RESUMO
Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded, RNA-dependent RNA polymerase in replication and transcription of the viral RNA genome in the cytoplasm of infected cells. The contribution of cellular proteins to these processes remains unclear. Here, we applied proximity proteomics to define the interactome of LASV polymerase in cells under conditions that recreate LASV RNA synthesis. We engineered a LASV polymerase-biotin ligase (TurboID) fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed the identification of 42 high-confidence LASV polymerase interactors. We subsequently performed a small interfering RNA (siRNA) screen to identify those interactors that have functional roles in authentic LASV infection. As proof of principle, we characterized eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we found to be a proviral factor that physically associates with LASV polymerase. Targeted degradation of GSPT1 by a small-molecule drug candidate, CC-90009, resulted in strong inhibition of LASV infection in cultured cells. Our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet-to-be-defined host-pathogen interactome, which can reveal new biology and uncover novel targets for the development of antivirals against highly pathogenic RNA viruses.
Assuntos
Acetamidas , Antivirais , Isoindóis , Vírus Lassa , Fatores de Terminação de Peptídeos , Piperidonas , RNA Polimerase Dependente de RNA , Proteínas Virais , Acetamidas/farmacologia , Acetamidas/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Linhagem Celular Tumoral , Humanos , Isoindóis/farmacologia , Isoindóis/uso terapêutico , Febre Lassa/tratamento farmacológico , Vírus Lassa/efeitos dos fármacos , Fatores de Terminação de Peptídeos/metabolismo , Piperidonas/metabolismo , Piperidonas/farmacologia , Piperidonas/uso terapêutico , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteoma , Proteômica , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismoRESUMO
Background: Ebola virus (EBOV) infection causes severe hemorrhagic fever. EBOV transcription is controlled by host protein phosphatase 1 (PP1), which dephosphorylates VP30 protein. We previously developed 1E7-03, a compound targeting a noncatalytic site of PP1 that induced VP30 phosphorylation and inhibited EBOV transcription. Here, we attempted to further improve 1E7-03, which was not stable in murine serum. Results: High-throughput screening with EBOV-green fluorescent protein was conducted on 72 1E7-03 analogs and identified 6 best inhibitory and the least toxic compounds. A parallel in silico screening of compounds from the ZINC database by docking to PP1 identified the best-binding compound C31, which was also present among the top 6 compounds found in the viral screen. C31 showed the best EBOV inhibitory activity among the top 6 compounds and also inhibited EBOV minigenome. C31 bound to the PP1 C-terminal groove in vitro and increased VP30 phosphorylation in cultured cells. C31 demonstrated improved stability in mouse plasma and cell permeability, compared with 1E7-03. It was also detected for 24 hours after injection in mice. Conclusion: C31 represents a novel PP1-targeting EBOV inhibitor with improved pharmacological properties that can be further evaluated for future antifiloviral therapy.
Assuntos
Antivirais/farmacologia , Ebolavirus/efeitos dos fármacos , Proteína Fosfatase 1/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Domínio Catalítico , Estabilidade de Medicamentos , Ebolavirus/fisiologia , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Fosforilação , Proteína Fosfatase 1/química , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismoRESUMO
Ebola virus (EBOV) infections are characterized by deficient T lymphocyte responses, T lymphocyte apoptosis, and lymphopenia in the absence of direct infection of T lymphocytes. In contrast, dendritic cells (DC) are infected but fail to mature appropriately, thereby impairing the T cell response. We investigated the contributions of EBOV proteins in modulating DC maturation by generating recombinant viruses expressing enhanced green fluorescent protein and carrying mutations affecting several potentially immunomodulating domains. They included envelope glycoprotein (GP) domains, as well as innate response antagonist domains (IRADs) previously identified in the VP24 and VP35 proteins. GP expressed by an unrelated vector, but not the wild-type EBOV, was found to strongly induce DC maturation, and infections with recombinant EBOV carrying mutations disabling GP functional domains did not restore DC maturation. In contrast, each of the viruses carrying mutations disabling any IRAD in VP35 induced a dramatic upregulation of DC maturation markers. This was dependent on infection, but not interaction with GP. Disabling of IRADs also resulted in up to a several hundredfold increase in secretion of cytokines and chemokines. Furthermore, these mutations induced formation of homotypic DC clusters, which represent close correlates of their maturation and presumably facilitate transfer of antigen from migratory DC to lymph node DC. Thus, an individual IRAD is insufficient to suppress DC maturation; rather, the suppression of DC maturation and the "immune paralysis" observed during EBOV infections results from a cooperative effect of two or more individual IRADs.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/virologia , Ebolavirus/genética , Proteínas do Envelope Viral/imunologia , Animais , Biomarcadores/metabolismo , Western Blotting , Chlorocebus aethiops , Citocinas/metabolismo , Células Dendríticas/citologia , Ebolavirus/metabolismo , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Mutação/genética , Células Vero , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
