RESUMO
Reactive oxygen species (ROS) are generated by virus-infected cells; however, the physiological importance of ROS generated under these conditions is unclear. Here we found that the inflammation and cell death induced by exposure of mice or cells to sources of ROS were not altered in the absence of canonical ROS-sensing pathways or known cell-death pathways. ROS-induced cell-death signaling involved interactions among the cellular ROS sensor and antioxidant factor KEAP1, the phosphatase PGAM5 and the proapoptotic factor AIFM1. Pgam5 -/- mice showed exacerbated lung inflammation and proinflammatory cytokines in an ozone-exposure model. Similarly, challenge with influenza A virus led to increased infiltration of the virus, lymphocytic bronchiolitis and reduced survival of Pgam5 -/- mice. This pathway, which we have called 'oxeiptosis', was a ROS-sensitive, caspase independent, non-inflammatory cell-death pathway and was important for protection against inflammation induced by ROS or ROS-generating agents such as viral pathogens.
Assuntos
Morte Celular/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Fator de Indução de Apoptose/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The emergence and global spread of SARS-CoV-2 has resulted in the urgent need for an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology1-10. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. Here we report a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactomes of both viruses, as well as their influence on the transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon infection with SARS-CoV-2 and SARS-CoV at different levels and enabled identification of distinct and common molecular mechanisms of these closely related coronaviruses. The TGF-ß pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy was specifically dysregulated by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org ) highlights many hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies, which we exemplify by identifying inhibitors of kinases and matrix metalloproteases with potent antiviral effects against SARS-CoV-2.
Assuntos
COVID-19/metabolismo , Interações Hospedeiro-Patógeno , Proteoma/metabolismo , Proteômica , SARS-CoV-2/patogenicidade , Síndrome Respiratória Aguda Grave/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Animais , Antivirais/farmacologia , Autofagia/efeitos dos fármacos , COVID-19/imunologia , COVID-19/virologia , Linhagem Celular , Conjuntos de Dados como Assunto , Avaliação Pré-Clínica de Medicamentos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Inibidores de Metaloproteinases de Matriz/farmacologia , Fosforilação , Mapas de Interação de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Proteoma/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , SARS-CoV-2/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/virologia , Fator de Crescimento Transformador beta/metabolismo , Ubiquitinação , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Viroporinas/metabolismoRESUMO
Cell-autonomous induction of type I interferon must be stringently regulated. Rapid induction is key to control virus infection, whereas proper limitation of signaling is essential to prevent immunopathology and autoimmune disease. Using unbiased kinome-wide RNAi screening followed by thorough validation, we identified 22 factors that regulate RIG-I/IRF3 signaling activity. We describe a negative-feedback mechanism targeting RIG-I activity, which is mediated by death associated protein kinase 1 (DAPK1). RIG-I signaling triggers DAPK1 kinase activation, and active DAPK1 potently inhibits RIG-I stimulated IRF3 activity and interferon-beta production. DAPK1 phosphorylates RIG-I in vitro at previously reported as well as other sites that limit 5'ppp-dsRNA sensing and virtually abrogate RIG-I activation.
