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Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah-/- mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease.
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Type I Interferons (IFN-Is) are a family of cytokines which play a major role in inhibiting viral infection. Resultantly, many viruses have evolved mechanisms in which to evade the IFN-I response. Here we tested the impact of expression of 27 different SARS-CoV-2 genes in relation to their effect on IFN production and activity using three independent experimental methods. We identified six gene products; NSP6, ORF6, ORF7b, NSP1, NSP5 and NSP15, which strongly (>10-fold) blocked MAVS-induced (but not TRIF-induced) IFNß production. Expression of the first three of these SARS-CoV-2 genes specifically blocked MAVS-induced IFNß-promoter activity, whereas all six genes induced a collapse in IFNß mRNA levels, corresponding with suppressed IFNß protein secretion. Five of these six genes furthermore suppressed MAVS-induced activation of IFNλs, however with no effect on IFNα or IFNγ production. In sharp contrast, SARS-CoV-2 infected cells remained extremely sensitive to anti-viral activity exerted by added IFN-Is. None of the SARS-CoV-2 genes were able to block IFN-I signaling, as demonstrated by robust activation of Interferon Stimulated Genes (ISGs) by added interferon. This, despite the reduced levels of STAT1 and phospho-STAT1, was likely caused by broad translation inhibition mediated by NSP1. Finally, we found that a truncated ORF7b variant that has arisen from a mutant SARS-CoV-2 strain harboring a 382-nucleotide deletion associating with mild disease (Δ382 strain identified in Singapore & Taiwan in 2020) lost its ability to suppress type I and type III IFN production. In summary, our findings support a multi-gene process in which SARS-CoV-2 blocks IFN-production, with ORF7b as a major player, presumably facilitating evasion of host detection during early infection. However, SARS-CoV-2 fails to suppress IFN-I signaling thus providing an opportunity to exploit IFN-Is as potential therapeutic antiviral drugs.
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Interferon beta/metabolismo , SARS-CoV-2/imunologia , Proteínas Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Chlorocebus aethiops , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HEK293 , Humanos , Interferon beta/genética , Interferon beta/farmacologia , SARS-CoV-2/efeitos dos fármacos , Fator de Transcrição STAT1/metabolismo , Células Vero , Proteínas Virais/genéticaRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1009800.].
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Wolbachia-infected mosquitoes are refractory to flavivirus infections, but the role of lipids in Wolbachia-mediated virus blocking remains to be elucidated. Here, we use liquid chromatography mass spectrometry to provide a comprehensive picture of the lipidome of Aedes aegypti (Aag2) cells infected with Wolbachia only, either dengue or Zika virus only, and Wolbachia-infected Aag2 cells superinfected with either dengue or Zika virus. This approach identifies a class of lipids, acyl-carnitines, as being down-regulated during Wolbachia infection. Furthermore, treatment with an acyl-carnitine inhibitor assigns a crucial role for acyl-carnitines in the replication of dengue and Zika viruses. In contrast, depletion of acyl-carnitines increases Wolbachia density while addition of commercially available acyl-carnitines impairs Wolbachia production. Finally, we show an increase in flavivirus infection of Wolbachia-infected cells with the addition of acyl-carnitines. This study uncovers a previously unknown role for acyl-carnitines in this tripartite interaction that suggests an important and broad mechanism that underpins Wolbachia-mediated pathogen blocking.
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Aedes/microbiologia , Aedes/virologia , Carnitina/metabolismo , Wolbachia/fisiologia , Zika virus/fisiologia , Aedes/química , Aedes/metabolismo , Animais , Carnitina/química , Feminino , Mosquitos Vetores/química , Mosquitos Vetores/metabolismo , Mosquitos Vetores/microbiologia , Mosquitos Vetores/virologiaRESUMO
Norovirus infection is the leading cause of food-borne gastroenteritis worldwide, being responsible for over 200,000 deaths annually. Studies with murine norovirus (MNV) showed that protective STAT1 signaling controls viral replication and pathogenesis, but the immune mechanisms that noroviruses exploit to induce pathology are elusive. Here, we show that gastrointestinal MNV infection leads to widespread IL-1ß maturation in MNV-susceptible STAT1-deficient mice. MNV activates the canonical Nlrp3 inflammasome in macrophages, leading to maturation of IL-1ß and to Gasdermin D (GSDMD)-dependent pyroptosis. STAT1-deficient macrophages displayed increased MAVS-mediated expression of pro-IL-1ß, facilitating elevated Nlrp3-dependent release of mature IL-1ß upon MNV infection. Accordingly, MNV-infected Stat1-/- mice showed Nlrp3-dependent maturation of IL-1ß as well as Nlrp3-dependent pyroptosis as assessed by in vivo cleavage of GSDMD to its active N-terminal fragment. While MNV-induced diarrheic responses were not affected, Stat1-/- mice additionally lacking either Nlrp3 or GSDMD displayed lower levels of the fecal inflammatory marker Lipocalin-2 as well as delayed lethality after gastrointestinal MNV infection. Together, these results uncover new insights into the mechanisms of norovirus-induced inflammation and cell death, thereby revealing Nlrp3 inflammasome activation and ensuing GSDMD-driven pyroptosis as contributors to MNV-induced immunopathology in susceptible STAT1-deficient mice.
