RESUMO
The NF-κB protein RelB controls dendritic cell (DC) maturation and may be targeted therapeutically to manipulate T cell responses in disease. Here we report that RelB promoted DC activation not as the expected RelB-p52 effector of the noncanonical NF-κB pathway, but as a RelB-p50 dimer regulated by canonical IκBs, IκBα and IκBÉ. IκB control of RelB minimized spontaneous maturation but enabled rapid pathogen-responsive maturation. Computational modeling of the NF-κB signaling module identified control points of this unexpected cell type-specific regulation. Fibroblasts that we engineered accordingly showed DC-like RelB control. Canonical pathway control of RelB regulated pathogen-responsive gene expression programs. This work illustrates the potential utility of systems analyses in guiding the development of combination therapeutics for modulating DC-dependent T cell responses.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Ativação Linfocitária , NF-kappa B/metabolismo , Fator de Transcrição RelB/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Quinase I-kappa B/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , Multimerização Proteica , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Fator de Transcrição RelB/genéticaRESUMO
The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy-termed the "R-clamp"-that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.
Assuntos
Evolução Molecular , Infecções por HIV/metabolismo , Lentivirus/genética , Proteínas Proto-Oncogênicas c-hck/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Animais , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Proteínas Proto-Oncogênicas c-hck/genética , Homologia de Sequência de Aminoácidos , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
Chronic immune activation is an important driver of human immunodeficiency virus type 1 (HIV-1) pathogenesis and has been associated with the presence of tumor necrosis factor-α converting enzyme (TACE) in extracellular vesicles (EVs) circulating in infected individuals. We have recently shown that activation of the Src-family tyrosine kinase hematopoietic cell kinase (Hck) by HIV-1 Nef can trigger the packaging of TACE into EVs via an unconventional protein secretion pathway. Using a panel of HIV-1 Nef mutants and natural HIV-2 and simian immunodeficiency virus (SIV) Nef alleles, we now show that the capacity to promote TACE secretion depends on the superior ability of HIV-1-like Nef alleles to induce Hck kinase activity, whereas other Nef effector functions are dispensable. Strikingly, among the numerous Src-family downstream effectors, serine/threonine kinase Raf-1 was found to be necessary and alone sufficient to trigger the secretion of TACE into EVs. These data reveal the involvement of Raf-1 in regulation of unconventional protein secretion and highlight the importance of Raf-1 as a cellular effector of Nef, thereby suggesting a novel rationale for testing pharmacological inhibitors of the Raf-MAPK pathway to treat HIV-associated immune activation.IMPORTANCE Chronic immune activation contributes to the immunopathogenesis of human immunodeficiency virus type 1 (HIV-1) infection and is associated with poor recovery of the immune system despite potent antiretroviral therapy, which is observed in 10% to 40% drug-treated patients depending on the definition of immune reconstitution. We have previously shown that the HIV pathogenicity factor Nef can promote loading of the proinflammatory protease TACE into extracellular vesicles (EVs), and the levels of such TACE-containing EVs circulating in the blood correlate with low CD4 lymphocyte counts in HIV patients receiving antiretroviral therapy. Here, we show that Nef promotes uploading of TACE into EVs by triggering unconventional secretion via activation of the Hck/Raf/mitogen-activated protein kinase (MAPK) cascade. We find that several pharmaceutical inhibitors of these kinases that are currently in clinical use for other diseases can potently suppress this pathogenic deregulation and could thus provide a novel strategy for treating HIV-associated immune activation.
