RESUMO
Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5' end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5' ends of positive- and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.
Assuntos
COVID-19 , RNA Viral , Humanos , COVID-19/metabolismo , Endonucleases/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Replicação ViralRESUMO
Interferon-stimulated genes (ISGs) form the backbone of the innate immune system and are important for limiting intra- and intercellular viral replication and spread. We conducted a mass-spectrometry-based survey to understand the fundamental organization of the innate immune system and to explore the molecular functions of individual ISGs. We identified interactions between 104 ISGs and 1,401 cellular binding partners engaging in 2,734 high-confidence interactions. 90% of these interactions are unreported so far, and our survey therefore illuminates a far wider activity spectrum of ISGs than is currently known. Integration of the resulting ISG-interaction network with published datasets and functional studies allowed us to identify regulators of immunity and processes related to the immune system. Given the extraordinary robustness of the innate immune system, this ISG network may serve as a blueprint for therapeutic targeting of cellular systems to efficiently fight viral infections.
Assuntos
Imunidade Inata , Interferons/fisiologia , Mapeamento de Interação de Proteínas , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Expressão Gênica , Glicoproteínas/metabolismo , Células HEK293 , Células HeLa , Humanos , Imunidade Inata/genética , Espectrometria de Massas , Receptores CCR4/metabolismo , Receptores de Peptídeos/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismoRESUMO
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 immune system is in place to assist in ensuring tissue homeostasis, which can be easily perturbed by invading pathogens or nonpathogenic stressors causing tissue damage. Extracellular nucleotides are well known to contribute to innate immune signaling specificity and strength, but how their signaling is relayed downstream of cell surface receptors and how this translates into antiviral immunity is only partially understood. Here, we systematically investigated the responses of human macrophages to extracellular nucleotides, focusing on the nucleotide-sensing GPRC receptors of the P2Y family. Time-resolved transcriptomic analysis showed that adenine- and uridine-based nucleotides induce a specific, immediate, and transient cytokine response through the MAPK signaling pathway that regulates transcriptional activation by AP-1. Using receptor trans-complementation, we identified a subset of P2Ys (P2Y1, P2Y2, P2Y6, and P2Y11) that govern inflammatory responses via cytokine induction, while others (P2Y4, P2Y11, P2Y12, P2Y13, and P2Y14) directly induce antiviral responses. Notably, P2Y11 combined both activities, and depletion or inhibition of this receptor in macrophages impaired both inflammatory and antiviral responses. Collectively, these results highlight the underappreciated functions of P2Y receptors in innate immune processes.
Assuntos
Nucleotídeos , Transdução de Sinais , Humanos , Citocinas , Imunidade , Macrófagos/metabolismo , Nucleotídeos/metabolismo , Replicação ViralRESUMO
In mammals, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide 2'3'-cGAMP in response to cytosolic DNA and this triggers an antiviral immune response. cGAS belongs to a large family of cGAS/DncV-like nucleotidyltransferases that is present in both prokaryotes1 and eukaryotes2-5. In bacteria, these enzymes synthesize a range of cyclic oligonucleotides and have recently emerged as important regulators of phage infections6-8. Here we identify two cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster. We show that cGLR1 and cGLR2 activate Sting- and NF-κB-dependent antiviral immunity in response to infection with RNA or DNA viruses. cGLR1 is activated by double-stranded RNA to produce the cyclic dinucleotide 3'2'-cGAMP, whereas cGLR2 produces a combination of 2'3'-cGAMP and 3'2'-cGAMP in response to an as-yet-unidentified stimulus. Our data establish cGAS as the founding member of a family of receptors that sense different types of nucleic acids and trigger immunity through the production of cyclic dinucleotides beyond 2'3'-cGAMP.
