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Background: Among the most common mucosal viral infections in infants are rotavirus, one of the main causes of severe gastroenteritis in infants and children up to 5 years, and respiratory syncytial virus (RSV), one of the leading causes of lower respiratory tract infections. Both human milk and bovine milk derived factors may provide protection against mucosal viral infections. More recently, a similar activity of milk derived proteins was suggested for SARS-CoV-2. The goal of the current study was to test antiviral activity of the bovine milkfat globule membrane (MFGM) against rotavirus, RSV and SARS-CoV-2 and to further characterize MFGM-enriched whey to identify which components in MFGM-enriched whey may contribute to the inhibitory activity. Methods: The effects of MFGM-enriched whey, its whey protein isolate counterpart (WPI, obtained from the same production process) and a conventional whey protein concentrate (WPC) on rotavirus (strains Wa and SA114F), RSV (strain RSV-A2) and SARS-CoV-2 (Alpha variant) infectivity were determined using MA104 cells, human alveolar basal epithelial (A549) cells and monkey kidney (Vero E6) cells, respectively. The compounds were characterized in detail by LC-MS/MS and 31P-NMR to determine protein and phospholipid composition, respectively. Results: Relative to its WPI counterpart, MFGM-enriched whey demonstrated a dose-dependent inhibition for both rotavirus and RSV whereas for SARS-CoV-2 inhibition was only observed at the highest concentration tested. Label-free quantification (LFQ) and intensity based absolute quantification (iBAQ) of identified proteins revealed a clear difference between MFGM-enriched whey and its controls including enrichment of known MFGM proteins and non-MFGM proteins that are enriched simultaneously, some of which have previously been demonstrated to display anti-viral activity. Although not completely absent from other whey protein preparations, MFGM-enriched whey had the highest specific and total phospholipid levels. Conclusion: MFGM-enriched whey displayed antiviral activity against multiple viruses of clinical importance. This study provides insights into the active components in MFGM-enriched whey and may contribute to previous clinical observations with MFGM-enriched formula demonstrating reduced respiratory and gastrointestinal infections in formula fed infants.
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Bone Marrow Stromal Cell Antigen 2 (BST-2)/tetherin inhibits the release of numerous enveloped viruses by physically tethering nascent particles to infected cells during the process of viral budding from the cell surface. Tetherin also restricts human immunodeficiency virus (HIV), and pandemic main (M) group HIV type 1s (HIV-1s) are thought to rely exclusively on their Vpu proteins to overcome tetherin-mediated restriction of virus release. However, at least one M group HIV-1 strain, the macrophage-tropic primary AD8 isolate, is unable to express Vpu due to a mutation in its translation initiation codon. Here, using primary monocyte-derived macrophages (MDMs), we show that AD8 Nef protein can compensate for the absence of Vpu and restore virus release to wild type levels. We demonstrate that HIV-1 AD8 Nef reduces endogenous cell surface tetherin levels, physically separating it from the site of viral budding, thus preventing HIV retention. Mechanistically, AD8 Nef enhances internalisation of the long isoform of human tetherin, leading to perinuclear accumulation of the restriction factor. Finally, we show that Nef proteins from other HIV strains also display varying degrees of tetherin antagonism. Overall, we show that M group HIV-1s can use an accessory protein other than Vpu to antagonise human tetherin.