Assuntos
Proteína ADAM17/metabolismo , Vesículas Extracelulares/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Células HEK293 , HIV-2/fisiologia , Humanos , Proteínas Proto-Oncogênicas c-hck/metabolismo , Vírus da Imunodeficiência Símia/fisiologia , Células THP-1 , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
When studying how HIV-1 Nef can promote packaging of the proinflammatory transmembrane protease TACE (tumor necrosis factor-α converting enzyme) into extracellular vesicles (EVs) we have revealed a novel tyrosine kinase-regulated unconventional protein secretion (UPS) pathway for TACE. When TACE was expressed without its trafficking cofactor iRhom allosteric Hck activation by Nef triggered translocation of TACE into EVs. This process was insensitive to blocking of classical secretion by inhibiting endoplasmic reticulum (ER) to Golgi transport, and involved a distinct form of TACE devoid of normal glycosylation and incompletely processed for prodomain removal. Like most other examples of UPS this process was Golgi reassembly stacking protein (GRASP)-dependent but was not associated with ER stress. These data indicate that Hck-activated UPS provides an alternative pathway for TACE secretion that can bypass iRhom-dependent ER to Golgi transfer, and suggest that tyrosine phosphorylation might have a more general role in regulating UPS.
Assuntos
Proteína ADAM17/metabolismo , Vesículas Extracelulares/metabolismo , Via Secretória , Produtos do Gene nef/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Proto-Oncogênicas c-hck/metabolismoRESUMO
MC159 is a viral FLIP (FLICE inhibitory protein) encoded by the molluscum contagiosum virus (MCV) enabling MCV to evade antiviral immunity and to establish persistent infections in humans. Here, we show that MC159 contains a functional SH3 binding motif, which mediates avid and selective binding to SH3BP4, a signaling protein known to regulate endocytic trafficking and suppress cellular autophagy. The capacity to bind SH3BP4 was dispensable for regulation of NF-κB-mediated transcription and suppression of proapoptotic caspase activation but contributed to inhibition of amino acid starvation-induced autophagy by MC159. These results provide new insights into the cellular functions of MC159 and reveal SH3BP4 as a novel host cell factor targeted by a viral immune evasion protein.IMPORTANCE After the eradication of smallpox, molluscum contagiosum virus (MCV) is the only poxvirus restricted to infecting humans. MCV infection is common and causes benign skin lesions that usually resolve spontaneously but may persist for years and grow large, especially in immunocompromised individuals. While not life threatening, MCV infections pose a significant global health burden. No vaccine or specific anti-MCV therapy is available. MCV encodes several proteins that enable it to evade antiviral immunity, a notable example of which is the MC159 protein. In this study, we describe a novel mechanism of action for MC159 involving hijacking of a host cell protein called SH3BP4 to suppress autophagy, a cellular recycling mechanism important for antiviral immunity. This study contributes to our understanding of the host cell interactions of MCV and the molecular function of MC159.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Vírus do Molusco Contagioso/metabolismo , Proteínas Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Apoptose/efeitos dos fármacos , Autofagia/fisiologia , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Evasão da Resposta Imune/efeitos dos fármacos , Evasão da Resposta Imune/fisiologia , Células MCF-7 , Molusco Contagioso/virologia , Vírus do Molusco Contagioso/patogenicidade , NF-kappa B/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transdução de Sinais , Proteínas Virais/fisiologia , Domínios de Homologia de src/fisiologiaRESUMO
The activation of the interferon (IFN) system, which is triggered largely by the recognition of viral nucleic acids, is one of the most important host defense reactions against viral infections. Although influenza A and B viruses, which both have segmented negative-strand RNA genomes, share major structural similarities, they have evolutionarily diverged, with total genetic incompatibility. Here we compare antiviral-inducing mechanisms during infections with type A and B influenza viruses in human dendritic cells. We observed that IFN responses are induced significantly faster in cells infected with influenza B virus than in cells infected with type A influenza virus and that the early induction of antiviral gene expression is mediated by the activation of the transcription factor IFN regulatory factor 3 (IRF3). We further demonstrate that influenza A virus infection activates IFN responses only after viral RNA (vRNA) synthesis, whereas influenza B virus induces IFN responses even if its infectivity is destroyed by UV treatment. Thus, initial viral transcription, replication, and viral protein synthesis are dispensable for influenza B virus-induced antiviral responses. Moreover, vRNA molecules from both type A and B viruses are equally potent activators of IFN induction, but incoming influenza B virus structures are recognized directly in the cytosol, while influenza A virus is able to evade early recognition. Collectively, our data provide new evidence of a novel antiviral evasion strategy for influenza A virus without a contribution of the viral NS1 protein, and this opens up new insights into different influenza virus pathogenicities.