Assuntos
Drosophila melanogaster/imunologia , Nucleotidiltransferases/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Vírus/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/virologia , Feminino , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Ligantes , Masculino , Proteínas de Membrana/metabolismo , Modelos Moleculares , NF-kappa B/metabolismo , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/classificação , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/metabolismo , RNA de Cadeia Dupla/análise , RNA de Cadeia Dupla/imunologia , RNA de Cadeia Dupla/metabolismo , Receptores de Reconhecimento de Padrão/classificação , Receptores de Reconhecimento de Padrão/deficiência , Receptores de Reconhecimento de Padrão/imunologiaRESUMO
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
The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Animais , Antivirais/farmacologia , Inflamação/tratamento farmacológico , Metiltransferases/metabolismo , Camundongos , Capuzes de RNA/metabolismo , RNA Viral/genética , Ribose , Proteínas não Estruturais Virais/genéticaRESUMO
RNA vaccines elicit protective immunity against SARS-CoV-2, but the use of mRNA as an antiviral immunotherapeutic is unexplored. Here, we investigate the activity of lipidoid nanoparticle (LNP)-formulated mRNA encoding human IFNλ1 (ETH47), which is a critical driver of innate immunity at mucosal surfaces protecting from viral infections. IFNλ1 mRNA administration promotes dose-dependent protein translation, induction of interferon-stimulated genes without relevant signs of unspecific immune stimulation, and dose-dependent inhibition of SARS-CoV-2 replication in vitro. Pulmonary administration of IFNλ1 mRNA in mice results in a potent reduction of virus load, virus-induced body weight loss and significantly increased survival. These data support the development of inhaled administration of IFNλ1 mRNA as a potential prophylactic option for individuals exposed to SARS-CoV-2 or at risk suffering from COVID-19. Based on the broad antiviral activity of IFNλ1 regardless of virus or variant, this approach might also be utilized for other respiratory viral infections or pandemic preparedness.
RESUMO
RIG-I is a key mediator of antiviral immunity, able to couple detection of infection by RNA viruses to the induction of interferons. Natural RIG-I stimulatory RNAs have variously been proposed to correspond to virus genomes, virus replication intermediates, viral transcripts, or self-RNA cleaved by RNase L. However, the relative contribution of each of these RNA species to RIG-I activation and interferon induction in virus-infected cells is not known. Here, we use three approaches to identify physiological RIG-I agonists in cells infected with influenza A virus or Sendai virus. We show that RIG-I agonists are exclusively generated by the process of virus replication and correspond to full-length virus genomes. Therefore, nongenomic viral transcripts, short replication intermediates, and cleaved self-RNA do not contribute substantially to interferon induction in cells infected with these negative strand RNA viruses. Rather, single-stranded RNA viral genomes bearing 5'-triphosphates constitute the natural RIG-I agonists that trigger cell-intrinsic innate immune responses during infection.
Assuntos
RNA Helicases DEAD-box/imunologia , Proteínas de Membrana/imunologia , Proteínas do Tecido Nervoso/imunologia , Infecções por Vírus de RNA/imunologia , RNA Viral/imunologia , Animais , Linhagem Celular , Proteína DEAD-box 58 , Cães , Humanos , Interferons/imunologia , Camundongos , Vírus de RNA/fisiologia , Receptores de Superfície Celular , Receptores Imunológicos , Replicação ViralRESUMO
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
Molluscum contagiosum virus (MCV) is a human-adapted poxvirus that causes a common and persistent yet mild infection characterized by distinct, contagious, papular skin lesions. These lesions are notable for having little or no inflammation associated with them and can persist for long periods without an effective clearance response from the host. Like all poxviruses, MCV encodes potent immunosuppressive proteins that perturb innate immune pathways involved in virus sensing, the interferon response, and inflammation, which collectively orchestrate antiviral immunity and clearance, with several of these pathways converging at common signaling nodes. One such node is the regulator of canonical nuclear factor kappa B (NF-κB) activation, NF-κB essential modulator (NEMO). Here, we report that the MCV protein MC008 specifically inhibits NF-κB through its interaction with NEMO, disrupting its early ubiquitin-mediated activation and subsequent downstream signaling. MC008 is the third NEMO-targeting inhibitor to be described in MCV to date, with each inhibiting NEMO activation in distinct ways, highlighting strong selective pressure to evolve multiple ways of disabling this key signaling protein. IMPORTANCE Inflammation lies at the heart of most human diseases. Understanding the pathways that drive this response is the key to new anti-inflammatory therapies. Viruses evolve to target inflammation; thus, understanding how they do this reveals how inflammation is controlled and, potentially, how to disable it when it drives disease. Molluscum contagiosum virus (MCV) has specifically evolved to infect humans and displays an unprecedented ability to suppress inflammation in our tissue. We have identified a novel inhibitor of human innate signaling from MCV, MC008, which targets NEMO, a core regulator of proinflammatory signaling. Furthermore, MC008 appears to inhibit early ubiquitination, thus interrupting later events in NEMO activation, thereby validating current models of IκB kinase (IKK) complex regulation.
