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1.
Nat Immunol ; 20(4): 493-502, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30833792

RESUMO

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/metabolismo
2.
Nature ; 594(7862): 246-252, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33845483

RESUMO

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/metabolismo
3.
EMBO J ; 41(17): e111608, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35833542

RESUMO

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/genética
4.
FASEB J ; 35(6): e21651, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34004056

RESUMO

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 , Humanos
5.
PLoS Pathog ; 15(12): e1008155, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31856218

RESUMO

Cellular response to environmental challenges requires immediate and precise regulation of transcriptional programs. During viral infections, this includes the expression of antiviral genes that are essential to combat the pathogen. Transcribed mRNAs are bound and escorted to the cytoplasm by the cap-binding complex (CBC). We recently identified a protein complex consisting of NCBP1 and NCBP3 that, under physiological conditions, has redundant function to the canonical CBC, consisting of NCBP1 and NCBP2. Here, we provide evidence that NCBP3 is essential to mount a precise and appropriate antiviral response. Ncbp3-deficient cells allow higher virus growth and elicit a reduced antiviral response, a defect happening on post-transcriptional level. Ncbp3-deficient mice suffered from severe lung pathology and increased morbidity after influenza A virus challenge. While NCBP3 appeared to be particularly important during viral infections, it may be more broadly involved to ensure proper protein expression.


Assuntos
Infecções por Orthomyxoviridae/imunologia , Proteínas de Ligação ao Cap de RNA/imunologia , Proteínas de Ligação ao Cap de RNA/metabolismo , Animais , Vírus da Influenza A/imunologia , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/metabolismo , Biossíntese de Proteínas/fisiologia
6.
Nat Commun ; 15(1): 6778, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39117661

RESUMO

Multiple omics analyzes of Vaccinia virus (VACV) infection have defined molecular characteristics of poxvirus biology. However, little is known about the monkeypox (mpox) virus (MPXV) in humans, which has a different disease manifestation despite its high sequence similarity to VACV. Here, we perform an in-depth multi-omics analysis of the transcriptome, proteome, and phosphoproteome signatures of MPXV-infected primary human fibroblasts to gain insights into the virus-host interplay. In addition to expected perturbations of immune-related pathways, we uncover regulation of the HIPPO and TGF-ß pathways. We identify dynamic phosphorylation of both host and viral proteins, which suggests that MAPKs are key regulators of differential phosphorylation in MPXV-infected cells. Among the viral proteins, we find dynamic phosphorylation of H5 that influenced the binding of H5 to dsDNA. Our extensive dataset highlights signaling events and hotspots perturbed by MPXV, extending the current knowledge on poxviruses. We use integrated pathway analysis and drug-target prediction approaches to identify potential drug targets that affect virus growth. Functionally, we exemplify the utility of this approach by identifying inhibitors of MTOR, CHUK/IKBKB, and splicing factor kinases with potent antiviral efficacy against MPXV and VACV.


Assuntos
Fibroblastos , Monkeypox virus , Mpox , Proteínas Virais , Humanos , Monkeypox virus/genética , Fosforilação , Mpox/virologia , Mpox/metabolismo , Fibroblastos/virologia , Fibroblastos/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais/genética , Proteoma/metabolismo , Interações Hospedeiro-Patógeno , Transdução de Sinais , Proteômica/métodos , Transcriptoma , Antivirais/farmacologia , Multiômica
7.
Nat Commun ; 14(1): 4906, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37582777

RESUMO

Changes of mRNA 3'UTRs by alternative polyadenylation (APA) have been associated to numerous pathologies, but the mechanisms and consequences often remain enigmatic. By combining transcriptomics, proteomics and recombinant viruses we show that all tested strains of IAV, including A/PR/8/34(H1N1) (PR8) and A/Cal/07/2009 (H1N1) (Cal09), cause APA. We mapped the effect to the highly conserved glycine residue at position 184 (G184) of the viral non-structural protein 1 (NS1). Unbiased mass spectrometry-based analyses indicate that NS1 causes APA by perturbing the function of CPSF4 and that this function is unrelated to virus-induced transcriptional shutoff. Accordingly, IAV strain PR8, expressing an NS1 variant with weak CPSF binding, does not induce host shutoff but only APA. However, recombinant IAV (PR8) expressing NS1(G184R) lacks binding to CPSF4 and thereby also the ability to cause APA. Functionally, the impaired ability to induce APA leads to an increased inflammatory cytokine production and an attenuated phenotype in a mouse infection model. Investigating diverse viral infection models showed that APA induction is a frequent ability of many pathogens. Collectively, we propose that targeting of the CPSF complex, leading to widespread alternative polyadenylation of host transcripts, constitutes a general immunevasion mechanism employed by a variety of pathogenic viruses.


Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Animais , Camundongos , Vírus da Influenza A/genética , Regiões 3' não Traduzidas/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Poliadenilação , Virulência/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
8.
Acta Chim Slov ; 59(3): 478-83, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24061300

RESUMO

A new mononuclear nickel(II) acetate with 2-pyridineethanol, Ni(ac)2(2-PyEtOH)2 has been prepared. The reaction product is a mixture of two polymorphic forms that crystallize concomitantly: triclinic (PI) and monoclinic (P21/c). Their structures have been determined at 150 K. The molecular structure of the mononuclear complex shows similar geometry in both polymorphic structures but they differ notably in the arrangement of mononuclear entities in space. The crystal densities are significantly different, nevertheless they behave as concomitant polymorphs.

9.
Viruses ; 14(7)2022 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-35891571

RESUMO

Human endogenous retrovirus (HERVs), normally silenced by methylation or mutations, can be reactivated by multiple environmental factors, including infections with exogenous viruses. In this work, we investigated the transcriptional activity of HERVs in human A549 cells infected by two wild-type (PR8M, SC35M) and one mutated (SC35MΔNS1) strains of Influenza A virus (IAVs). We found that the majority of differentially expressed HERVs (DEHERVS) and genes (DEGs) were up-regulated in the infected cells, with the most significantly enriched biological processes associated with the genes differentially expressed exclusively in SC35MΔNS1 being linked to the immune system. Most DEHERVs in PR8M and SC35M are mammalian apparent LTR retrotransposons, while in SC35MΔNS1, more HERV loci from the HERVW9 group were differentially expressed. Furthermore, up-regulated pairs of HERVs and genes in close chromosomal proximity to each other tended to be associated with immune responses, which implies that specific HERV groups might have the potential to trigger specific gene networks and influence host immunological pathways.


Assuntos
Retrovirus Endógenos , Vírus da Influenza A , Animais , Antivirais , Retrovirus Endógenos/genética , Humanos , Sistema Imunitário , Vírus da Influenza A/genética , Mamíferos , Retroelementos
10.
Cell Rep ; 37(2): 109803, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34644581

RESUMO

Human respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.


Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Células Dendríticas/metabolismo , Células Epiteliais/metabolismo , Pulmão/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sincicial Respiratório Humano/metabolismo , Transcrição Gênica , Proteínas não Estruturais Virais/metabolismo , Células A549 , Animais , Sítios de Ligação , Núcleo Celular/virologia , Cromatina/genética , Cromatina/virologia , Células Dendríticas/virologia , Células Epiteliais/virologia , Feminino , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Pulmão/virologia , Complexo Mediador/genética , Complexo Mediador/metabolismo , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/patogenicidade , Proteínas não Estruturais Virais/genética
11.
Vaccines (Basel) ; 9(5)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925446

RESUMO

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and ß-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small ß-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The ß-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the ß-annulus peptide for vaccine design.

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