Assuntos
Células Dendríticas/virologia , Vírus da Influenza A/imunologia , Vírus da Influenza A/patogenicidade , Vírus da Influenza B/imunologia , Vírus da Influenza B/patogenicidade , Fator Regulador 3 de Interferon/metabolismo , Interferons/biossíntese , Animais , Linhagem Celular , Células Dendríticas/imunologia , Cães , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune , Influenza Humana/imunologia , Interferons/imunologia , Células Madin Darby de Rim Canino , RNA Viral/biossíntese , Internalização do VírusRESUMO
The emergence of increasingly immunoevasive SARS-CoV-2 variants emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Intranasal administration of neutralizing antibodies has shown encouraging protective potential but there remains a need for SARS-CoV-2 blocking agents that are less vulnerable to mutational viral variation and more economical to produce in large scale. Here we describe TriSb92, a highly manufacturable and stable trimeric antibody-mimetic sherpabody targeted against a conserved region of the viral spike glycoprotein. TriSb92 potently neutralizes SARS-CoV-2, including the latest Omicron variants like BF.7, XBB, and BQ.1.1. In female Balb/c mice intranasal administration of just 5 or 50 micrograms of TriSb92 as early as 8 h before but also 4 h after SARS-CoV-2 challenge can protect from infection. Cryo-EM and biochemical studies reveal triggering of a conformational shift in the spike trimer as the inhibitory mechanism of TriSb92. The potency and robust biochemical properties of TriSb92 together with its resistance against viral sequence evolution suggest that TriSb92 could be useful as a nasal spray for protecting susceptible individuals from SARS-CoV-2 infection.
Assuntos
COVID-19 , SARS-CoV-2 , Feminino , Animais , Camundongos , Humanos , Administração Intranasal , COVID-19/prevenção & controle , Pandemias , Anticorpos Neutralizantes , Camundongos Endogâmicos BALB C , Anticorpos Antivirais , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
The ongoing SARS-CoV-2 pandemic is controlled but not halted by public health measures and mass vaccination strategies which have exclusively relied on intramuscular vaccines. Intranasal vaccines can prime or recruit to the respiratory epithelium mucosal immune cells capable of preventing infection. Here we report a comprehensive series of studies on this concept using various mouse models, including HLA class II-humanized transgenic strains. We found that a single intranasal (i.n.) dose of serotype-5 adenoviral vectors expressing either the receptor binding domain (Ad5-RBD) or the complete ectodomain (Ad5-S) of the SARS-CoV-2 spike protein was effective in inducing i) serum and bronchoalveolar lavage (BAL) anti-spike IgA and IgG, ii) robust SARS-CoV-2-neutralizing activity in the serum and BAL, iii) rigorous spike-directed T helper 1 cell/cytotoxic T cell immunity, and iv) protection of mice from a challenge with the SARS-CoV-2 beta variant. Intramuscular (i.m.) Ad5-RBD or Ad5-S administration did not induce serum or BAL IgA, and resulted in lower neutralizing titers in the serum. Moreover, prior immunity induced by an intramuscular mRNA vaccine could be potently enhanced and modulated towards a mucosal IgA response by an i.n. Ad5-S booster. Notably, Ad5 DNA was found in the liver or spleen after i.m. but not i.n. administration, indicating a lack of systemic spread of the vaccine vector, which has been associated with a risk of thrombotic thrombocytopenia. Unlike in otherwise genetically identical HLA-DQ6 mice, in HLA-DQ8 mice Ad5-RBD vaccine was inferior to Ad5-S, suggesting that the RBD fragment does not contain a sufficient collection of helper-T cell epitopes to constitute an optimal vaccine antigen. Our data add to previous promising preclinical results on intranasal SARS-CoV-2 vaccination and support the potential of this approach to elicit mucosal immunity for preventing transmission of SARS-CoV-2.