The first-ever recent Marburg virus (MARV) outbreak in Ghana, West Africa and Equatorial Guinea has refocused efforts towards the development of therapeutics since no vaccine or treatment has been approved. mRNA vaccines were proven successful in a pandemic-response to severe acute respiratory syndrome coronavirus-2, making it an appealing vaccine platform to target highly pathogenic emerging viruses. Here, 1-methyl-pseudouridine-modified mRNA vaccines formulated in lipid nanoparticles (LNP) were developed against MARV and the closely-related Ravn virus (RAVV), which were based on sequences of the glycoproteins (GP) of the two viruses. Vaccination of guinea pigs with both vaccines elicited robust binding and neutralizing antibodies and conferred complete protection against virus replication, disease and death. The study characterized antibody responses to identify disparities in the binding and functional profiles between the two viruses and regions in GP that are broadly reactive. For the first time, the glycan cap is highlighted as an immunoreactive site for marburgviruses, inducing both binding and neutralizing antibody responses that are dependent on the virus. Profiling the antibody responses against the two viruses provided an insight into how antigenic differences may affect the response towards conserved GP regions which would otherwise be predicted to be cross-reactive and has implications for the future design of broadly protective vaccines. The results support the use of mRNA-LNPs against pathogens of high consequence.
RESUMO
Ebola virus (EBOV) and Bundibugyo virus (BDBV) belong to the family Filoviridae and cause a severe disease in humans. We previously isolated a large panel of monoclonal antibodies from B cells of human survivors from the 2007 Uganda BDBV outbreak, 16 survivors from the 2014 EBOV outbreak in the Democratic Republic of the Congo, and one survivor from the West African 2013-2016 EBOV epidemic. Here, we demonstrate that EBOV and BDBV are capable of spreading to neighboring cells through intercellular connections in a process that depends upon actin and T cell immunoglobulin and mucin 1 protein. We quantify spread through intercellular connections by immunofluorescence microscopy and flow cytometry. One of the antibodies, BDBV223, specific to the membrane-proximal external region, induces virus accumulation at the plasma membrane. The inhibiting activity of BDBV223 depends on BST2/tetherin.
Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Antígeno 2 do Estroma da Médula Óssea , Ebolavirus , Doença pelo Vírus Ebola , Humanos , Antígenos CD , Antígeno 2 do Estroma da Médula Óssea/imunologia , Ebolavirus/imunologia , Proteínas Ligadas por GPI , Doença pelo Vírus Ebola/virologiaRESUMO
The Ebola virus (EBOV) transcriptional regulation involves host protein phosphatases PP1 and PP2A, which dephosphorylate the transcriptional cofactor of EBOV polymerase VP30. The 1E7-03 compound, which targets PP1, induces VP30 phosphorylation and inhibits EBOV infection. This study aimed to investigate the role of PP1 in EBOV replication. When EBOV-infected cells were continuously treated with 1E7-03, the NP E619K mutation was selected. This mutation moderately reduced EBOV minigenome transcription, which was restored by the treatment with 1E7-03. Formation of EBOV capsids, when NP was co-expressed with VP24 and VP35, was impaired with NPE 619K. Treatment with 1E7-03 restored capsid formation by NP E619K mutation, but inhibited capsids formed by WT NP. The dimerization of NP E619K, tested in a split NanoBiT assay, was significantly decreased (~ 15-fold) compared to WT NP. NP E619K bound more efficiently to PP1 (~ 3-fold) but not B56 subunit of PP2A or VP30. Cross-linking and co-immunoprecipitation experiments showed fewer monomers and dimers for NP E619K which were increased with 1E7-03 treatment. NP E619K showed increased co-localization with PP1α compared to WT NP. Mutations of potential PP1 binding sites and NP deletions disrupted its interaction with PP1. Collectively, our findings suggest that PP1 binding to the NP regulates NP dimerization and capsid formation, and that NP E619K mutation, which has the enhanced PP1 binding, disrupts these processes. Our results point to a new role for PP1 in EBOV replication in which NP binding to PP1 may facilitate viral transcription by delaying capsid formation and EBOV replication.