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Proteínas Reguladoras de Apoptose/metabolismo , Infecções por Caliciviridae/patologia , Trato Gastrointestinal/patologia , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose/fisiologia , Fator de Transcrição STAT1/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/virologia , Células Cultivadas , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/virologia , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Norovirus/imunologia , Norovirus/patogenicidade , Proteínas de Ligação a FosfatoRESUMO
BACKGROUND: The uncontrollable spread of Zika virus (ZIKV) in the Americas during 2015-2017, and its causal link to microcephaly in newborns and Guillain-Barré syndrome in adults, led the World Health Organisation to declare it a global public health emergency. One of the most notable features of ZIKV pathogenesis was the ability of the virus to pass the placental barrier to infect the growing foetus. This pathogenic trait had not been observed previously for medically important flaviviruses, including dengue and yellow fever viruses. METHODS: In this study we evaluated the replication kinetics of ZIKV and the related encephalitic flavivirus West Nile strain Kunjin virus (WNVKUN) in early-term placental cell lines. RESULTS: We have observed that WNVKUN in fact replicates with a greater rate and to higher titres that ZIKV in these cell lines. CONCLUSIONS: These results would indicate the potential for all flaviviruses to replicate in placental tissue but it is the ability to cross the placenta itself that is the restrictive factor in the clinical progression and presentation of congenital Zika syndrome.
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Flavivirus , Infecção por Zika virus , Zika virus , Linhagem Celular , Feminino , Humanos , Recém-Nascido , Cinética , Placenta/patologia , GravidezRESUMO
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.
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Retículo Endoplasmático/virologia , Rim/enzimologia , Lipídeos de Membrana/metabolismo , Fosfolipases A2/metabolismo , Replicação Viral , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/patogenicidade , Animais , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Retículo Endoplasmático/enzimologia , Rim/virologia , Células Vero , Febre do Nilo Ocidental/enzimologiaRESUMO
BACKGROUND: Extracellular vesicles (EVs) are small membrane vesicles secreted by the cells that mediate intercellular transfer of molecules and contribute to transduction of various signals. Viral infection and action of pro-inflammatory cytokines has been shown to alter molecular composition of EV content. Transfer of antiviral proteins by EVs is thought to contribute to the development of inflammation and antiviral state. Altered incorporation of selected host RNAs into EVs in response to infection has also been demonstrated for several viruses, but not for WNV. Considering the medical significance of flaviviruses and the importance of deeper knowledge about the mechanisms of flavivirus-host interactions we assessed the ability of West Nile virus (WNV) and type I interferon (IFN), the main cytokine regulating antiviral response to WNV, to alter the composition of EV RNA cargo. RESULTS: We employed next generation sequencing to perform transcriptome-wide profiling of RNA cargo in EVs produced by cells infected with WNV or exposed to IFN-alpha. RNA profile of EVs secreted by uninfected cells was also determined and used as a reference. We found that WNV infection significantly changed the levels of certain host microRNAs (miRNAs), small noncoding RNAs (sncRNAs) and mRNAs incorporated into EVs. Treatment with IFN-alpha also altered miRNA and mRNA profiles in EV but had less profound effect on sncRNAs. Functional classification of RNAs differentially incorporated into EVs upon infection and in response to IFN-alpha treatment demonstrated association of enriched in EVs mRNAs and miRNAs with viral processes and pro-inflammatory pathways. Further analysis revealed that WNV infection and IFN-alpha treatment changed the levels of common and unique mRNAs and miRNAs in EVs and that IFN-dependent and IFN-independent processes are involved in regulation of RNA sorting into EVs during infection. CONCLUSIONS: WNV infection and IFN-alpha treatment alter the spectrum and the levels of mRNAs, miRNAs and sncRNAs in EVs. Differentially incorporated mRNAs and miRNAs in EVs produced in response to WNV infection and to IFN-alpha treatment are associated with viral processes and host response to infection. WNV infection affects composition of RNA cargo in EVs via IFN-dependent and IFN-independent mechanisms.