Assuntos
COVID-19 , Vacinas Virais , Humanos , Animais , Camundongos , Glicoproteína da Espícula de Coronavírus/genética , Vacinas contra COVID-19 , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , SARS-CoV-2 , Administração Intranasal , Modelos Animais de Doenças , Imunoglobulina ARESUMO
BACKGROUND: Influenza A virus non-structural protein 1 (NS1) is a virulence factor, which is targeted into the cell cytoplasm, nucleus and nucleolus. NS1 is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. Previously, we have shown that the NS1 protein of the H3N2 subtype influenza viruses possesses a C-terminal nuclear localization signal (NLS) that also functions as a nucleolar localization signal (NoLS) and targets the protein into the nucleolus. RESULTS: Here, we show that the NS1 protein of the human H3N2 virus subtype interacts in vitro primarily via its C-terminal NLS2/NoLS and to a minor extent via its N-terminal NLS1 with the nucleolar proteins, nucleolin and fibrillarin. Using chimeric green fluorescence protein (GFP)-NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions. CONCLUSION: NS1 protein of the human H3N2 virus interacts primarily via the C-terminal NLS2/NoLS and to a minor extent via the N-terminal NLS1 with the main nucleolar proteins, nucleolin, B23 and fibrillarin.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H3N2/fisiologia , Sinais de Localização Nuclear , Fosfoproteínas/metabolismo , Mapeamento de Interação de Proteínas , Proteínas de Ligação a RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular , Células Epiteliais/química , Células Epiteliais/virologia , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Microscopia Confocal , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Coloração e Rotulagem , NucleolinaRESUMO
Class I SH3 domain-binding motifs generally comply with the consensus sequence [R/K]xØPxxP, the hydrophobic residue Ø being proline or leucine. We have studied the unusual Ø = Ala-specificity of SNX9 SH3 by determining its complex structure with a peptide present in eastern equine encephalitis virus (EEEV) nsP3. The structure revealed the length and composition of the n-Src loop as important factors determining specificity. We also compared the affinities of EEEV nsP3 peptide, its mutants, and cellular ligands to SNX9 SH3. These data suggest that nsP3 has evolved to minimize reduction of conformational entropy upon binding, hence acquiring stronger affinity, enabling takeover of SNX9. The RxAPxxP motif was also found in human T cell leukemia virus-1 (HTLV-1) Gag polyprotein. We found that this motif was required for efficient HTLV-1 infection, and that the specificity of SNX9 SH3 for the RxAPxxP core binding motif was importantly involved in this process.
Assuntos
Alanina , Domínios de Homologia de src , Animais , Sítios de Ligação , Cavalos , Ligantes , Peptídeos/química , Ligação ProteicaRESUMO
Hypoxia-inducible factors are the key elements in the essential process of oxygen homeostasis of vertebrate cells. Stabilisation and subsequent nuclear localisation of HIF-alpha subunits results in the activation of target genes such as vegf, epo and glut1. The passage of transcription factors e.g. HIF-1alpha into the nucleus through the nuclear pore complex is regulated by nuclear transport receptors. Therefore nucleocytoplasmic shuttling can regulate transcriptional activity by facilitating the cellular traffic of transcription factors between both compartments. Here, we report on the identification of specific interactions of hypoxia-inducible factors with nuclear transport receptors importin alpha/beta. HIF-1alpha, -1beta, and HIF-2alpha are binding to importin alpha1, alpha3, alpha5, and alpha7. The direct interaction of HIF-1alpha to alpha importins is dependent on a functional nuclear localisation signal within the C-terminal region of the protein. In contrast, the supposed N-terminal NLS is not effective. Our findings provide new insight into the mechanism of the regulation of nuclear transport of hypoxia-inducible factors.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , alfa Carioferinas/fisiologia , beta Carioferinas/fisiologia , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Sítios de Ligação , Células Cultivadas , Células HeLa , Humanos , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: The viral genome of hepatitis C virus constitutes a 9.6-kb single-stranded positive-sense RNA which encodes altogether 11 viral proteins. In order to study the humoral immune responses against different HCV proteins in patients suffering from chronic HCV infection, we produced three structural (core, E1 and E2) and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) in Sf9 insect cells by using the baculovirus expression system. RESULTS: The recombinant HCV core, E1, E2, NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins were purified and used in Western blot analysis to determine antibody responses against individual HCV protein in 68 HCV RNA and antibody positive human sera that were obtained from patients suffering from genotype 1, 2, 3 or 4 infection. These sera were also analysed with INNO-LIA Score test for HCV antibodies against core, NS3, NS4AB and NS5A, and the results were similar to the ones obtained by Western blot method. Based on our Western blot analyses we found that the major immunogenic HCV antigens were the core, NS4B, NS3 and NS5A proteins which were recognized in 97%, 86%, 68% and 53% of patient sera, respectively. There were no major genotype specific differences in antibody responses to individual HCV proteins. A common feature within the studied sera was that all except two sera recognized the core protein in high titers, whereas none of the sera recognized NS2 protein and only three sera (from genotype 3) recognised NS5B. CONCLUSION: The data shows significant variation in the specificity in humoral immunity in chronic HCV patients.
