-2 family member, A1, controls activation of the essential intrinsic apoptotic effectors BAX/BAK in macrophages and monocytes following bacterial lipopolysaccharide (LPS) sensing. We show that, due to its tight transcriptional and post-translational regulation, A1 acts as a molecular rheostat to regulate BAX/BAK-dependent apoptosis and the subsequent NLRP3 inflammasome-dependent and inflammasome-independent maturation of the inflammatory cytokine IL-1ß. Furthermore, induction of A1 expression in inflammatory monocytes limits cell death modalities and IL-1ß activation triggered by Neisseria gonorrhoeae-derived outer membrane vesicles (NOMVs). Consequently, A1-deficient mice exhibit heightened IL-1ß production in response to NOMV injection. These findings reveal that bacteria can induce A1 expression to delay myeloid cell death and inflammatory responses, which has implications for the development of host-directed antimicrobial therapeutics.


Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Ratones , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Células Mieloides/metabolismo , Muerte Celular , Interleucina-1beta/metabolismo
3.
Nat Commun ; 14(1): 5666, 2023 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-37723181

RESUMEN

TANK-binding kinase 1 (TBK1) is a key signalling component in the production of type-I interferons, which have essential antiviral activities, including against SARS-CoV-2. TBK1, and its homologue IκB kinase-ε (IKKε), can also induce pro-inflammatory responses that contribute to pathogen clearance. While initially protective, sustained engagement of type-I interferons is associated with damaging hyper-inflammation found in severe COVID-19 patients. The contribution of TBK1/IKKε signalling to these responses is unknown. Here we find that the small molecule idronoxil inhibits TBK1/IKKε signalling through destabilisation of TBK1/IKKε protein complexes. Treatment with idronoxil, or the small molecule inhibitor MRT67307, suppresses TBK1/IKKε signalling and attenuates cellular and molecular lung inflammation in SARS-CoV-2-challenged mice. Our findings additionally demonstrate that engagement of STING is not the major driver of these inflammatory responses and establish a critical role for TBK1/IKKε signalling in SARS-CoV-2 hyper-inflammation.


Asunto(s)
COVID-19 , Interferón Tipo I , Animales , Ratones , Quinasa I-kappa B , Modelos Animales de Enfermedad , SARS-CoV-2 , Inflamación
4.
Nat Commun ; 14(1): 6046, 2023 09 28.
Artículo en Inglés | MEDLINE | ID: mdl-37770424

RESUMEN

Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKLS132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-ß induced death in non-hematopoietic cells. In vivo, MlklS131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease.


Asunto(s)
Apoptosis , Proteínas Quinasas , Humanos , Animales , Ratones , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Membrana Celular/metabolismo , Mutación , Factores de Transcripción/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo
5.
Viruses ; 15(2)2023 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-36851669

RESUMEN

Bovine respiratory disease (BRD) is a major health problem within the global cattle industry. This disease has a complex aetiology, with viruses playing an integral role. In this study, metagenomics was used to sequence viral nucleic acids in the nasal swabs of BRD-affected cattle. The viruses detected included those that are well known for their association with BRD in Australia (bovine viral diarrhoea virus 1), as well as viruses known to be present but not fully characterised (bovine coronavirus) and viruses that have not been reported in BRD-affected cattle in Australia (bovine rhinitis, bovine influenza D, and bovine nidovirus). The nasal swabs from a case-control study were subsequently tested for 10 viruses, and the presence of at least one virus was found to be significantly associated with BRD. Some of the more recently detected viruses had inconsistent associations with BRD. Full genome sequences for bovine coronavirus, a virus increasingly associated with BRD, and bovine nidovirus were completed. Both viruses belong to the Coronaviridae family, which are frequently associated with disease in mammals. This study has provided greater insights into the viral pathogens associated with BRD and highlighted the need for further studies to more precisely elucidate the roles viruses play in BRD.