RESUMO
Intranasal vaccination represents a promising approach for preventing disease caused by respiratory pathogens by eliciting a mucosal immune response in the respiratory tract that may act as an early barrier to infection and transmission. This study investigated immunogenicity and protective efficacy of intranasally administered messenger RNA (mRNA)-lipid nanoparticle (LNP) encapsulated vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Syrian golden hamsters. Intranasal mRNA-LNP vaccination systemically induced spike-specific binding [immunoglobulin G (IgG) and IgA] and neutralizing antibodies. Intranasally vaccinated hamsters also had decreased viral loads in the respiratory tract, reduced lung pathology, and prevented weight loss after SARS-CoV-2 challenge. Together, this study demonstrates successful immunogenicity and protection against respiratory viral infection by an intranasally administered mRNA-LNP vaccine.
Assuntos
COVID-19 , Animais , Cricetinae , COVID-19/prevenção & controle , SARS-CoV-2 , Vacinação , Anticorpos Neutralizantes , RNA Mensageiro/genéticaRESUMO
Ebola virus (EBOV) critically depends on the viral polymerase to replicate and transcribe the viral RNA genome in the cytoplasm of host cells, where cellular factors can antagonize or facilitate the virus life cycle. Here we leverage proximity proteomics and conduct a small interfering RNA (siRNA) screen to define the functional interactome of EBOV polymerase. As a proof of principle, we validate two cellular mRNA decay factors from 35 identified host factors: eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1) and up-frameshift protein 1 (UPF1). Our data suggest that EBOV can subvert restrictions of cellular mRNA decay and repurpose GSPT1 and UPF1 to promote viral replication. Treating EBOV-infected human hepatocytes with a drug candidate that targets GSPT1 for degradation significantly reduces viral RNA load and particle production. Our work demonstrates the utility of proximity proteomics to capture the functional host interactome of the EBOV polymerase and to illuminate host-dependent regulation of viral RNA synthesis.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Ebolavirus/genética , Interações Hospedeiro-Patógeno , Humanos , Proteômica , RNA Helicases/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , Transativadores , Replicação ViralRESUMO
Although substantial progress has been made with Ebola virus (EBOV) vaccine measures, the immune correlates of vaccine-mediated protection remain uncertain. Here, five mucosal vaccine vectors based on human and avian paramyxoviruses provided nonhuman primates with varying degrees of protection, despite expressing the same EBOV glycoprotein (GP) immunogen. Each vaccine produced antibody responses that differed in Fc-mediated functions and isotype composition, as well as in magnitude and coverage toward GP and its conformational and linear epitopes. Differences in the degree of protection and comprehensive characterization of the response afforded the opportunity to identify which features and functions were elevated in survivors and could therefore serve as vaccine correlates of protection. Pairwise network correlation analysis of 139 immune- and vaccine-related parameters was performed to demonstrate relationships with survival. Total GP-specific antibodies, as measured by biolayer interferometry, but not neutralizing IgG or IgA titers, correlated with survival. Fc-mediated functions and the amount of receptor binding domain antibodies were associated with improved survival outcomes, alluding to the protective mechanisms of these vaccines. Therefore, functional qualities of the antibody response, particularly Fc-mediated effects and GP specificity, rather than simply magnitude of the response, appear central to vaccine-induced protection against EBOV. The heterogeneity of the response profile between the vaccines indicates that each vaccine likely exhibits its own protective signature and the requirements for an efficacious EBOV vaccine are complex.
Assuntos
Vacinas contra Ebola , Ebolavirus , Doença pelo Vírus Ebola , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Especificidade de Anticorpos , Doença pelo Vírus Ebola/prevenção & controle , Humanos , PrimatasRESUMO
The mRNA-1273 vaccine is effective against SARS-CoV-2 and was granted emergency use authorization by the FDA. Clinical studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibits severe SARS-CoV-2 disease similar to that in hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and nonhuman primates, low-level virus replication in mRNA-1273-vaccinated hamsters coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high-resolution analysis that is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock-vaccinated hamsters. Our results support the use of mRNA-1273 in a 2-dose schedule and provide insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2.