Assuntos
Anticorpos Antivirais/sangue , Hepatite C Crônica/imunologia , Proteínas do Core Viral/imunologia , Proteínas não Estruturais Virais/imunologia , Antígenos Virais/imunologia , Western Blotting , Hepacivirus/imunologia , HumanosRESUMO
In resting cells NF-kappaB transcription factors are retained in the cytoplasm as latent inactive complexes, until they are activated and rapidly transported into the nucleus. We show that all NF-kappaB proteins are imported into the nucleus via a subset of importin alpha isoforms. Our data indicate that the NF-kappaB components of the classical and alternative pathways have somewhat different specifities to importin alpha molecules. Based on the results from binding experiments of in vitro-translated and Sendai virus infection-induced or TNF-alpha-stimulated endogenous NF-kappaB proteins, it can be predicted that the specifity of NF-kappaB proteins to importin alpha molecules is different and changes upon the composition of the imported dimer. p52 protein binds directly to importin alpha3, alpha4, alpha5 and alpha6 and c-Rel binds to importin alpha5, alpha6 and alpha7 via a previously described monopartite nuclear localization signals (NLSs). Here we show that RelB, instead, has a bipartite arginine/lysine-rich NLS that mediates the binding of RelB to importin alpha5 and alpha6 and subsequent nuclear translocation of the protein. Moreover, we show that the nuclear import of p52/RelB heterodimers is mediated exclusively by the NLS of RelB. In addition, we found that the NLS of p52 mediates the nuclear import of p52/p65 heterodimers.
Assuntos
Núcleo Celular/metabolismo , Subunidade p52 de NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Fator de Transcrição RelB/metabolismo , alfa Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Dimerização , Humanos , Cinética , Subunidade p52 de NF-kappa B/química , Sinais de Localização Nuclear , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-rel/química , Fator de Transcrição RelB/químicaRESUMO
The non-structural protein-1 (NS1) of many influenza A strains, especially those of avian origin, contains an SH3 ligand motif, which binds tightly to the cellular adaptor proteins Crk (Chicken tumor virus number 10 (CT10) regulator of kinase) and Crk-like adapter protein (CrkL). This interaction has been shown to potentiate NS1-induced activation of the phosphatidylinositol 3-kinase (PI3K), but additional effects on the host cell physiology may exist. Here we show that NS1 can induce an efficient translocation of Crk proteins from the cytoplasm into the nucleus, which results in an altered pattern of nuclear protein tyrosine phosphorylation. This was not observed using NS1 proteins deficient in SH3 binding or engineered to be exclusively cytoplasmic, indicating a physical role for NS1 as a carrier in the nuclear translocation of Crk. These data further emphasize the role of Crk proteins as host cell interaction partners of NS1, and highlight the potential for host cell manipulation gained by a viral protein simply via acquiring a short SH3 binding motif.