Asunto(s)
Enfermedades de los Bovinos , Coronavirus Bovino , Nidovirales , Enfermedades Respiratorias , Animales , Bovinos , Estudios de Casos y Controles , Viroma , Tráquea , Nariz , Coronavirus Bovino/genética , Mamíferos
6.
mBio ; 13(4): e0206422, 2022 08 30.
Artículo en Inglés | MEDLINE | ID: mdl-35924852

RESUMEN

Cytoplasmic detection of DNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) is an essential component of antiviral responses. Upon synthesis, cGAMP binds to the stimulator of interferon (IFN) genes (STING) in infected and adjacent cells through intercellular transfer by connexins forming gap-junctions, eliciting a strong IFN-ß-driven antiviral response. We demonstrate here that Genistein, a flavonoid compound naturally occurring in soy-based foods, inhibits cGAS-STING antiviral signaling at two levels. First, Genistein pretreatment of cGAMP-producing cells inhibited gap-junction intercellular communication, resulting in reduced STING responses in adjacent cells. In addition, Genistein directly blocked STING activation by the murine agonist DMXAA, by decreasing the interaction of STING with TBK1 and IKKε. As a result, Genistein attenuated STING signaling in human and mouse cells, dampening antiviral activity against Semliki Forest Virus infection. Collectively, our findings identify a previously unrecognized proviral activity of Genistein mediated via its inhibitory effects at two levels of cGAS-STING signaling. IMPORTANCE Several reports suggest that Genistein exhibits antiviral activities against DNA viruses. Our work uncovers a previously unrecognized proviral effect of Genistein, through inhibition of the cGAS-STING pathway at the level of cGAMP transfer and its sensing by STING. This suggests that the use of Genistein as an antiviral should be taken with caution as it may reduce the protective antiviral effects elicited by host STING activation.


Asunto(s)
Genisteína , Proteínas de la Membrana , Animales , Antivirales/farmacología , Genisteína/farmacología , Humanos , Inmunidad Innata/genética , Interferón beta/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Nucleotidiltransferasas/genética
7.
iScience ; 25(7): 104632, 2022 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-35800780

RESUMEN

Pathogen recognition and TNF receptors signal via receptor interacting serine/threonine kinase-3 (RIPK3) to cause cell death, including MLKL-mediated necroptosis and caspase-8-dependent apoptosis. However, the post-translational control of RIPK3 is not fully understood. Using mass-spectrometry, we identified that RIPK3 is ubiquitylated on K469. The expression of mutant RIPK3 K469R demonstrated that RIPK3 ubiquitylation can limit both RIPK3-mediated apoptosis and necroptosis. The enhanced cell death of overexpressed RIPK3 K469R and activated endogenous RIPK3 correlated with an overall increase in RIPK3 ubiquitylation. Ripk3 K469R/K469R mice challenged with Salmonella displayed enhanced bacterial loads and reduced serum IFNγ. However, Ripk3 K469R/K469R macrophages and dermal fibroblasts were not sensitized to RIPK3-mediated apoptotic or necroptotic signaling suggesting that, in these cells, there is functional redundancy with alternate RIPK3 ubiquitin-modified sites. Consistent with this idea, the mutation of other ubiquitylated RIPK3 residues also increased RIPK3 hyper-ubiquitylation and cell death. Therefore, the targeted ubiquitylation of RIPK3 may act as either a brake or accelerator of RIPK3-dependent killing.

8.
Sci Adv ; 8(19): eabh2332, 2022 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-35544574

RESUMEN

Tumor necrosis factor (TNF) is a key component of the innate immune response. Upon binding to its receptor, TNFR1, it promotes production of other cytokines via a membrane-bound complex 1 or induces cell death via a cytosolic complex 2. To understand how TNF-induced cell death is regulated, we performed mass spectrometry of complex 2 and identified tankyrase-1 as a native component that, upon a death stimulus, mediates complex 2 poly-ADP-ribosylation (PARylation). PARylation promotes recruitment of the E3 ligase RNF146, resulting in proteasomal degradation of complex 2, thereby limiting cell death. Expression of the ADP-ribose-binding/hydrolyzing severe acute respiratory syndrome coronavirus 2 macrodomain sensitizes cells to TNF-induced death via abolishing complex 2 PARylation. This suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