Assuntos
Vacinas contra COVID-19/farmacologia , COVID-19/imunologia , COVID-19/prevenção & controle , Pulmão/imunologia , SARS-CoV-2 , Vacina de mRNA-1273 contra 2019-nCoV , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Imunização Secundária , Pulmão/patologia , Pulmão/virologia , Ativação Linfocitária , Mesocricetus , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Análise de Célula Única , Replicação ViralRESUMO
We investigated serological responses following a SARS-CoV-2 outbreak in spring 2020 on a US Marine recruit training base. 147 participants that were isolated during an outbreak of respiratory illness were enrolled in this study, with visits approximately 6 and 10 weeks post-outbreak (PO). This cohort is comprised of young healthy adults, ages 18-26, with a high rate of asymptomatic infection or mild symptoms, and therefore differs from previously reported longitudinal studies on humoral responses to SARS-CoV-2, which often focus on more diverse age populations and worse clinical presentation. 80.9% (119/147) of the participants presented with circulating IgG antibodies against SARS-CoV-2 spike (S) receptor-binding domain (RBD) at 6 weeks PO, of whom 97.3% (111/114) remained positive, with significantly decreased levels, at 10 weeks PO. Neutralizing activity was detected in all sera from SARS-CoV-2 IgG positive participants tested (n=38) at 6 and 10 weeks PO, without significant loss between time points. IgG and IgA antibodies against SARS-CoV-2 RBD, S1, S2, and the nucleocapsid (N) protein, as well neutralization activity, were generally comparable between those participants that had asymptomatic infection or mild disease. A multiplex assay including S proteins from SARS-CoV-2 and related zoonotic and human endemic betacoronaviruses revealed a positive correlation for polyclonal cross-reactivity to S after SARS-CoV-2 infection. Overall, young adults that experienced asymptomatic or mild SARS-CoV-2 infection developed comparable humoral responses, with no decrease in neutralizing activity at least up to 10 weeks after infection.
Assuntos
Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , COVID-19/imunologia , Militares , SARS-CoV-2/fisiologia , Adolescente , Adulto , Formação de Anticorpos , Doenças Assintomáticas , Estudos de Coortes , Surtos de Doenças , Progressão da Doença , Feminino , Humanos , Masculino , Glicoproteína da Espícula de Coronavírus/imunologia , Estados Unidos/epidemiologia , Adulto JovemRESUMO
The mRNA-1273 vaccine was recently determined to be effective against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from interim Phase 3 results. Human studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibit more severe SARS-CoV-2 disease similar to hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and non-human primates, mRNA-1273- mediated immunity was non-sterilizing and coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high resolution analysis which is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock-vaccinated hamsters. Our results support the use of mRNA-1273 in a two-dose schedule and provides insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2.
RESUMO
Marburg virus (MARV) causes severe disease in humans and is known to activate nuclear factor erythroid 2-related factor 2 (Nrf2), the major transcription factor of the antioxidant response. Canonical activation of Nrf2 involves oxidative or electrophilic stress that prevents Kelch-like ECH-associated protein 1 (Keap1) targeted degradation of Nrf2, leading to Nrf2 stabilization and activation of the antioxidant response. MARV activation of Nrf2 is noncanonical with the MARV VP24 protein (mVP24) interacting with Keap1, freeing Nrf2 from degradation. A high-throughput screening (HTS) assay was developed to identify inhibitors of mVP24-induced Nrf2 activity and used to screen more than 55,000 compounds. Hit compounds were further screened against secondary HTS assays for the inhibition of antioxidant activity induced by additional canonical and noncanonical mechanisms. This pipeline identified 14 compounds that suppress the response, dependent on the inducer, with 50% inhibitory concentrations below 5 µM and selectivity index values greater than 10. Notably, several of the identified compounds specifically inhibit mVP24-induced Nrf2 activity.
Assuntos
Expressão Gênica/efeitos dos fármacos , Marburgvirus/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Oxirredução/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Antioxidantes , Regulação da Expressão Gênica , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Fator 2 Relacionado a NF-E2/genética , Ligação Proteica , Proteínas Virais/metabolismoRESUMO
Based on genome-scale loss-of-function screens we discovered that Topoisomerase III-ß (TOP3B), a human topoisomerase that acts on DNA and RNA, is required for yellow fever virus and dengue virus-2 replication. Remarkably, we found that TOP3B is required for efficient replication of all positive-sense-single stranded RNA viruses tested, including SARS-CoV-2. While there are no drugs that specifically inhibit this topoisomerase, we posit that TOP3B is an attractive anti-viral target.