Assuntos
Antígenos CD/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Macrófagos/metabolismo , Macrófagos/virologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Antígenos CD/genética , Linhagem Celular , Imunofluorescência , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Expressão Gênica , Infecções por HIV/imunologia , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Produtos do Gene nef do Vírus da Imunodeficiência Humana/químicaRESUMO
Host interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral restriction factors. Of these, IFITM3 potently inhibits viruses that enter cells through acidic endosomes, many of which are zoonotic and emerging viruses with bats (order Chiroptera) as their natural hosts. We previously demonstrated that microbat IFITM3 is antiviral. Here, we show that bat IFITMs are characterized by strong adaptive evolution and identify a highly variable and functionally important site-codon 70-within the conserved CD225 domain of IFITMs. Mutation of this residue in microbat IFITM3 impairs restriction of representatives of four different virus families that enter cells via endosomes. This mutant shows altered subcellular localization and reduced S-palmitoylation, a phenotype copied by mutation of conserved cysteine residues in microbat IFITM3. Furthermore, we show that microbat IFITM3 is S-palmitoylated on cysteine residues C71, C72, and C105, mutation of each cysteine individually impairs virus restriction, and a triple C71A-C72A-C105A mutant loses all restriction activity, concomitant with subcellular re-localization of microbat IFITM3 to Golgi-associated sites. Thus, we propose that S-palmitoylation is critical for Chiropteran IFITM3 function and identify a key molecular determinant of IFITM3 S-palmitoylation.
Assuntos
Quirópteros/genética , Lipoilação/genética , Proteínas de Membrana/genética , Polimorfismo Genético , Domínios Proteicos/genética , Proteínas de Ligação a RNA/genética , Células A549 , Animais , Antígenos de Diferenciação/genética , Códon/genética , Códon/metabolismo , Endossomos/metabolismo , Endossomos/virologia , Evolução Molecular , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/metabolismo , Influenza Humana/virologia , Proteínas de Membrana/metabolismo , Filogenia , Proteínas de Ligação a RNA/metabolismo , Transdução Genética , Internalização do Vírus , Zika virus/fisiologia , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologiaRESUMO
The process of entry into a host cell is a key step in the life cycle of most viruses. In recent years, there has been a significant increase in our understanding of the routes and mechanisms of entry for a number of these viruses. This has led to the development of novel broad-spectrum antiviral approaches that target host cell proteins and pathways, in addition to strategies focused on individual viruses or virus families. Here we consider a number of these approaches and their broad-spectrum potential.
Assuntos
Antivirais/farmacologia , Internalização do Vírus , Vírus/efeitos dos fármacos , Humanos , Receptores de Superfície Celular , Receptores ViraisRESUMO
CpG dinucleotides are suppressed in most vertebrate RNA viruses, including HIV-1, and introducing CpGs into RNA virus genomes inhibits their replication. The zinc finger antiviral protein (ZAP) binds regions of viral RNA containing CpGs and targets them for degradation. ZAP does not have enzymatic activity and recruits other cellular proteins to inhibit viral replication. We found that KHNYN, a protein with no previously known function, interacts with ZAP. KHNYN overexpression selectively inhibits HIV-1 containing clustered CpG dinucleotides and this requires ZAP and its cofactor TRIM25. KHNYN requires both its KH-like domain and NYN endonuclease domain for antiviral activity. Crucially, depletion of KHNYN eliminated the deleterious effect of CpG dinucleotides on HIV-1 RNA abundance and infectious virus production and also enhanced the production of murine leukemia virus. Overall, we have identified KHNYN as a novel cofactor for ZAP to target CpG-containing retroviral RNA for degradation.
Assuntos
Ilhas de CpG/genética , HIV-1/genética , Proteínas de Ligação a RNA/metabolismo , Células HEK293 , Células HeLa , Humanos , Domínios Proteicos , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Deleção de Sequência/genética , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/metabolismoRESUMO
Viruses are a major threat to human health and economic well-being. In recent years Ebola, Zika, influenza, and chikungunya virus epidemics have raised awareness that infections can spread rapidly before vaccines or specific antagonists can be made available. Broad-spectrum antivirals are drugs with the potential to inhibit infection by viruses from different groups or families, which may be deployed during outbreaks when specific diagnostics, vaccines or directly acting antivirals are not available. While pathogen-directed approaches are generally effective against a few closely related viruses, targeting cellular pathways used by multiple viral agents can have broad-spectrum efficacy. Virus entry, particularly clathrin-mediated endocytosis, constitutes an attractive target as it is used by many viruses. Using a phenotypic screening strategy where the inhibitory activity of small molecules was sequentially tested against different viruses, we identified 12 compounds with broad-spectrum activity, and found a subset blocking viral internalisation and/or fusion. Importantly, we show that compounds identified with this approach can reduce viral replication in a mouse model of Zika infection. This work provides proof of concept that it is possible to identify broad-spectrum inhibitors by iterative phenotypic screenings, and that inhibition of host-pathways critical for viral life cycles can be an effective antiviral strategy.