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Vesículas Extracelulares/genética , Interferon-alfa/farmacologia , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Vírus do Nilo Ocidental/fisiologia , Animais , Linhagem Celular , Vesículas Extracelulares/efeitos dos fármacos , Perfilação da Expressão Gênica , HumanosRESUMO
Globally, hepatitis E virus (HEV) causes significant morbidity and mortality each year. Despite this burden, there are no specific antivirals available to treat HEV patients, and the only licensed vaccine is not available outside China. Ribavirin and alpha interferon are used to treat chronic HEV infections; however, severe side effects and treatment failure are commonly reported. Therefore, this study aimed to identify potential antivirals for further development to combat HEV infection. We selected 16 compounds from the nucleoside and nonnucleoside antiviral classes that range in developmental status from late preclinical to FDA approved and evaluated them as potential antivirals for HEV infection, using genotype 1 replicon luminescence studies and replicon RNA quantification. Two potent inhibitors of HEV replication included NITD008 (half-maximal effective concentration [EC50], 0.03 µM; half-maximal cytotoxic concentration [CC50], >100 µM) and GPC-N114 (EC50, 1.07 µM, CC50, >100 µM), and both drugs reduced replicon RNA levels in cell culture (>50% reduction with either 10 µM GPC-N114 or 2.50 µM NITD008). Furthermore, GPC-N114 and NITD008 were synergistic in combinational treatment (combination index, 0.4) against HEV replication, allowing for dose reduction indices of 20.42 and 8.82 at 50% inhibition, respectively. Sofosbuvir has previously exhibited mixed results against HEV as an antiviral, both in vitro and in a few clinical applications; however, in this study it was effective against the HEV genotype 1 replicon (EC50, 1.97 µM; CC50, >100 µM) and reduced replicon RNA levels (47.2% reduction at 10 µM). Together these studies indicate drug repurposing may be a promising pathway for development of antivirals against HEV infection.
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Adenosina/análogos & derivados , Antivirais/farmacologia , Vírus da Hepatite E/efeitos dos fármacos , Hepatite E/tratamento farmacológico , Nitrilas/farmacologia , Nitrocompostos/farmacologia , Adenosina/farmacologia , Antivirais/uso terapêutico , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Genes Reporter , Hepatite E/virologia , Humanos , RNA Viral/análise , Replicon/efeitos dos fármacosRESUMO
Human noroviruses are highly infectious single-stranded RNA (ssRNA) viruses and the major cause of nonbacterial gastroenteritis worldwide. With the discovery of murine norovirus (MNV) and the introduction of an effective model for norovirus infection and replication, knowledge about infection mechanisms and their impact on the host immune response has progressed. A major player in the immune response against viral infections is the group of major histocompatibility complex (MHC) class I proteins, which present viral antigen to immune cells. We have observed that MNV interferes with the antigen presentation pathway in infected cells by reducing the surface expression of MHC class I proteins. We have shown that MNV-infected dendritic cells or macrophages have lower levels of surface expression of MHC class I proteins than uninfected and bystander cells. Transcriptional analysis revealed that this defect is not due to a decreased amount of mRNA but is reflected at the protein level. We have determined that this defect is mediated via the MNV NS3 protein. Significantly, treatment of MNV-infected cells with the endocytic recycling inhibitor dynasore completely restored the surface expression of MHC class I proteins, whereas treatment with the proteasome inhibitor MG132 partly restored such expression. These observations indicate a role for endocytic recycling and proteasome-mediated degradation of these proteins. Importantly, we show that due to the reduced surface expression of MHC class I proteins, antigen presentation is inhibited, resulting in the inability of CD8+ T cells to become activated in the presence of MNV-infected cells.IMPORTANCE Human noroviruses (HuNoVs) are the major cause of nonbacterial gastroenteritis worldwide and impose a great burden on patients and health systems every year. So far, no antiviral treatment or vaccine is available. We show that MNV evades the host immune response by reducing the amount of MHC class I proteins displayed on the cell surface. This reduction leads to a decrease in viral antigen presentation and interferes with the CD8+ T cell response. CD8+ T cells respond to foreign antigen by activating cytotoxic pathways and inducing immune memory to the infection. By evading this immune response, MNV is able to replicate efficiently in the host, and the ability of cells to respond to consecutive infections is impaired. These findings have a major impact on our understanding of the ways in which noroviruses interact with the host immune response and manipulate immune memory.