RESUMO
Based on genome-scale loss-of-function screens we discovered that Topoisomerase III-ß (TOP3B), a human topoisomerase that acts on DNA and RNA, is required for yellow fever virus and dengue virus-2 replication. Remarkably, we found that TOP3B is required for efficient replication of all positive-sense-single stranded RNA viruses tested, including SARS-CoV-2. While there are no drugs that specifically inhibit this topoisomerase, we posit that TOP3B is an attractive anti-viral target.
Assuntos
Betacoronavirus/fisiologia , DNA Topoisomerases Tipo I/metabolismo , Vírus de RNA/metabolismo , Replicação Viral/fisiologia , Linhagem Celular , Vírus da Dengue/fisiologia , Ebolavirus/fisiologia , Técnicas de Inativação de Genes , Humanos , Vírus da Influenza A/fisiologia , SARS-CoV-2 , Vírus da Febre Amarela/fisiologia , Zika virus/fisiologiaRESUMO
Marburg virus (MARV) and Ebola virus (EBOV) belong to the family Filoviridae. MARV causes severe disease in humans with high fatality. We previously isolated a large panel of monoclonal antibodies (mAbs) from B cells of a human survivor with previous naturally acquired MARV infection. Here, we characterized functional properties of these mAbs and identified non-neutralizing mAbs targeting the glycoprotein (GP) 2 portion of the mucin-like domain (MLD) of MARV GP, termed the wing region. One mAb targeting the GP2 wing, MR228, showed therapeutic protection in mice and guinea pigs infected with MARV. The protection was mediated by the Fc fragment functions of MR228. Binding of another GP2 wing-specific non-neutralizing mAb, MR235, to MARV GP increased accessibility of epitopes in the receptor-binding site (RBS) for neutralizing mAbs, resulting in enhanced virus neutralization by these mAbs. These findings highlight an important role for non-neutralizing mAbs during natural human MARV infection.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Doença do Vírus de Marburg/imunologia , Marburgvirus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linfócitos B , Chlorocebus aethiops , Modelos Animais de Doenças , Ebolavirus/imunologia , Epitopos/imunologia , Feminino , Glicoproteínas/imunologia , Cobaias , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Sobreviventes , Células THP-1 , Células Vero , Proteínas do Envelope Viral/imunologiaRESUMO
Ebola virus (EBOV) is a non-segmented negative-sense RNA virus that causes a severe human disease. The ongoing EBOV outbreak in the Eastern part of Democratic Republic of the Congo has resulted to date in over 2500 confirmed cases including over 1500 deaths. Difficulties with vaccine administration indicate the necessity for development of new general drugs and therapeutic strategies against EBOV. Host Ser/Thr protein phosphatases, particularly PP1 and PP2A, facilitate EBOV transcription by dephosphorylating the EBOV VP30 protein and switching activity of the polymerase complex toward replication. Previously, we developed small molecule 1E7-03 that targeted host protein phosphatase-1 (PP1) and induces phosphorylation of EBOV VP30 protein thus shifting transcription-replication balance and inhibiting EBOV replication. Here, we developed a new EBOV inhibitor, 1E7-07, that potently inhibits EBOV replication and displays significantly improved metabolic stability when compared to previously described 1E7-03. Proteome analysis of VP30 shows that 1E7-07 increases its phosphorylation on Thr-119 and Ser-124 over 3-fold with p < 0.001, which likely contributes to EBOV inhibition. We analyzed 1E7-07 binding to PP1 using a mass spectrometry-based protein painting approach. Combined with computational docking, protein painting shows that 1E7-07 binds to several PP1 sites including the RVxF site, C-terminal groove and NIPP1-helix binding pocket. Further analysis using surface plasmon resonance and a split NanoBiT system demonstrates that 1E7-07 binds primarily to the RVxF site. Together, detailed analysis of 1E7-07 binding to PP1 and identification of the RVxF site as the main binding site opens up an opportunity for future development of PP1-targeting EBOV inhibitors.