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Antivirais/isolamento & purificação , Antivirais/farmacologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Internalização do Vírus/efeitos dos fármacos , Vírus/efeitos dos fármacos , Animais , Células HeLa , Humanos , Concentração Inibidora 50 , Camundongos , RNA Viral/genética , Replicação Viral/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Infecção por Zika virus/tratamento farmacológicoRESUMO
Members of the Flaviviridae family constitute a severe risk to human health. Whilst effective drugs have been developed against the hepacivirus HCV, no antiviral therapy is currently available for any other viruses, including the flaviviruses dengue (DENV), West Nile and Zika viruses. The RNA-dependent RNA polymerase (RdRp) is responsible for viral replication and represents an excellent therapeutic target with no homologue found in mammals. The identification of compounds targeting the RdRp of other flaviviruses is an active area of research. One of the main factors hampering further developments in the field is the difficulty in obtaining high-quality crystal information that could aid a structure-based drug discovery approach. To address this, we have developed a convenient and economical 96-well screening platform. We validated the screen by successfully obtaining crystals of both native DENV serotype 2 and 3 RdRps under several conditions included in the screen. In addition, we have obtained crystal structures of RdRp3 in complex with a previously identified fragment using both soaking and co-crystallization techniques. This work will streamline and accelerate the generation of crystal structures of viral RdRps and provide the community with a valuable tool to aid the development of structure-based antiviral design.
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Two-pore channels (TPCs) are Ca2+-permeable ion channels localised to the endo-lysosomal system where they regulate trafficking of various cargoes including viruses. As a result, TPCs are emerging as important drug targets. However, their pharmacology is ill-defined. There are no approved drugs to target them. And their mechanism of ligand activation is largely unknown. Here, we identify a number of FDA-approved drugs as TPC pore blockers. Using a model of the pore of human TPC2 based on recent structures of mammalian TPCs, we virtually screened a database of ~1500 approved drugs. Because TPCs have recently emerged as novel host factors for Ebola virus entry, we reasoned that Ebola virus entry inhibitors may exert their effects through inhibition of TPCs. Cross-referencing hits from the TPC virtual screen with two recent high throughput anti-Ebola screens yielded approved drugs targeting dopamine and estrogen receptors as common hits. These compounds inhibited endogenous NAADP-evoked Ca2+ release from sea urchin egg homogenates, NAADP-mediated channel activity of TPC2 re-routed to the plasma membrane, and PI(3,5)P2-mediated channel activity of TPC2 expressed in enlarged lysosomes. Mechanistically, single channel analyses showed that the drugs reduced mean open time consistent with a direct action on the pore. Functionally, drug potency in blocking TPC2 activity correlated with inhibition of Ebola virus-like particle entry. Our results expand TPC pharmacology through the identification of approved drugs as novel blockers, support a role for TPCs in Ebola virus entry, and provide insight into the mechanisms underlying channel regulation. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
Assuntos
Antivirais/farmacologia , Canais de Cálcio/metabolismo , Ebolavirus/metabolismo , Lisossomos/metabolismo , Internalização do Vírus/efeitos dos fármacos , Animais , Antivirais/química , Canais de Cálcio/genética , Avaliação de Medicamentos , Ebolavirus/genética , Células HEK293 , Humanos , Lisossomos/genética , Lisossomos/virologia , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Ouriços-do-MarRESUMO
Virus infection is sensed by pattern recognition receptors (PRRs) detecting virus nucleic acids and initiating an innate immune response. DNA-dependent protein kinase (DNA-PK) is a PRR that binds cytosolic DNA and is antagonized by vaccinia virus (VACV) protein C16. Here, VACV protein C4 is also shown to antagonize DNA-PK by binding to Ku and blocking Ku binding to DNA, leading to a reduced production of cytokines and chemokines in vivo and a diminished recruitment of inflammatory cells. C4 and C16 share redundancy in that a double deletion virus has reduced virulence not seen with single deletion viruses following intradermal infection. However, non-redundant functions exist because both single deletion viruses display attenuated virulence compared to wild-type VACV after intranasal infection. It is notable that VACV expresses two proteins to antagonize DNA-PK, but it is not known to target other DNA sensors, emphasizing the importance of this PRR in the response to infection in vivo.