Assuntos
Proteínas Quinases Associadas com Morte Celular/metabolismo , RNA Interferente Pequeno/genética , Receptores do Ácido Retinoico/metabolismo , Células A549 , Animais , Células Cultivadas , Retroalimentação Fisiológica , Células HEK293 , Humanos , Camundongos , Fosforilação , Proteínas Quinases/metabolismo , Transdução de SinaisRESUMO
Molluscum contagiosum virus (MCV) is a human-specific poxvirus that causes a highly common but mild infection characterized by distinctive and persistent papular skin lesions. These lesions can persist for long periods without an effective clearance response from the host. MCV, like all poxviruses, encodes multiple known immunosuppressive proteins which target innate immune signalling pathways involved in viral nucleic acid sensing, interferon production and inflammation which should trigger antiviral immunity leading to clearance. Two major families of transcription factors responsible for driving the immune response to viruses are the NF-κB and the interferon regulatory factor (IRF) families. While NF-κB broadly drives pro-inflammatory gene expression and IRFs chiefly drive interferon induction, both collaborate in transactivating many of the same genes in a concerted immune response to viral infection. Here, we report that the MCV protein MC089 specifically inhibits IRF activation from both DNA- and RNA-sensing pathways, making it the first characterized MCV inhibitor to selectively target IRF activation to date. MC089 interacts with proteins required for IRF activation, namely IKKε, TBKBP1 and NAP1. Additionally, MC089 targets RNA sensing by associating with the RNA-sensing adaptor protein mitochondrial antiviral-signalling protein on mitochondria. MC089 displays specificity in its inhibition of IRF3 activation by suppressing immunostimulatory nucleic acid-induced serine 396 phosphorylation without affecting the phosphorylation of serine 386. The selective interaction of MC089 with IRF-regulatory proteins and site-specific inhibition of IRF3 phosphorylation may offer a tool to provide novel insights into the biology of IRF3 regulation.
Assuntos
Fator Regulador 3 de Interferon , Vírus do Molusco Contagioso , Proteínas Virais , Humanos , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Vírus do Molusco Contagioso/imunologia , Vírus do Molusco Contagioso/genética , Proteínas Virais/metabolismo , Proteínas Virais/genética , Proteínas Virais/imunologia , Transdução de Sinais , Imunidade Inata , Células HEK293 , Interações Hospedeiro-Patógeno/imunologiaRESUMO
Zika virus (ZIKV) has recently emerged as a global health concern owing to its widespread diffusion and its association with severe neurological symptoms and microcephaly in newborns1. However, the molecular mechanisms that are responsible for the pathogenicity of ZIKV remain largely unknown. Here we use human neural progenitor cells and the neuronal cell line SK-N-BE2 in an integrated proteomics approach to characterize the cellular responses to viral infection at the proteome and phosphoproteome level, and use affinity proteomics to identify cellular targets of ZIKV proteins. Using this approach, we identify 386 ZIKV-interacting proteins, ZIKV-specific and pan-flaviviral activities as well as host factors with known functions in neuronal development, retinal defects and infertility. Moreover, our analysis identified 1,216 phosphorylation sites that are specifically up- or downregulated after ZIKV infection, indicating profound modulation of fundamental signalling pathways such as AKT, MAPK-ERK and ATM-ATR and thereby providing mechanistic insights into the proliferation arrest elicited by ZIKV infection. Functionally, our integrative study identifies ZIKV host-dependency factors and provides a comprehensive framework for a system-level understanding of ZIKV-induced perturbations at the levels of proteins and cellular pathways.
Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Proteoma/análise , Proteômica , Zika virus/patogenicidade , Animais , Diferenciação Celular , Linhagem Celular , Chlorocebus aethiops , Interações Hospedeiro-Patógeno/genética , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/virologia , Fosfoproteínas/análise , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Mapas de Interação de Proteínas , Proteoma/genética , Proteoma/metabolismo , RNA Interferente Pequeno/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Zika virus/genética , Zika virus/metabolismoRESUMO
Viruses have evolved a plethora of mechanisms to target host antiviral responses. Here, we propose a yet uncharacterized mechanism of immune regulation by the orthomyxovirus Thogoto virus (THOV) ML protein through engaging general transcription factor TFIIB. ML generates a TFIIB depleted nuclear environment by re-localizing it into the cytoplasm. Although a broad effect on gene expression would be anticipated, ML expression, delivery of an ML-derived functional domain or experimental depletion of TFIIB only leads to altered expression of a limited number of genes. Our data indicate that TFIIB is critically important for the de novo recruitment of Pol II to promoter start sites and that TFIIB may not be required for regulated gene expression from paused promoters. Since many immune genes require de novo recruitment of Pol II, targeting of TFIIB by THOV represents a neat mechanism to affect immune responses while keeping other cellular transcriptional activities intact. Thus, interference with TFIIB activity may be a favourable site for therapeutic intervention to control undesirable inflammation.