9.
Nat Commun ; 12(1): 4786, 2021 08 09.
Artículo en Inglés | MEDLINE | ID: mdl-34373455

RESUMEN

Salmonella enterica serovar 4,[5],12:i:- (Salmonella 4,[5],12:i:-) is a monophasic variant of Salmonella Typhimurium that has emerged as a global cause of multidrug resistant salmonellosis. We used Bayesian phylodynamics, genomic epidemiology, and phenotypic characterization to describe the emergence and evolution of Salmonella 4,[5],12:i:- in Australia. We show that the interruption of the genetic region surrounding the phase II flagellin, FljB, causing a monophasic phenotype, represents a stepwise evolutionary event through the accumulation of mobile resistance elements with minimal impairment to bacterial fitness. We identify three lineages with different population dynamics and discrete antimicrobial resistance profiles emerged, likely reflecting differential antimicrobial selection pressures. Two lineages are associated with travel to South-East Asia and the third lineage is endemic to Australia. Moreover antimicrobial-resistant Salmonella 4,[5],12:i- lineages efficiently infected and survived in host phagocytes and epithelial cells without eliciting significant cellular cytotoxicity, suggesting a suppression of host immune response that may facilitate the persistence of Salmonella 4,[5],12:i:-.


Asunto(s)
Farmacorresistencia Bacteriana Múltiple/genética , Evolución Molecular , Salmonella enterica/clasificación , Salmonella enterica/genética , Serogrupo , Antibacterianos/farmacología , Australia , Teorema de Bayes , Línea Celular , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Flagelina/genética , Humanos , Inmunidad , Metales Pesados/farmacología , Filogenia , Salmonella enterica/efectos de los fármacos , Salmonella typhimurium , Células THP-1 , Secuenciación Completa del Genoma
10.
Front Cell Dev Biol ; 9: 655606, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34055786

RESUMEN

Flavivirus replication is intimately associated with re-organized cellular membranes. These virus-induced changes in membrane architecture form three distinct membranous "organelles" that have specific functions during the flavivirus life cycle. One of these structures is the replication complex in which the flaviviral RNA is replicated to produce progeny genomes. We have previously observed that this process is strictly dependent on cellular cholesterol. In this study we have identified a putative cholesterol recognition/interaction amino acid consensus (CRAC) motif within the West Nile virus strain Kunjin virus (WNVKUN) NS4A protein. Site-directed mutagenesis of this motif within a WNVKUN infectious clone severely attenuated virus replication and the capacity of the mutant viruses to form the replication complex. Replication of the mutant viruses also displayed reduced co-localization with cellular markers recruited to replication sites during wild-type virus replication. In addition, we observed that the mutant viruses were significantly impaired in their ability to remodel cytoplasmic membranes. However, after extensive analysis we are unable to conclusively reveal a role for the CRAC motif in direct cholesterol binding to NS4A, suggesting additional complex lipid-protein and protein-protein interactions. We believe this study highlights the crucial role for this region within NS4A protein in recruitment of cellular and viral proteins to specialized subdomains on membrane platforms to promote efficient virus replication.

11.
Viruses ; 12(6)2020 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-32517260

RESUMEN

The Australasian Virology Society (AVS) aims to promote, support and advocate for the discipline of virology in the Australasian region. The society was incorporated in 2011 after 10 years operating as the Australian Virology Group (AVG) founded in 2001, coinciding with the inaugural biennial scientific meeting. AVS conferences aim to provide a forum for the dissemination of all aspects of virology, foster collaboration, and encourage participation by students and post-doctoral researchers. The tenth Australasian Virology Society (AVS10) scientific meeting was held on 2-5 December 2019 in Queenstown, New Zealand. This report highlights the latest research presented at the meeting, which included cutting-edge virology presented by our international plenary speakers Ana Fernandez-Sesma and Benjamin tenOever, and keynote Richard Kuhn. AVS10 honoured female pioneers in Australian virology, Lorena Brown and Barbara Coulson. We report outcomes from the AVS10 career development session on "Successfully transitioning from post-doc to lab head", winners of best presentation awards, and the AVS gender equity policy, initiated in 2013. Plans for the 2021 meeting are underway which will celebrate the 20th anniversary of AVS where it all began, in Fraser Island, Queensland, Australia.