Assuntos
Proteína Quinase Ativada por DNA/metabolismo , DNA/metabolismo , Vaccinia virus/metabolismo , Proteínas Virais/metabolismo , Administração Intranasal , Animais , Citocinas/metabolismo , Proteína Quinase Ativada por DNA/química , Feminino , Células HEK293 , Células HeLa , Humanos , Imunidade Inata , Autoantígeno Ku/metabolismo , Ativação Linfocitária/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Ligação Proteica , Multimerização Proteica , Linfócitos T/imunologia , Vaccinia virus/patogenicidade , VirulênciaRESUMO
Virus reprogramming of cellular metabolism is recognised as a critical determinant for viral growth. While most viruses appear to activate central energy metabolism, different viruses have been shown to rely on alternative mechanisms of metabolic activation. Whether related viruses exploit conserved mechanisms and induce similar metabolic changes is currently unclear. In this work we investigate how two alphaviruses, Semliki Forest virus and Ross River virus, reprogram host metabolism and define the molecular mechanisms responsible. We demonstrate that in both cases the presence of a YXXM motif in the viral protein nsP3 is necessary for binding to the PI3K regulatory subunit p85 and for activating AKT. This leads to an increase in glucose metabolism towards the synthesis of fatty acids, although additional mechanisms of metabolic activation appear to be involved in Ross River virus infection. Importantly, a Ross River virus mutant that fails to activate AKT has an attenuated phenotype in vivo, suggesting that viral activation of PI3K/AKT contributes to virulence and disease.
Assuntos
Infecções por Alphavirus/metabolismo , Infecções por Alphavirus/virologia , Alphavirus/fisiologia , Glucose/metabolismo , Interações Hospedeiro-Patógeno , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Alphavirus/patogenicidade , Animais , Células Cultivadas , Cricetinae , Ativação Enzimática , Glicólise/fisiologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ross River virus/fisiologia , Vírus da Floresta de Semliki/fisiologia , VirulênciaRESUMO
Vaccinia virus (VACV) is the prototypic orthopoxvirus and the vaccine used to eradicate smallpox. Here we show that VACV strain Western Reserve protein 169 is a cytoplasmic polypeptide expressed early during infection that is excluded from virus factories and inhibits the initiation of cap-dependent and cap-independent translation. Ectopic expression of protein 169 causes the accumulation of 80S ribosomes, a reduction of polysomes, and inhibition of protein expression deriving from activation of multiple innate immune signaling pathways. A virus lacking 169 (vΔ169) replicates and spreads normally in cell culture but is more virulent than parental and revertant control viruses in intranasal and intradermal murine models of infection. Intranasal infection by vΔ169 caused increased pro-inflammatory cytokines and chemokines, infiltration of pulmonary leukocytes, and lung weight. These alterations in innate immunity resulted in a stronger CD8+ T-cell memory response and better protection against virus challenge. This work illustrates how inhibition of host protein synthesis can be a strategy for virus suppression of innate and adaptive immunity.