Assuntos
Vírus do Molusco Contagioso , NF-kappa B , Humanos , NF-kappa B/metabolismo , Vírus do Molusco Contagioso/metabolismo , Proteínas Virais/metabolismo , Transdução de Sinais , Ubiquitinação , Quinase I-kappa B/metabolismoRESUMO
The severe-acute-respiratory-syndrome-coronavirus-2 (SARS-CoV-2) is the causative agent of COVID-19, but host cell factors contributing to COVID-19 pathogenesis remain only partly understood. We identify the host metalloprotease ADAM17 as a facilitator of SARS-CoV-2 cell entry and the metalloprotease ADAM10 as a host factor required for lung cell syncytia formation, a hallmark of COVID-19 pathology. ADAM10 and ADAM17, which are broadly expressed in the human lung, cleave the SARS-CoV-2 spike protein (S) in vitro, indicating that ADAM10 and ADAM17 contribute to the priming of S, an essential step for viral entry and cell fusion. ADAM protease-targeted inhibitors severely impair lung cell infection by the SARS-CoV-2 variants of concern alpha, beta, delta, and omicron and also reduce SARS-CoV-2 infection of primary human lung cells in a TMPRSS2 protease-independent manner. Our study establishes ADAM10 and ADAM17 as host cell factors for viral entry and syncytia formation and defines both proteases as potential targets for antiviral drug development.
Assuntos
COVID-19 , SARS-CoV-2 , Proteína ADAM10/genética , Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide/genética , Enzima de Conversão de Angiotensina 2 , Fusão Celular , Humanos , Pulmão , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloproteases , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do VírusRESUMO
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
A promising approach to tackle the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. So far it is unclear, which viral replication steps can be efficiently inhibited with siRNAs. Here, we report that siRNAs can target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, and thereby terminate replication before start of transcription and prevent virus-induced cell death. Coronaviruses replicate via negative sense RNA intermediates using a unique discontinuous transcription process. As a result, each viral RNA contains identical sequences at the 5' and 3' end. Surprisingly, siRNAs were not active against intermediate negative sense transcripts. Targeting common sequences shared by all viral transcripts allowed simultaneous suppression of gRNA and subgenomic (sg)RNAs by a single siRNA. The most effective suppression of viral replication and spread, however, was achieved by siRNAs that targeted open reading frame 1 (ORF1) which only exists in gRNA. In contrast, siRNAs that targeted the common regions of transcripts were outcompeted by the highly abundant sgRNAs leading to an impaired antiviral efficacy. Verifying the translational relevance of these findings, we show that a chemically modified siRNA that targets a highly conserved region of ORF1, inhibited SARS-CoV-2 replication ex vivo in explants of the human lung. Our work encourages the development of siRNA-based therapies for COVID-19 and suggests that early therapy start, or prophylactic application, together with specifically targeting gRNA, might be key for high antiviral efficacy.