Asunto(s)
Virología/organización & administración , Australia , Distinciones y Premios , Procesos de Grupo , Sociedades Científicas
12.
Heliyon ; 6(6): e04115, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32518853

RESUMEN

Regulation of type-I interferon (IFN) production is essential to the balance between antimicrobial defence and autoimmune disorders. The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domains, previously C6orf106) was recently characterised as an inhibitor of antiviral and proinflammatory cytokine (interferon-alpha/beta and tumor necrosis factor alpha) transcription. Currently there is a paucity of information about the molecular characteristics of ILRUN, despite it being associated with several diseases including virus infection, coronary artery disease, obesity and cancer. Here, we characterise ILRUN as a highly phylogenetically conserved protein containing UBA-like and a NBR1-like domains that are both essential for inhibition of type-I interferon and tumor necrosis factor alpha) transcription in human cells. We also solved the crystal structure of the NBR1-like domain, providing insights into its potential role in ILRUN function. This study provides critical information for future investigations into the role of ILRUN in health and disease.

13.
J Virol ; 92(21)2018 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-30111567

RESUMEN

Dengue virus (DENV) is the most prevalent mosquito-transmitted viral pathogen in humans. The recently licensed dengue vaccine has major weaknesses. Therefore, there is an urgent need to develop improved dengue vaccines. Here, we report a virion assembly-defective DENV as a vaccine platform. DENV containing an amino acid deletion (K188) in nonstructural protein 2A (NS2A) is fully competent in viral RNA replication but is completely defective in virion assembly. When trans-complemented with wild-type NS2A protein, the virion assembly defect could be rescued, generating pseudoinfectious virus (PIVNS2A) that could initiate single-round infection. The trans-complementation efficiency could be significantly improved through selection for adaptive mutations, leading to high-yield PIVNS2A production, with titers of >107 infectious-focus units (IFU)/ml. Mice immunized with a single dose of PIVNS2A elicited strong T cell immune responses and neutralization antibodies and were protected from wild-type-virus challenge. Collectively, the results proved the concept of using assembly-defective virus as a vaccine approach. The study also solved the technical bottleneck in producing high yields of PIVNS2A vaccine. The technology could be applicable to vaccine development for other viral pathogens.IMPORTANCE Many flaviviruses are significant human pathogens that pose global threats to public health. Although licensed vaccines are available for yellow fever, Japanese encephalitis, tick-borne encephalitis, and dengue viruses, new approaches are needed to develop improved vaccines. Using dengue virus as a model, we developed a vaccine platform using a virion assembly-defective virus. We show that such an assembly-defective virus could be rescued to higher titers and infect cells for a single round. Mice immunized with the assembly-defective virus were protected from wild-type-virus infection. This vaccine approach could be applicable to other viral pathogens.


Asunto(s)
Virus Defectuosos/patogenicidad , Vacunas contra el Dengue/inmunología , Virus del Dengue/patogenicidad , Dengue/virología , Proteínas no Estructurales Virales/inmunología , Ensamble de Virus , Replicación Viral , Animales , Anticuerpos Neutralizantes/inmunología , Virus Defectuosos/genética , Dengue/genética , Dengue/inmunología , Virus del Dengue/genética , Femenino , Humanos , Masculino , Ratones , Mutación , ARN Viral , Proteínas no Estructurales Virales/genética
14.
J Biol Chem ; 293(27): 10561-10573, 2018 07 06.
Artículo en Inglés | MEDLINE | ID: mdl-29802199

RESUMEN

Host recognition of intracellular viral RNA and subsequent induction of cytokine signaling are tightly regulated at the cellular level and are a target for manipulation by viruses and therapeutics alike. Here, we characterize chromosome 6 ORF 106 (C6orf106) as an evolutionarily conserved inhibitor of the innate antiviral response. C6orf106 suppresses the synthesis of interferon (IFN)-α/ß and proinflammatory tumor necrosis factor (TNF) α in response to the dsRNA mimic poly(I:C) and to Sendai virus infection. Unlike canonical inhibitors of antiviral signaling, C6orf106 blocks interferon-regulatory factor 3 (IRF3) and, to a lesser extent, NF-κB activity without modulating their activation, nuclear translocation, cellular expression, or degradation. Instead, C6orf106 interacts with IRF3 and inhibits IRF3 recruitment to type I IFN promoter sequences while also reducing the nuclear levels of the coactivator proteins p300 and CREB-binding protein (CBP). In summary, we have defined C6orf106 as a negative regulator of antiviral immunity that blocks IRF3-dependent cytokine production via a noncanonical and poorly defined mechanism. This work presents intriguing implications for antiviral immunity, autoimmune disorders, and cancer.