Assuntos
Imunidade Adaptativa , Interações Hospedeiro-Patógeno , Imunidade Inata , Iniciação Traducional da Cadeia Peptídica , Vaccinia virus/fisiologia , Vacínia/virologia , Proteínas Virais/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Linhagem Celular , Feminino , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Memória Imunológica , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Vacínia/imunologia , Vacínia/metabolismo , Vacínia/patologia , Vaccinia virus/imunologia , Vaccinia virus/patogenicidade , Proteínas Virais/genética , VirulênciaRESUMO
Vaccinia virus (VACV) is a large DNA virus that replicates in the cytoplasm and encodes about 200 proteins of which approximately 50â% may be non-essential for viral replication. These proteins enable VACV to suppress transcription and translation of cellular genes, to inhibit the innate immune response, to exploit microtubule- and actin-based transport for virus entry and spread, and to subvert cellular metabolism for the benefit of the virus. VACV strain WR protein C16 induces stabilization of the hypoxia-inducible transcription factor (HIF)-1α by binding to the cellular oxygen sensor prolylhydroxylase domain-containing protein (PHD)2. Stabilization of HIF-1α is induced by several virus groups, but the purpose and consequences are unclear. Here, (1)H-NMR spectroscopy and liquid chromatography-mass spectrometry are used to investigate the metabolic alterations during VACV infection in HeLa and 2FTGH cells. The role of C16 in such alterations was examined by comparing infection to WT VACV (strain WR) and a derivative virus lacking gene C16L (vΔC16). Compared with uninfected cells, VACV infection caused increased nucleotide and glutamine metabolism. In addition, there were increased concentrations of glutamine derivatives in cells infected with WT VACV compared with vΔC16. This indicates that C16 contributes to enhanced glutamine metabolism and this may help preserve tricarboxylic acid cycle activity. These data show that VACV infection reprogrammes cellular energy metabolism towards increased synthesis of the metabolic precursors utilized during viral replication, and that C16 contributes to this anabolic reprogramming of the cell, probably via the stabilization of HIF-1α.
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Metabolismo Energético , Interações Hospedeiro-Patógeno , Vaccinia virus/fisiologia , Proteínas Virais/metabolismo , Linhagem Celular , Deleção de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estabilidade Proteica , Vaccinia virus/genética , Proteínas Virais/genéticaRESUMO
For entry and infection viruses have developed numerous strategies to subjugate indispensable cellular factors and functions. Host cell lipids and cellular lipid synthesis machinery are no exception. Not only do viruses exploit existing lipid signalling and modifications for virus entry and trafficking, they also reprogram lipid synthesis, metabolism, and compartmentalization for assembly and egress. Here we review these various concepts and highlight recent progress in understanding viral interactions with host cell lipids during entry and assembly.
Assuntos
Membrana Celular/virologia , Interações Hospedeiro-Patógeno/fisiologia , Metabolismo dos Lipídeos/fisiologia , Lipídeos de Membrana/metabolismo , Internalização do Vírus , Humanos , Transdução de Sinais , Viroses/metabolismo , Replicação ViralRESUMO
The innate immune system is critical in the response to infection by pathogens and it is activated by pattern recognition receptors (PRRs) binding to pathogen associated molecular patterns (PAMPs). During viral infection, the direct recognition of the viral nucleic acids, such as the genomes of DNA viruses, is very important for activation of innate immunity. Recently, DNA-dependent protein kinase (DNA-PK), a heterotrimeric complex consisting of the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs was identified as a cytoplasmic PRR for DNA that is important for the innate immune response to intracellular DNA and DNA virus infection. Here we show that vaccinia virus (VACV) has evolved to inhibit this function of DNA-PK by expression of a highly conserved protein called C16, which was known to contribute to virulence but by an unknown mechanism. Data presented show that C16 binds directly to the Ku heterodimer and thereby inhibits the innate immune response to DNA in fibroblasts, characterised by the decreased production of cytokines and chemokines. Mechanistically, C16 acts by blocking DNA-PK binding to DNA, which correlates with reduced DNA-PK-dependent DNA sensing. The C-terminal region of C16 is sufficient for binding Ku and this activity is conserved in the variola virus (VARV) orthologue of C16. In contrast, deletion of 5 amino acids in this domain is enough to knockout this function from the attenuated vaccine strain modified vaccinia virus Ankara (MVA). In vivo a VACV mutant lacking C16 induced higher levels of cytokines and chemokines early after infection compared to control viruses, confirming the role of this virulence factor in attenuating the innate immune response. Overall this study describes the inhibition of DNA-PK-dependent DNA sensing by a poxvirus protein, adding to the evidence that DNA-PK is a critical component of innate immunity to DNA viruses.