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Linfócitos T CD8-Positivos/imunologia , Infecções por Caliciviridae/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Norovirus/patogenicidade , Animais , Apresentação de Antígeno , Infecções por Caliciviridae/virologia , Células Dendríticas/imunologia , Ativação Linfocitária , Macrófagos/imunologia , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas não Estruturais Virais/metabolismoRESUMO
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.
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Vírus Defeituosos/patogenicidade , Vacinas contra Dengue/imunologia , Vírus da Dengue/patogenicidade , Dengue/virologia , Proteínas não Estruturais Virais/imunologia , Montagem de Vírus , Replicação Viral , Animais , Anticorpos Neutralizantes/imunologia , Vírus Defeituosos/genética , Dengue/genética , Dengue/imunologia , Vírus da Dengue/genética , Feminino , Humanos , Masculino , Camundongos , Mutação , RNA Viral , Proteínas não Estruturais Virais/genéticaRESUMO
Flavivirus replication is intimately involved with remodelled membrane organelles that are compartmentalised for different functions during their life cycle. Recent advances in lipid analyses and gene depletion have identified a number of host components that enable efficient virus replication in infected cells. Here, we describe the current understanding on the role and contribution of host lipids and membrane bending proteins to flavivirus replication, with a particular focus on the components that bend and shape the membrane bilayer to induce the flavivirus-induced organelles characteristic of infection.
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Membrana Celular/metabolismo , Membrana Celular/virologia , Flavivirus/fisiologia , Interações Hospedeiro-Patógeno , Replicação Viral , Animais , Humanos , Metabolismo dos LipídeosRESUMO
West Nile virus (WNV) is a single-stranded, positive sense RNA virus of the family Flaviviridae and is a significant pathogen of global medical importance. Flavivirus replication is known to be exclusively cytoplasmic, but we show here for the first time that access to the nucleus of the WNV strain Kunjin (WNVKUN ) RNA-dependent RNA polymerase (protein NS5) is central to WNVKUN virus production. We show that treatment of cells with the specific nuclear export inhibitor leptomycin B (LMB) results in increased NS5 nuclear accumulation in WNVKUN -infected cells and NS5-transfected cells, indicative of nucleocytoplasmic shuttling under normal conditions. We used site-directed mutagenesis to identify the nuclear localisation sequence (NLS) responsible for WNVKUN NS5 nuclear targeting, observing that mutation of this NLS resulted in exclusively cytoplasmic accumulation of NS5 even in the presence of leptomycin B. Introduction of NS5 NLS mutations into FLSDX, an infectious clone of WNVKUN , resulted in lethality, suggesting that the ability of NS5 to traffic into the nucleus in integral to WNVKUN replication. This study thus shows for the first time that NLS-dependent trafficking into the nucleus during infection of WNVKUN NS5 is critical for viral replication. Excitingly, specific inhibitors of NS5 nuclear import reduce WNVKUN virus production, proving the principle that inhibition of WNVKUN NS5 nuclear import is a viable therapeutic avenue for antiviral drug development in the future.