Asunto(s)
Antivirales/farmacología , Inmunidad Innata/inmunología , Factor 3 Regulador del Interferón/antagonistas & inhibidores , Proteínas de Neoplasias/farmacología , Infecciones por Respirovirus/prevención & control , Virus Sendai/inmunología , Animales , Antivirales/administración & dosificación , Chlorocebus aethiops , Regulación de la Expresión Génica , Células HeLa , Humanos , Inmunidad Innata/efectos de los fármacos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas de Neoplasias/administración & dosificación , Infecciones por Respirovirus/inmunología , Infecciones por Respirovirus/virología , Virus Sendai/efectos de los fármacos , Transducción de Señal , Células Vero
15.
PLoS Pathog ; 14(4): e1007029, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29709018

RESUMEN

Positive-sense RNA virus intracellular replication is intimately associated with membrane platforms that are derived from host organelles and comprised of distinct lipid composition. For flaviviruses, such as West Nile virus strain Kunjin virus (WNVKUN) we have observed that these membrane platforms are derived from the endoplasmic reticulum and are rich in (at least) cholesterol. To extend these studies and identify the cellular lipids critical for WNVKUN replication we utilized a whole cell lipidomics approach and revealed an elevation in phospholipase A2 (PLA2) activity to produce lyso-phosphatidylcholine (lyso-PChol). We observed that the PLA2 enzyme family is activated in WNVKUN-infected cells and the generated lyso-PChol lipid moieties are sequestered to the subcellular sites of viral replication. The requirement for lyso-PChol was confirmed using chemical inhibition of PLA2, where WNVKUN replication and production of infectious virus was duly affected in the presence of the inhibitors. Importantly, we could rescue chemical-induced inhibition with the exogenous addition of lyso-PChol species. Additionally, electron microscopy results indicate that lyso-PChol appears to contribute to the formation of the WNVKUN membranous replication complex (RC); particularly affecting the morphology and membrane curvature of vesicles comprising the RC. These results extend our current understanding of how flaviviruses manipulate lipid homeostasis to favour their own intracellular replication.


Asunto(s)
Retículo Endoplásmico/virología , Riñón/enzimología , Lípidos de la Membrana/metabolismo , Fosfolipasas A2/metabolismo , Replicación Viral , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/patogenicidad , Animales , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Retículo Endoplásmico/enzimología , Riñón/virología , Células Vero , Fiebre del Nilo Occidental/enzimología
16.
Pathogens ; 7(1)2018 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-29351201

RESUMEN

Bovine viral diarrhoea virus 1 (BVDV-1) is strongly associated with several important diseases of cattle, such as bovine respiratory disease, diarrhoea and haemoragic lesions. To date many subgenotypes have been reported for BVDV-1, currently ranging from subgenotype 1a to subgenotype 1u. While BVDV-1 has a world-wide distribution, the subgenotypes have a more restricted geographical distribution. As an example, BVDV-1 subgenotypes 1a and 1b are frequently detected in North America and Europe, while the subgenotype 1c is rarely detected. In contrast, BVDV-1 subgenotype 1c is by far the most commonly reported in Australia. Despite this, uneven distribution of the biological importance of the subgenotypes remains unclear. The aim of this study was to characterise the in vivo properties of five strains of BVDV-1 subgenotype 1c in cattle infection studies. No overt respiratory signs were reported in any of the infected cattle regardless of strain. Consistent with other subgenotypes, transient pyrexia and leukopenia were commonly identified, while thrombocytopenia was not. The quantity of virus detected in the nasal secretions of transiently infected animals suggested the likelihood of horizontal transmission was very low. Further studies are required to fully understand the variability and importance of the BVDV-1 subgenotype 1c.