Assuntos
Proteína Quinase Ativada por DNA/imunologia , Proteínas de Ligação a DNA/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Imunidade Inata , Proteínas Nucleares/imunologia , Vaccinia virus/imunologia , Vacínia/imunologia , Proteínas Virais/imunologia , Animais , Antígenos Nucleares/genética , Antígenos Nucleares/imunologia , Antígenos Nucleares/metabolismo , Linhagem Celular , Proteína Quinase Ativada por DNA/biossíntese , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Autoantígeno Ku , Camundongos Endogâmicos BALB C , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Ligação Proteica , Vacínia/genética , Vacínia/metabolismo , Vaccinia virus/genética , Vaccinia virus/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Virus infection of mammalian cells is sensed by pattern recognition receptors and leads to an innate immune response that restricts virus replication and induces adaptive immunity. In response, viruses have evolved many countermeasures that enable them to replicate and be transmitted to new hosts, despite the host innate immune response. Poxviruses, such as vaccinia virus (VACV), have large DNA genomes and encode many proteins that are dedicated to host immune evasion. Some of these proteins are secreted from the infected cell, where they bind and neutralize complement factors, interferons, cytokines and chemokines. Other VACV proteins function inside cells to inhibit apoptosis or signalling pathways that lead to the production of interferons and pro-inflammatory cytokines and chemokines. In this review, these VACV immunomodulatory proteins are described and the potential to create more immunogenic VACV strains by manipulation of the gene encoding these proteins is discussed.
Assuntos
Evasão da Resposta Imune/imunologia , Vaccinia virus/imunologia , Vaccinia virus/patogenicidade , Proteínas Virais/metabolismo , Animais , Humanos , Imunomodulação , Vacínia/imunologia , Vacínia/virologia , Vaccinia virus/metabolismo , Proteínas Virais/genética , VirulênciaRESUMO
Viruses have evolved sophisticated strategies to exploit host cell function for their benefit. Here we show that under physiologically normal oxygen levels (normoxia) vaccinia virus (VACV) infection leads to a rapid stabilization of hypoxia-inducible factor (HIF)-1α, its translocation into the nucleus and the activation of HIF-responsive genes, such as vascular endothelial growth factor (VEGF), glucose transporter-1, and pyruvate dehydrogenase kinase-1. HIF-1α stabilization is mediated by VACV protein C16 that binds the human oxygen sensing enzyme prolyl-hydroxylase domain containing protein (PHD)2 and thereby inhibits PHD2-dependent hydroxylation of HIF-1α. The binding between C16 and PHD2 is direct and specific, and ectopic expression of C16 alone induces transcription of HIF-1α responsive genes. Conversely, a VACV strain lacking the gene for C16, C16L, is unable to induce HIF-1α stabilization. Interestingly, the N-terminal region of C16 is predicted to have a PHD2-like structural fold but lacks the catalytic active site residues of PHDs. The induction of a hypoxic response by VACV is reminiscent of the biochemical consequences of solid tumor formation, and illustrates a poxvirus strategy for manipulation of cellular gene expression and biochemistry.