Assuntos
Proteínas Proto-Oncogênicas c-crk/metabolismo , Proteínas não Estruturais Virais/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Influenza Humana/virologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Proteínas não Estruturais Virais/química , Domínios de Homologia de srcRESUMO
Whereas ubiquitin-dependent degrons have been characterized in some detail, how proteins may be targeted to ubiquitin-independent proteasomal degradation remains unclear. Here we show that IκBα contains an ubiquitin-independent degron whose activity is portable to heterologous proteins such as the globular protein GFP (green fluorescent protein) via a proteasome-dependent, ubiquitin-independent, non-lysosomal pathway. The ubiquitin-independent degradation signal resides in an 11-amino-acid sequence, which is not only sufficient but also required for IκBα's short half-life. Finally, we show that this degron's activity is regulated by the interaction with NFκB, which controls its solvent exposure, and we demonstrate that this regulation of the degron's activity is critical for IκBα's signaling functions.
Assuntos
Repetição de Anquirina/genética , Proteínas I-kappa B/metabolismo , Transdução de Sinais/genética , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Linhagem Celular , Cloroquina/farmacologia , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde , Células HEK293 , Humanos , Lisossomos/metabolismo , Macrolídeos/farmacologia , Camundongos , Inibidor de NF-kappaB alfa , Proteólise , Ubiquitina/metabolismoRESUMO
The magnitude, duration and oscillation of cellular signalling pathway responses are often limited by negative feedback loops, defined as an 'activator-induced inhibitor' regulatory motif. Within the NFκB signalling pathway, a key negative feedback regulator is IκBα. We show here that, contrary to current understanding, NFκB-inducible expression is not sufficient for providing effective negative feedback. We then employ computational simulations of NFκB signalling to identify IκBα molecular properties that are critical for proper negative feedback control and test the resulting predictions in biochemical and single-cell live-imaging studies. We identified nuclear import and nuclear export of IκBα and the IκBα-NFκB complex, as well as the free IκBα half-life, as key determinants of post-induction repression of NFκB and the potential for subsequent reactivation. Our work emphasizes that negative feedback is an emergent systems property determined by multiple molecular and biophysical properties in addition to the required 'activator-induced inhibitor' relationship.
Assuntos
Simulação por Computador , Fibroblastos/metabolismo , Regulação da Expressão Gênica/fisiologia , Quinase I-kappa B/metabolismo , Modelos Biológicos , Transdução de Sinais/fisiologia , Motivos de Aminoácidos , Animais , Fibroblastos/citologia , Quinase I-kappa B/genética , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismoRESUMO
Recognition of viral nucleic acids leads to type I and type III IFN gene expression and activation of host antiviral responses. At present, type III IFN genes are the least well-characterized IFN types. Here, we demonstrate that the p38 MAPK signaling pathway is involved in regulating IFN-λ1 gene expression in response to various types of RNA molecules in human moDCs. Inhibition of p38 MAPK strongly reduced IFN gene expression, and overexpression of p38α MAPK enhanced IFN-λ1 gene expression in RNA-stimulated moDCs. The regulation of IFN gene expression by p38 MAPK signaling was independent of protein synthesis and thus, a direct result of RNA stimulation. Moreover, the RIG-I/MDA5-MAVS-IRF3 pathway was required for p38α MAPK to up-regulate IFN-λ1 promoter activation, whereas the MyD88-IRF7 pathway was not needed, and the regulation was not involved directly in IRF7-dependent IFN-α1 gene expression. The stimulatory effect of p38α MAPK on IFN-λ1 mRNA expression in human moDCs did not take place directly via the activating TBK1/IKKε complex, but rather, it occurred through some other parallel pathways. Furthermore, mutations in ISRE and NF-κB binding sites in the promoter region of the IFN-λ1 gene led to a significant reduction in p38α MAPK-mediated IFN responses after RNA stimulation. Altogether, our data suggest that the p38α MAPK pathway is linked with RLR signaling pathways and regulates the expression of early IFN genes after RNA stimulation cooperatively with IRF3 and NF-κB to induce antiviral responses further.