17.
J Virol ; 88(21): 12276-95, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25122779

RESUMEN

UNLABELLED: Hepatitis C virus (HCV) assembles its replication complex on cytosolic membrane vesicles often clustered in a membranous web (MW). During infection, HCV NS5A protein activates PI4KIIIα enzyme, causing massive production and redistribution of phosphatidylinositol 4-phosphate (PI4P) lipid to the replication complex. However, the role of PI4P in the HCV life cycle is not well understood. We postulated that PI4P recruits host effectors to modulate HCV genome replication or virus particle production. To test this hypothesis, we generated cell lines for doxycycline-inducible expression of short hairpin RNAs (shRNAs) targeting the PI4P effector, four-phosphate adaptor protein 2 (FAPP2). FAPP2 depletion attenuated HCV infectivity and impeded HCV RNA synthesis. Indeed, FAPP2 has two functional lipid-binding domains specific for PI4P and glycosphingolipids. While expression of the PI4P-binding mutant protein was expected to inhibit HCV replication, a marked drop in replication efficiency was observed unexpectedly with the glycosphingolipid-binding mutant protein. These data suggest that both domains are crucial for the role of FAPP2 in HCV genome replication. We also found that HCV significantly increases the level of some glycosphingolipids, whereas adding these lipids to FAPP2-depleted cells partially rescued replication, further arguing for the importance of glycosphingolipids in HCV RNA synthesis. Interestingly, FAPP2 is redistributed to the replication complex (RC) characterized by HCV NS5A, NS4B, or double-stranded RNA (dsRNA) foci. Additionally, FAPP2 depletion disrupts the RC and alters the colocalization of HCV replicase proteins. Altogether, our study implies that HCV coopts FAPP2 for virus genome replication via PI4P binding and glycosphingolipid transport to the HCV RC. IMPORTANCE: Like most viruses with a positive-sense RNA genome, HCV replicates its RNA on remodeled host membranes composed of lipids hijacked from various internal membrane compartments. During infection, HCV induces massive production and retargeting of the PI4P lipid to its replication complex. However, the role of PI4P in HCV replication is not well understood. In this study, we have shown that FAPP2, a PI4P effector and glycosphingolipid-binding protein, is recruited to the HCV replication complex and is required for HCV genome replication and replication complex formation. More importantly, this study demonstrates, for the first time, the crucial role of glycosphingolipids in the HCV life cycle and suggests a link between PI4P and glycosphingolipids in HCV genome replication.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Glicoesfingolípidos/metabolismo , Hepacivirus/fisiología , Interacciones Huésped-Patógeno , Fosfatos de Fosfatidilinositol/metabolismo , Replicación Viral/efectos de los fármacos , Humanos
18.
Virol J ; 10: 339, 2013 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-24245822

RESUMEN

BACKGROUND: Flavivirus NS1 is a non-structural glycoprotein that is expressed on the cell surface and secreted into the extracellular space, where it acts as an antagonist of complement pathway activation. Despite its transit through the secretory pathway and intracellular localization in the lumen of the endoplasmic reticulum and Golgi vesicles, NS1 is as an essential gene for flavivirus replication. How NS1 modulates infection remains uncertain given that the viral RNA replication complex localizes to the cytosolic face of the endoplasmic reticulum. METHODS AND RESULTS: Using a trans-complementation assay, we show that viruses deleted for NS1 (∆-NS1) can be rescued by transgenic expression of NS1 from West Nile virus (WNV) or heterologous flaviviruses in the absence of adaptive mutations. In viral lifecycle experiments, we demonstrate that WNV NS1 was not required for virus attachment or input strand translation of the infectious viral RNA, but was necessary for negative and positive strand RNA synthesis and formation of the endoplasmic reticulum-associated replication complex. CONCLUSIONS: WNV RNA lacking intact NS1 genes was efficiently translated but failed to form canonical replication complexes at early times after infection, which resulted in an inability to replicate viral RNA. These results expand on prior studies with yellow fever and Kunjin viruses to show that flavivirus NS1 has an essential co-factor role in regulating replication complex formation and viral RNA synthesis.