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Hipóxia Celular/fisiologia , Vaccinia virus/fisiologia , Sequência de Aminoácidos , Células HEK293 , Humanos , Hidroxilação , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia , Dados de Sequência Molecular , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Type I interferons (interferon [IFN]-alpha/beta) are key mediators of innate antiviral responses. Inhibition of IFN-mediated signal transduction by dengue viruses (DENVs), mosquito-borne flaviviruses of immense global health importance, probably plays a crucial role in determining the outcome of the virus-host interaction. Understanding the molecular basis of IFN antagonism by DENV would therefore provide critical insight into disease pathogenesis and new opportunities for development of antiviral therapies and rationally attenuated vaccines. Here we examine the effects of expression of DENV nonstructural proteins on cellular IFN responses. We show that expression of nonstructural protein 5 (NS5) alone inhibits IFN-alpha, but not IFN-gamma, signaling. Expression of the polymerase domain of NS5 is sufficient to inhibit IFN-alpha signaling. NS5 binds signal transducer and activator of transcription 2 (STAT2) and inhibits its phosphorylation. NS5 alone did not, however, induce degradation of STAT2, which occurs when all nonstructural proteins are expressed together. We conclude that DENV NS5 is a potent and specific type I IFN antagonist.
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Vírus da Dengue/fisiologia , Interferon-alfa/metabolismo , Fator de Transcrição STAT2/antagonistas & inibidores , Transdução de Sinais/fisiologia , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular Tumoral , Regulação da Expressão Gênica/fisiologia , Humanos , Fosforilação , Estrutura Terciária de ProteínaRESUMO
Flaviviruses, such as the dengue virus and the West Nile virus, are emerging arthropod-borne viruses that represent an immense global health problem. Considerable progress has been made in understanding flavivirus structure and replication strategies, but only now are the complex molecular interactions between the virus and host cell starting to be unraveled. In this Review, we discuss the ongoing efforts toward elucidating the molecular mechanisms that allow flaviviruses to manipulate host cell functions for successful infection. We draw attention to the importance of these studies in defining the pathogenesis of flaviviral diseases.
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Infecções por Flavivirus/virologia , Flavivirus/patogenicidade , Interações Hospedeiro-Patógeno , Vírus da Dengue/patogenicidade , Modelos Biológicos , Vírus do Nilo Ocidental/patogenicidadeRESUMO
Many viruses escape the cellular immune response by downregulating cell surface expression of major histocompatibility complex (MHC) class I molecules. However, infection of cells with flaviviruses can upregulate the expression of these molecules. In this study we analyzed the expression of MHC class I in K562 and THP-1 human cell lines that were stably transfected with self-replicating subgenomic dengue virus RNA (replicons) and express all the dengue virus nonstructural proteins together. We show that MHC class I expression is upregulated in the dengue virus replicon-expressing cells and that the binding of natural killer (NK) inhibitory receptors to these cells is augmented. This upregulation results in reduced susceptibility of the dengue virus replicon-expressing cells to NK lysis, indicating a possible mechanism for evasion of the dengue virus from NK cell recognition. Visualizing MHC class I expression in replicon-containing K562 and THP-1 cells by confocal microscopy demonstrated aggregation of MHC class I molecules on the cell surface. Finally, replicon-expressing K562 cells manifested increased TAP (transporter associated with antigen processing) and LMP (low-molecular-mass protein) gene transcription, while replicon-expressing THP-1 cells manifested increased NF-kappaB activity and MHC class I transcription. We suggest that expression of dengue virus nonstructural proteins is sufficient to induce MHC class I upregulation through both TAP-dependent and -independent mechanisms. Additionally, aggregation of MHC class I molecules on the cell membrane also contributes to significantly higher binding of low-affinity NK inhibitory receptors, resulting in lower sensitivity to lysis by NK cells.