Assuntos
Antivirais/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Influenza Humana/imunologia , Thogotovirus/imunologia , Fator de Transcrição TFIIB/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas Virais/metabolismo , Células HeLa , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/metabolismo , Regiões Promotoras Genéticas , Thogotovirus/efeitos dos fármacos , Fator de Transcrição TFIIB/genéticaRESUMO
Molluscum contagiosum virus (MCV), the only known extant human-adapted poxvirus, causes a long-duration infection characterized by skin lesions that typically display an absence of inflammation despite containing high titers of live virus. Despite this curious presentation, MCV is very poorly characterized in terms of host-pathogen interactions. The absence of inflammation around MCV lesions suggests the presence of potent inhibitors of human antiviral immunity and inflammation. However, only a small number of MCV immunomodulatory genes have been characterized in detail. It is likely that many more remain to be discovered, given the density of such sequences in other poxvirus genomes. NF-κB activation occurs in response to both virus-induced pattern recognition receptor (PRR) signaling and cellular activation by virus-induced proinflammatory cytokines like tumor necrosis factor and interleukin-1. Activated NF-κB drives cytokine and interferon gene expression, leading to inflammation and virus clearance. We report that MC005, which has no orthologs in other poxvirus genomes, is a novel inhibitor of PRR- and cytokine-stimulated NF-κB activation. MC005 inhibited NF-κB proximal to the IκB kinase (IKK) complex, and unbiased affinity purification revealed that MC005 interacts with the IKK subunit NEMO (NF-κB essential modulator). MC005 binding to NEMO prevents the conformational priming of the IKK complex that occurs when NEMO binds to ubiquitin chains during pathway activation. These data reveal a novel mechanism of poxvirus inhibition of human innate immunity, validate current dynamic models of NEMO-dependent IKK complex activation, and further clarify how the human-adapted poxvirus MCV can so effectively evade antiviral immunity and suppress inflammation to persist in human skin lesions.IMPORTANCE Poxviruses adapt to specific hosts over time, evolving and tailoring elegantly precise inhibitors of the rate-limiting steps within the signaling pathways that control innate immunity and inflammation. These inhibitors reveal new features of the antiviral response, clarify existing models of signaling regulation while offering potent new tools for approaching therapeutic intervention in autoimmunity and inflammatory disease. Molluscum contagiosum virus (MCV) is the only known extant poxvirus specifically adapted to human infection and appears adept at evading normal human antiviral responses, yet it remains poorly characterized. We report the identification of MCV protein MC005 as an inhibitor of the pathways leading to the activation of NF-κB, an essential regulator of innate immunity. Further, identification of the mechanism of inhibition of NF-κB by MC005 confirms current models of the complex way in which NF-κB is regulated and greatly expands our understanding of how MCV so effectively evades human immunity.
Assuntos
Interações Hospedeiro-Patógeno , Quinase I-kappa B/antagonistas & inibidores , Evasão da Resposta Imune , Vírus do Molusco Contagioso/patogenicidade , NF-kappa B/antagonistas & inibidores , Proteínas Virais/metabolismo , Animais , Linhagem Celular , HumanosRESUMO
Molluscum contagiosum virus (MCV) is unique in being the only known extant, human-adapted poxvirus, yet to date, it is very poorly characterized in terms of host-pathogen interactions. MCV causes persistent skin lesions filled with live virus, but these are generally immunologically silent, suggesting the presence of potent inhibitors of human antiviral immunity and inflammation. Fewer than five MCV immunomodulatory genes have been characterized in detail, but it is likely that many more remain to be discovered given the density of such sequences in all well-characterized poxviruses. Following virus infection, NF-B activation occurs in response to both pattern recognition receptor (PRR) signaling and cellular activation by virus-elicited proinflammatory cytokines, such as tumor necrosis factor (TNF). As such, NF-B activation is required for virus detection, antiviral signaling, inflammation, and clearance of viral infection. Hence, we screened a library of MCV genes for effects on TNF-stimulated NF-B activation. This revealed MC132, a unique protein with no orthologs in other poxviral genomes, as a novel inhibitor of NF-B. Interestingly, MC132 also inhibited PRR- and virus-activated NF-B, since MC132 interacted with the NF-B subunit p65 and caused p65 degradation. Unbiased affinity purification to identify host targets of MC132 revealed that MC132 acted by targeting NF-B p65 for ubiquitin-dependent proteasomal degradation by recruiting p65 to a host Cullin-5/Elongin B/Elongin C complex. These data reveal a novel mechanism for poxviral inhibition of human innate immunity and further clarify how the human-adapted poxvirus MCV can so effectively evade antiviral immunity to persist in skin lesions.