Assuntos
COVID-19/virologia , Pulmão/virologia , RNA Interferente Pequeno , RNA Viral , SARS-CoV-2/genética , Replicação Viral , Regiões 3' não Traduzidas , Animais , Antivirais/farmacologia , Sobrevivência Celular , Bases de Dados Genéticas , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Oligonucleotídeos , Fases de Leitura Aberta , RNA Interferente Pequeno/metabolismo , Tratamento Farmacológico da COVID-19RESUMO
A notable signalling mechanism employed by mammalian innate immune signalling pathways uses nucleotide-based second messengers such as 2'3'-cGAMP and 2'-5'-oligoadenylates (OAs), which bind and activate STING and RNase L, respectively. Interestingly, the involvement of nucleotide second messengers to activate antiviral responses is evolutionarily conserved, as evidenced by the identification of an antiviral cGAMP-dependent pathway in Drosophila. Using a mass spectrometry approach, we identified several members of the ABCF family in human, mouse and Drosophila cell lysates as 2'-5' OA-binding proteins, suggesting an evolutionarily conserved function. Biochemical characterization of these interactions demonstrates high-affinity binding of 2'-5' OA to ABCF1, dependent on phosphorylated 2'-5' OA and an intact Walker A/B motif of the ABC cassette of ABCF1. As further support for species-specific interactions with 2'-5' OA, we additionally identified that the metabolic enzyme Decr1 from mouse, but not human or Drosophila cells, forms a high-affinity complex with 2'-5' OA. A 1.4 Å co-crystal structure of the mouse Decr1-2'-5' OA complex explains high-affinity recognition of 2'-5' OA and the mechanism of species specificity. Despite clear evidence of physical interactions, we could not identify profound antiviral functions of ABCF1, ABCF3 or Decr1 or 2'-5' OA-dependent regulation of cellular translation rates, as suggested by the engagement of ABCF proteins. Thus, although the biological consequences of the here identified interactions need to be further studied, our data suggest that 2'-5' OA can serve as a signalling hub to distribute a signal to different recipient proteins.
Assuntos
Antivirais , Drosophila , Animais , Camundongos , Nucleotídeos , MamíferosRESUMO
Antiviral innate immunity relies on the recognition of microbial structures. One such structure is viral RNA that carries a triphosphate group on its 5' terminus (PPP-RNA). By an affinity proteomics approach with PPP-RNA as the 'bait', we found that the antiviral protein IFIT1 (interferon-induced protein with tetratricopeptide repeats 1) mediated binding of a larger protein complex containing other IFIT family members. IFIT1 bound PPP-RNA with nanomolar affinity and required the arginine at position 187 in a highly charged carboxy-terminal groove of the protein. In the absence of IFIT1, the growth and pathogenicity of viruses containing PPP-RNA was much greater. In contrast, IFIT proteins were dispensable for the clearance of pathogens that did not generate PPP-RNA. On the basis of this specificity and the great abundance of IFIT proteins after infection, we propose that the IFIT complex antagonizes viruses by sequestering specific viral nucleic acids.
Assuntos
Arginina/imunologia , Proteínas de Transporte/imunologia , RNA Viral/imunologia , Vírus/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Arginina/química , Arginina/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Feminino , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNARESUMO
Hepatitis B virus (HBV) persists by depositing a covalently closed circular DNA (cccDNA) in the nucleus of infected cells that cannot be targeted by available antivirals. Interferons can diminish HBV cccDNA via APOBEC3-mediated deamination. Here, we show that overexpression of APOBEC3A alone is not sufficient to reduce HBV cccDNA that requires additional treatment of cells with interferon indicating involvement of an interferon-stimulated gene (ISG) in cccDNA degradation. Transcriptome analyses identify ISG20 as the only type I and II interferon-induced, nuclear protein with annotated nuclease activity. ISG20 localizes to nucleoli of interferon-stimulated hepatocytes and is enriched on deoxyuridine-containing single-stranded DNA that mimics transcriptionally active, APOBEC3A-deaminated HBV DNA. ISG20 expression is detected in human livers in acute, self-limiting but not in chronic hepatitis B. ISG20 depletion mitigates the interferon-induced loss of cccDNA, and co-expression with APOBEC3A is sufficient to diminish cccDNA. In conclusion, non-cytolytic HBV cccDNA decline requires the concerted action of a deaminase and a nuclease. Our findings highlight that ISGs may cooperate in their antiviral activity that may be explored for therapeutic targeting.