Asunto(s)
ARN Viral/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Virus del Nilo Occidental/fisiología , Animales , Línea Celular , Cricetinae , Prueba de Complementación Genética , Virus del Nilo Occidental/genética
19.
J Virol ; 87(4): 2206-14, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23221566

RESUMEN

West Nile virus strain Kunjin (WNV(KUN)) is an enveloped, positive-sense RNA virus within the virus family Flaviviridae. Many flaviviruses have been shown to manipulate multiple signaling pathways, including autophagic, innate immune, and stress responses, in order to benefit replication. In particular, we have demonstrated that WNV(KUN) regulates the unfolded protein response (UPR), skewing the downstream effectors toward chaperone expression and Xbp-1 activation while preventing PERK-mediated translation attenuation and C/EBP homologous protein (CHOP) upregulation. WNV(KUN)-regulated UPR signaling can then be hijacked in order to affect type I interferon (IFN) responses, preventing IFN-mediated STAT1 phosphorylation and nuclear translocation. To extend our previous observations, we aimed to investigate the contribution of ATF6- and IRE1-mediated signaling during WNV(KUN) replication and how the two sensors contribute to the inhibition of IFN signaling. ATF6-deficient cells infected with WNV(KUN) showed decreased protein and virion production. These cells also demonstrated increased eIF2α phosphorylation and CHOP transcription, absent in infected matched control cells. Thus, we propose that in the absence of ATF6, WNV(KUN) is incapable of manipulating the PERK-mediated response to infection. In contrast, infection of IRE1(-/-) knockout cells showed no discernible differences compared to IRE1(+/+) cells. However, both ATF6 and IRE1 were required for WNV(KUN)-induced inhibition of STAT1 phosphorylation. We suggest that the combination of abhorrent UPR signaling, promotion of cell death, and increased innate immune responses contributes to the replication defects in ATF6-deficient cells, thus demonstrating the dual importance of ATF6 in maintaining cell viability and modulating immune responses during WNV(KUN) infection.


Asunto(s)
Factor de Transcripción Activador 6/metabolismo , Interacciones Huésped-Patógeno , Evasión Inmune , Inmunidad Innata , Transducción de Señal , Virus del Nilo Occidental/patogenicidad , Factor de Transcripción Activador 6/genética , Animales , Línea Celular , Supervivencia Celular , Eliminación de Gen , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/metabolismo
20.
J Neurochem ; 118(5): 749-59, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21682723

RESUMEN

Exploring the mechanisms of serotonin [5-hydroxytryptamine (5-HT)] in the brain requires an in vivo method that combines fast temporal resolution with chemical selectivity. Fast-scan cyclic voltammetry is a technique with sufficient temporal and chemical resolution for probing dynamic 5-HT neurotransmission events; however, traditionally it has not been possible to probe in vivo 5-HT mechanisms. Recently, we optimized fast-scan cyclic voltammetry for measuring 5-HT release and uptake in vivo in the substantia nigra pars reticulata (SNR) with electrical stimulation of the dorsal raphe nucleus (DRN) in the rat brain. Here, we address technical challenges associated with rat DRN surgery by electrically stimulating 5-HT projections in the medial forebrain bundle (MFB), a more accessible anatomical location. MFB stimulation elicits 5-HT in the SNR; furthermore, we find simultaneous release of an additional species. We use electrochemical and pharmacological methods and describe physiological, anatomical and independent chemical analyses to identify this species as histamine. We also show pharmacologically that increasing the lifetime of extracellular histamine significantly decreases 5-HT release, most likely because of increased activation of histamine H-3 receptors that inhibit 5-HT release. Despite this, under physiological conditions, we find by kinetic comparisons of DRN and MFB stimulations that the simultaneous release of histamine does not interfere with the quantitative 5-HT concentration profile. We therefore present a novel and robust electrical stimulation of the MFB that is technically less challenging than DRN stimulation to study 5-HT and histamine release in the SNR.


Asunto(s)
Electroquímica/métodos , Histamina/metabolismo , Haz Prosencefálico Medial/fisiología , Serotonina/metabolismo , Sustancia Negra/metabolismo , Animales , Dimaprit/análogos & derivados , Dimaprit/farmacología , Estimulación Eléctrica/métodos , Histamina/farmacología , Agonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos H3/farmacología , Modelos Lineales , Masculino , Vías Nerviosas/fisiología , Piperidinas/farmacología , Núcleos del Rafe/fisiología , Ratas , Ratas Sprague-Dawley , Serotonina/farmacología
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