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Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Vírus do Nilo Ocidental/enzimologia , Vírus do Nilo Ocidental/fisiologia , Animais , Chlorocebus aethiops , Inibidores Enzimáticos/metabolismo , Ácidos Graxos Insaturados/metabolismo , Mutagênese Sítio-Dirigida , Sinais de Localização Nuclear , Transporte Proteico , Células Vero , Proteínas não Estruturais Virais/genética , Ensaio de Placa ViralRESUMO
Norovirus infections are a significant health and economic burden globally, accounting for hundreds of millions of cases of acute gastroenteritis every year. In the absence of an approved norovirus vaccine, there is an urgent need to develop antivirals to treat chronic infections and provide prophylactic therapy to limit viral spread during epidemics and pandemics. Toll-like receptor (TLR) agonists have been explored widely for their antiviral potential, and several are progressing through clinical trials for the treatment of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) and as adjuvants for norovirus viruslike particle (VLP) vaccines. However, norovirus therapies in development are largely direct-acting antivirals (DAAs) with fewer compounds that target the host. Our aim was to assess the antiviral potential of TLR7 agonist immunomodulators on norovirus infection using the murine norovirus (MNV) and human Norwalk replicon models. TLR7 agonists R-848, Gardiquimod, GS-9620, R-837, and loxoribine were screened using a plaque reduction assay, and each displayed inhibition of MNV replication (50% effective concentrations [EC50s], 23.5 nM, 134.4 nM, 0.59 µM, 1.5 µM, and 79.4 µM, respectively). RNA sequencing of TLR7-stimulated cells revealed a predominant upregulation of innate immune response genes and interferon (IFN)-stimulated genes (ISGs) that are known to drive an antiviral state. Furthermore, the combination of R-848 and the nucleoside analogue (NA) 2'C-methylcytidine elicited a synergistic antiviral effect against MNV, demonstrating that combinational therapy of host modulators and DAAs might be used to reduce drug cytotoxicity. In summary, we have identified that TLR7 agonists display potent inhibition of norovirus replication and are a therapeutic option to combat norovirus infections.
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Antivirais/uso terapêutico , Infecções por Caliciviridae/tratamento farmacológico , Receptor 7 Toll-Like/metabolismo , Aminoquinolinas/uso terapêutico , Animais , Linhagem Celular , Guanosina/análogos & derivados , Guanosina/uso terapêutico , Humanos , Imidazóis/uso terapêutico , Imiquimode/uso terapêutico , Camundongos , Pteridinas/uso terapêutico , Células RAW 264.7 , Receptor 7 Toll-Like/agonistas , Replicação Viral/efeitos dos fármacosRESUMO
Norovirus (NoV) infections are a significant health burden to society, yet the lack of reliable tissue culture systems has hampered the development of appropriate antiviral therapies. Here we show that the NoV NS3 protein, derived from murine NoV (MNV), is intimately associated with the MNV replication complex and the viral replication intermediate double-stranded RNA (dsRNA). We observed that when expressed individually, MNV NS3 and NS3 encoded by human Norwalk virus (NV) induced the formation of distinct vesicle-like structures that did not colocalize with any particular protein markers to cellular organelles but localized to cellular membranes, in particular those with a high cholesterol content. Both proteins also showed some degree of colocalization with the cytoskeleton marker ß-tubulin. Although the distribution of MNV and NV NS3s were similar, NV NS3 displayed a higher level of colocalization with the Golgi apparatus and the endoplasmic reticulum (ER). However, we observed that although both proteins colocalized in membranes counterstained with filipin, an indicator of cholesterol content, MNV NS3 displayed a greater association with flotillin and stomatin, proteins known to associate with sphingolipid- and cholesterol-rich microdomains. Utilizing time-lapse epifluorescence microscopy, we observed that the membrane-derived vesicular structures induced by MNV NS3 were highly motile and dynamic in nature, and their movement was dependent on intact microtubules. These results begin to interrogate the functions of NoV proteins during virus replication and highlight the conserved properties of the NoV NS3 proteins among the seven Norovirus genogroups. IMPORTANCE: Many mechanisms involved in the replication of norovirus still remain unclear, including the role for the NS3 protein, one of seven nonstructural viral proteins, which remains to be elucidated. This study reveals that murine norovirus (MNV) NS3 is intimately associated with the viral replication complex and dsRNA. We observed that the NS3 proteins of both MNV and Norwalk virus (NV) induce prominent vesicular structures and that this formation is dependent on microtubules and cellular cholesterol. Thus, this study contributes to our understanding of protein function within different Norovirus genogroups and expands a growing knowledge base on the interaction between positive-strand RNA [(+)RNA] viruses and cellular membranes that contribute to the biogenesis of virus-induced membrane organelles. This study contributes to our understanding of viral protein function and the ability of a viral protein to recruit specific cellular organelles and lipids that enable replication.