Assuntos
Células Dendríticas/imunologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 14 Ativada por Mitógeno/imunologia , Monócitos/imunologia , RNA/farmacologia , Células 3T3 , Animais , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/imunologia , Células Dendríticas/citologia , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/imunologia , Células HEK293 , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Helicase IFIH1 Induzida por Interferon , Interferon-alfa , Interferons , Interleucinas/genética , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Camundongos Knockout , Proteína Quinase 14 Ativada por Mitógeno/genética , Monócitos/citologia , NF-kappa B/genética , NF-kappa B/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Receptores Imunológicos , Elementos de Resposta/imunologiaRESUMO
Influenza A virus nonstructural protein 1 (NS1A protein) is a virulence factor which is targeted into the nucleus. It is a multifunctional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. We show that the NS1A protein can interact with all six human importin alpha isoforms, indicating that the nuclear translocation of NS1A protein is mediated by the classical importin alpha/beta pathway. The NS1A protein of the H1N1 (WSN/33) virus has only one N-terminal arginine- or lysine-rich nuclear localization signal (NLS1), whereas the NS1A protein of the H3N2 subtype (Udorn/72) virus also has a second C-terminal NLS (NLS2). NLS1 is mapped to residues 35 to 41, which also function in the double-stranded RNA-binding activity of the NS1A protein. NLS2 was created by a 7-amino-acid C-terminal extension (residues 231 to 237) that became prevalent among human influenza A virus types isolated between the years 1950 to 1987. NLS2 includes basic amino acids at positions 219, 220, 224, 229, 231, and 232. Surprisingly, NLS2 also forms a functional nucleolar localization signal NoLS, a function that was retained in H3N2 type virus NS1A proteins even without the C-terminal extension. It is likely that the evolutionarily well-conserved nucleolar targeting function of NS1A protein plays a role in the pathogenesis of influenza A virus.
Assuntos
Nucléolo Celular/metabolismo , Vírus da Influenza A Subtipo H3N2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Nucléolo Celular/virologia , Evolução Molecular , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Dados de Sequência Molecular , Transporte Proteico/fisiologiaRESUMO
The pneumococcal genes encoding for the surface associated proteins have been proposed to be important for pneumococcal protein vaccine development. We cloned the full-length putative proteinase maturation protein A gene SP098l/ppmA (as published by Tettelin et al. in 2001) and produced the encoded protein in high levels in E. coli. The purified recombinant PpmA was used as an antigen in Western blotting to study systemic antibody responses to PpmA in animals and in children with acute otitis media (AOM). In children, the geometric mean titers of serum IgG antibodies against PpmA increased with age and differed significantly in relation to pneumococcal findings in middle ear fluid and/or nasopharyngeal aspirate. The serum IgG antibody titers against PpmA were low in children with Streptococcus pneumoniae cultured in the middle ear, and the highest in children with pneumococci in the nasopharynx, without them being found in the middle ear fluid. We conclude that PpmA is immunogenic in humans, and therefore an interesting antigen to study further in developing pneumococcal multicomponent protein vaccines.
Assuntos
Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/imunologia , Otite Média/imunologia , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Fatores Etários , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Western Blotting , Criança , Pré-Escolar , Clonagem Molecular , Escherichia coli/genética , Cobaias , Humanos , Imunoglobulina G/sangue , Lactente , Nasofaringe/microbiologia , Otite Média com Derrame/microbiologia , Coelhos , Estatística como Assunto , Streptococcus pneumoniae/genéticaRESUMO
Lung epithelial cells are the primary cellular targets for respiratory virus pathogens such as influenza and parainfluenza viruses. Here, we have analyzed influenza A, influenza B and Sendai virus-induced chemokine response in human A549 lung epithelial cells. Influenza virus infection resulted in low CCL2/MCP-1, CCL5/RANTES, CXCL8/IL-8 and CXCL10/IP-10 production at late times of infection. However, when cells were pretreated with TNF-alpha or IFN-alpha, influenza-A-virus-induced chemokine production was greatly enhanced. Cytokine pretreatment resulted in enhanced expression of RIG-I, IKKepsilon, interferon regulatory factor (IRF)1, IRF7 and p50 proteins. Most importantly, influenza-A-virus-induced DNA binding of IRF1, IRF3, IRF7 and NF-kappaB onto CXCL10 ISRE and NF-kappaB elements, respectively, was markedly enhanced in cytokine-pretreated cells. Our results suggest that IFN-alpha and TNF-alpha have a significant role in priming epithelial cells for higher cytokine and chemokine production in influenza A virus infection.