Assuntos
Tolerância Imunológica/imunologia , Imunidade Inata/imunologia , Vírus do Molusco Contagioso/imunologia , NF-kappa B/metabolismo , Proteínas Virais/imunologia , eIF-2 Quinase/metabolismo , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Imunoprecipitação , Microscopia Confocal , Vírus do Molusco Contagioso/metabolismo , Oligonucleotídeos/genética , Proteólise , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Reconhecimento de Padrão/metabolismo , Espectrometria de Massas em Tandem , Proteínas Virais/metabolismoRESUMO
Kaposi's sarcoma (KS) is a mesenchymal tumour, which is caused by Kaposi's sarcoma herpesvirus (KSHV) and develops under inflammatory conditions. KSHV-infected endothelial spindle cells, the neoplastic cells in KS, show increased invasiveness, attributed to the elevated expression of metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2). The majority of these spindle cells harbour latent KSHV genomes, while a minority undergoes lytic reactivation with subsequent production of new virions and viral or cellular chemo- and cytokines, which may promote tumour invasion and dissemination. In order to better understand KSHV pathogenesis, we investigated cellular mechanisms underlying the lytic reactivation of KSHV. Using a combination of small molecule library screening and siRNA silencing we found a STE20 kinase family member, MAP4K4, to be involved in KSHV reactivation from latency and to contribute to the invasive phenotype of KSHV-infected endothelial cells by regulating COX-2, MMP-7, and MMP-13 expression. This kinase is also highly expressed in KS spindle cells in vivo. These findings suggest that MAP4K4, a known mediator of inflammation, is involved in KS aetiology by regulating KSHV lytic reactivation, expression of MMPs and COX-2, and, thereby modulating invasiveness of KSHV-infected endothelial cells.
Assuntos
Células Endoteliais/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Herpesvirus Humano 8/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteínas de Neoplasias/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Sarcoma de Kaposi/metabolismo , Ativação Viral/fisiologia , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Células Endoteliais/patologia , Células Endoteliais/virologia , Feminino , Células HEK293 , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Inflamação/virologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Metaloproteinase 13 da Matriz/biossíntese , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 7 da Matriz/biossíntese , Metaloproteinase 7 da Matriz/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/patologiaRESUMO
Kaposi's Sarcoma (KS), caused by Kaposi's Sarcoma Herpesvirus (KSHV), is a highly vascularised angiogenic tumor of endothelial cells, characterized by latently KSHV-infected spindle cells and a pronounced inflammatory infiltrate. Several KSHV proteins, including LANA-1 (ORF73), vCyclin (ORF72), vGPCR (ORF74), vIL6 (ORF-K2), vCCL-1 (ORF-K6), vCCL-2 (ORF-K4) and K1 have been shown to exert effects that can lead to the proliferation and atypical differentiation of endothelial cells and/or the secretion of cytokines with angiogenic and inflammatory properties (VEGF, bFGF, IL6, IL8, GROα, and TNFß). To investigate a role of the KSHV K15 protein in KSHV-mediated angiogenesis, we carried out a genome wide gene expression analysis on primary endothelial cells infected with KSHV wildtype (KSHVwt) and a KSHV K15 deletion mutant (KSHVΔK15). We found RCAN1/DSCR1 (Regulator of Calcineurin 1/Down Syndrome critical region 1), a cellular gene involved in angiogenesis, to be differentially expressed in KSHVwt- vs KSHVΔK15-infected cells. During physiological angiogenesis, expression of RCAN1 in endothelial cells is regulated by VEGF (vascular endothelial growth factor) through a pathway involving the activation of PLCγ1, Calcineurin and NFAT1. We found that K15 directly recruits PLCγ1, and thereby activates Calcineurin/NFAT1-dependent RCAN1 expression which results in the formation of angiogenic tubes. Primary endothelial cells infected with KSHVwt form angiogenic tubes upon activation of the lytic replication cycle. This effect is abrogated when K15 is deleted (KSHVΔK15) or silenced by an siRNA targeting the K15 expression. Our study establishes K15 as one of the KSHV proteins that contribute to KSHV-induced angiogenesis.