Assuntos
DNA Circular , Vírus da Hepatite B , Antivirais/farmacologia , Citidina Desaminase , DNA Circular/genética , DNA Viral/genética , DNA Viral/farmacologia , Exorribonucleases , Vírus da Hepatite B/genética , Humanos , Interferons , Proteínas , Replicação ViralRESUMO
The SARS-CoV-2 pandemic imposed a large burden on health and society. Therapeutics targeting different components and processes of the viral infection replication cycle are being investigated, particularly to repurpose already approved drugs. Spike protein is an important target for both vaccines and therapeutics. Insights into the mechanisms of spike-ACE2 binding and cell fusion could support the identification of compounds with inhibitory effects. Here, we demonstrate that the integrity of disulfide bonds within the receptor-binding domain (RBD) plays an important role in the membrane fusion process although their disruption does not prevent binding of spike protein to ACE2. Several reducing agents and thiol-reactive compounds are able to inhibit viral entry. N-acetyl cysteine amide, L-ascorbic acid, JTT-705, and auranofin prevented syncytia formation, viral entry into cells, and infection in a mouse model, supporting disulfides of the RBD as a therapeutically relevant target.
Assuntos
Acetilcisteína/análogos & derivados , Amidas/farmacologia , Ácido Ascórbico/farmacologia , Auranofina/farmacologia , Tratamento Farmacológico da COVID-19 , COVID-19 , Dissulfetos/metabolismo , Ésteres/farmacologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Compostos de Sulfidrila/farmacologia , Internalização do Vírus/efeitos dos fármacos , Acetilcisteína/farmacologia , COVID-19/metabolismo , COVID-19/patologia , Células HEK293 , HumanosRESUMO
As the COVID-19 pandemic continues to spread, thousands of scientists around the globe have changed research direction to understand better how the virus works and to find out how it may be tackled. The number of manuscripts on preprint servers is soaring and peer-reviewed publications using MS-based proteomics are beginning to emerge. To facilitate proteomic research on SARS-CoV-2, the virus that causes COVID-19, this report presents deep-scale proteomes (10,000 proteins; >130,000 peptides) of common cell line models, notably Vero E6, Calu-3, Caco-2, and ACE2-A549 that characterize their protein expression profiles including viral entry factors such as ACE2 or TMPRSS2. Using the 9 kDa protein SRP9 and the breast cancer oncogene BRCA1 as examples, we show how the proteome expression data can be used to refine the annotation of protein-coding regions of the African green monkey and the Vero cell line genomes. Monitoring changes of the proteome on viral infection revealed widespread expression changes including transcriptional regulators, protease inhibitors, and proteins involved in innate immunity. Based on a library of 98 stable-isotope labeled synthetic peptides representing 11 SARS-CoV-2 proteins, we developed PRM (parallel reaction monitoring) assays for nano-flow and micro-flow LC-MS/MS. We assessed the merits of these PRM assays using supernatants of virus-infected Vero E6 cells and challenged the assays by analyzing two diagnostic cohorts of 24 (+30) SARS-CoV-2 positive and 28 (+9) negative cases. In light of the results obtained and including recent publications or manuscripts on preprint servers, we critically discuss the merits of MS-based proteomics for SARS-CoV-2 research and testing.