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Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/virologia , Metabolismo dos Lipídeos , Microtúbulos/metabolismo , Norovirus/fisiologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Sequência de Aminoácidos , Animais , Linhagem Celular , Chlorocebus aethiops , Colesterol/metabolismo , Interações Hospedeiro-Patógeno , Espaço Intracelular , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Ligação Proteica , Transporte Proteico , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Células Vero , Proteínas não Estruturais Virais/químicaRESUMO
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.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Glicoesfingolipídeos/metabolismo , Hepacivirus/fisiologia , Interações Hospedeiro-Patógeno , Fosfatos de Fosfatidilinositol/metabolismo , Replicação Viral/efeitos dos fármacos , HumanosRESUMO
BACKGROUND: Dengue virus (DENV) is estimated to cause 390 million infections each year, but there is no licensed vaccine or therapeutic currently available. METHODS: We describe a novel, high-throughput screen to identify compounds inhibiting the interaction between DENV nonstructural protein 5 and host nuclear transport proteins. We document the antiviral properties of a lead compound against all 4 serotypes of DENV, antibody-dependent enhanced (ADE) infection, and ex vivo and in vivo DENV infections. In addition, we use quantitative reverse-transcription polymerase chain reaction to examine cellular effects upon compound addition. RESULTS: We identify N-(4-hydroxyphenyl) retinamide (4-HPR) as effective in protecting against DENV-1-4 and DENV-1 ADE infections, with 50% effective concentrations in the low micromolar range. 4-HPR but not the closely related N-(4-methoxyphenyl) retinamide (4-MPR) could reduce viral RNA levels and titers when applied to an established infection. 4-HPR but not 4-MPR was found to specifically upregulate the protein kinase R-like endoplasmic reticulum kinase arm of the unfolded protein response. Strikingly, 4-HPR but not 4-MPR restricted infection in peripheral blood mononuclear cells and in a lethal ADE-infection mouse model. CONCLUSIONS: 4-HPR is a novel antiviral that modulates the unfolded protein response, effective against DENV1-4 at concentrations achievable in the plasma in a clinical setting, and provides protection in a lethal mouse model.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/metabolismo , Dengue/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Dengue/tratamento farmacológico , Dengue/virologia , Vírus da Dengue/classificação , Modelos Animais de Doenças , Fenretinida/farmacologia , Humanos , Camundongos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais , Tretinoína/análogos & derivados , Tretinoína/farmacologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , eIF-2 Quinase/metabolismoRESUMO
Norovirus (NoV) is the leading cause of acute gastroenteritis worldwide, causing over 200,000 deaths a year. NoV is nonenveloped, with a single-stranded RNA genome, and is primarily transmitted person to person. The viral RNA-dependent RNA polymerase (RdRp) is critical for the production of genomic and subgenomic RNA and is therefore a prime target for antiviral therapies. Using high-throughput screening, nearly 20,000 "lead-like" compounds were tested for inhibitory activity against the NoV genogroup II, genotype 4 (GII.4) RdRp. The four most potent hits demonstrated half-maximal inhibitory concentrations (IC50s) between 5.0 µM and 9.8 µM against the target RdRp. Compounds NIC02 and NIC04 revealed a mixed mode of inhibition, while NIC10 and NIC12 were uncompetitive RdRp inhibitors. When examined using enzymes from related viruses, NIC02 demonstrated broad inhibitory activity while NIC04 was the most specific GII.4 RdRp inhibitor. The antiviral activity was examined using available NoV cell culture models; the GI.1 replicon and the infectious GV.1 murine norovirus (MNV). NIC02 and NIC04 inhibited the replication of the GI.1 replicon, with 50% effective concentrations (EC50s) of 30.1 µM and 71.1 µM, respectively, while NIC10 and NIC12 had no observable effect on the NoV GI.1 replicon. In the MNV model, NIC02 reduced plaque numbers, size, and viral RNA levels in a dose-dependent manner (EC50s between 2.3 µM and 4.8 µM). The remaining three compounds also reduced MNV replication, although with higher EC50s, ranging from 32 µM to 38 µM. In summary, we have identified novel nonnucleoside inhibitor scaffolds that will provide a starting framework for the development and future optimization of targeted antivirals against NoV.
Assuntos
Antivirais/farmacologia , Infecções por Caliciviridae/tratamento farmacológico , Gastroenterite/tratamento farmacológico , Norovirus/efeitos dos fármacos , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Antivirais/química , Infecções por Caliciviridae/virologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Gastroenterite/virologia , Genótipo , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Norovirus/enzimologia , RNA Viral/genética , Proteínas Recombinantes , Replicon , Relação Estrutura-AtividadeRESUMO
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.