Assuntos
Herpesvirus Humano 8/metabolismo , Células Endoteliais da Veia Umbilical Humana/virologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Musculares/metabolismo , Neovascularização Patológica/virologia , Fosfolipase C gama/metabolismo , Proteínas Virais/metabolismo , Indutores da Angiogênese , Animais , Calcineurina/metabolismo , Linhagem Celular , Chlorocebus aethiops , Proteínas de Ligação a DNA , Células HEK293 , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/crescimento & desenvolvimento , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Dados de Sequência Molecular , Proteínas Musculares/genética , Fatores de Transcrição NFATC/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Sarcoma de Kaposi/virologia , Deleção de Sequência , Células Vero , Proteínas Virais/genéticaRESUMO
Persistent virus infection continuously produces non-self nucleic acids that activate cell-intrinsic immune responses. However, the antiviral defense evolved as a transient, acute phase response and the effects of persistently ongoing stimulation onto cellular homeostasis are not well understood. To study the consequences of long-term innate immune activation, we expressed the NS5B polymerase of Hepatitis C virus (HCV), which in absence of viral genomes continuously produces immune-stimulatory RNAs. Surprisingly, within 3 weeks, NS5B expression declined and the innate immune response ceased. Proteomics and functional analyses indicated a reduced proliferation of those cells most strongly stimulated, which was independent of interferon signaling but required mitochondrial antiviral signaling protein (MAVS) and interferon regulatory factor 3 (IRF3). Depletion of MAVS or IRF3, or overexpression of the MAVS-inactivating HCV NS3/4A protease not only blocked interferon responses but also restored cell growth in NS5B expressing cells. However, pan-caspase inhibition could not rescue the NS5B-induced cytostasis. Our results underline an active counter selection of cells with prolonged innate immune activation, which likely constitutes a cellular strategy to prevent persistent virus infections.
Assuntos
Portador Sadio/imunologia , Caspases/imunologia , Hepatite C/imunologia , Imunidade Inata , Fator Regulador 3 de Interferon/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Portador Sadio/virologia , Caspases/genética , Hepacivirus/genética , Hepacivirus/fisiologia , Hepatite C/genética , Hepatite C/virologia , Humanos , Fator Regulador 3 de Interferon/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
The flow of genetic information from DNA to protein requires polymerase-II-transcribed RNA characterized by the presence of a 5'-cap. The cap-binding complex (CBC), consisting of the nuclear cap-binding protein (NCBP) 2 and its adaptor NCBP1, is believed to bind all capped RNA and to be necessary for its processing and intracellular localization. Here we show that NCBP1, but not NCBP2, is required for cell viability and poly(A) RNA export. We identify C17orf85 (here named NCBP3) as a cap-binding protein that together with NCBP1 forms an alternative CBC in higher eukaryotes. NCBP3 binds mRNA, associates with components of the mRNA processing machinery and contributes to poly(A) RNA export. Loss of NCBP3 can be compensated by NCBP2 under steady-state conditions. However, NCBP3 becomes pivotal under stress conditions, such as virus infection. We propose the existence of an alternative CBC involving NCBP1 and NCBP3 that plays a key role in mRNA biogenesis.