RESUMO
Specific host pathways that may be targeted therapeutically to inhibit the replication of Ebola virus (EBOV) and other emerging viruses remain incompletely defined. A screen of 200,000 compounds for inhibition of an EBOV minigenome (MG) assay that measures the function of the viral polymerase complex identified as hits several compounds with an amino-tetrahydrocarbazole scaffold. This scaffold was structurally similar to GSK983, a compound previously described as having broad-spectrum antiviral activity due to its impairing de novo pyrimidine biosynthesis through inhibition of dihydroorotate dehydrogenase (DHODH). We generated compound SW835, the racemic version of GSK983 and demonstrated that SW835 and brequinar, another DHODH inhibitor, potently inhibit the MG assay and the replication of EBOV, vesicular stomatitis virus (VSV) and Zika (ZIKV) in vitro. Nucleoside and deoxynucleoside supplementation studies demonstrated that depletion of pyrimidine pools contributes to antiviral activity of these compounds. As reported for other DHODH inhibitors, SW835 and brequinar also induced expression of interferon stimulated genes (ISGs). ISG induction was demonstrated to occur without production of IFNα/ß and independently of the IFNα receptor and was not blocked by EBOV-encoded suppressors of IFN signaling pathways. Furthermore, we demonstrated that transcription factor IRF1 is required for this ISG induction, and that IRF1 induction requires the DNA damage response kinase ATM. Therefore, de novo pyrimidine biosynthesis is critical for the replication of EBOV and other RNA viruses and inhibition of this pathway activates an ATM and IRF1-dependent innate immune response that subverts EBOV immune evasion functions.
Assuntos
Ebolavirus/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Nucleosídeos/farmacologia , Pirimidinas/antagonistas & inibidores , Pirimidinas/biossíntese , Replicação Viral/efeitos dos fármacos , Células A549 , Antivirais/farmacologia , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Carbazóis/química , Carbazóis/farmacologia , Dano ao DNA , Di-Hidro-Orotato Desidrogenase , Células HEK293 , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Evasão da Resposta Imune , Imunidade Inata/genética , Fator Regulador 1 de Interferon/metabolismo , Fator Regulador 1 de Interferon/farmacologia , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Vírus de RNA/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Vesiculovirus/efeitos dos fármacos , Zika virus/efeitos dos fármacosRESUMO
Ebola virus (EBOV) genome and mRNAs contain long, structured regions that could hijack host RNA-binding proteins to facilitate infection. We performed RNA affinity chromatography coupled with mass spectrometry to identify host proteins that bind to EBOV RNAs and identified four high-confidence proviral host factors, including Staufen1 (STAU1), which specifically binds both 3' and 5' extracistronic regions of the EBOV genome. We confirmed that EBOV infection rate and production of infectious particles were significantly reduced in STAU1-depleted cells. STAU1 was recruited to sites of EBOV RNA synthesis upon infection and enhanced viral RNA synthesis. Furthermore, STAU1 interacts with EBOV nucleoprotein (NP), virion protein 30 (VP30), and VP35; the latter two bridge the viral polymerase to the NP-coated genome, forming the viral ribonucleoprotein (RNP) complex. Our data indicate that STAU1 plays a critical role in EBOV replication by coordinating interactions between the viral genome and RNA synthesis machinery.IMPORTANCE Ebola virus (EBOV) is a negative-strand RNA virus with significant public health importance. Currently, no therapeutics are available for Ebola, which imposes an urgent need for a better understanding of EBOV biology. Here we dissected the virus-host interplay between EBOV and host RNA-binding proteins. We identified novel EBOV host factors, including Staufen1, which interacts with multiple viral factors and is required for efficient viral RNA synthesis.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Ebolavirus/genética , Interações Hospedeiro-Patógeno , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Linhagem Celular , Proteínas do Citoesqueleto/genética , Genoma Viral , Humanos , Ligação Proteica , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
Ebola virus (EBOV) is an enveloped negative-sense, single-stranded RNA virus of the filovirus family that causes severe disease in humans. Approved therapies for EBOV disease are lacking. EBOV RNA synthesis is carried out by a virus-encoded complex with RNA-dependent RNA polymerase activity that is required for viral propagation. This complex and its activities are therefore potential antiviral targets. To identify potential lead inhibitors of EBOV RNA synthesis, a library of small molecule compounds was screened against a previously established assay of EBOV RNA synthesis, the EBOV minigenome assay (MGA), in 384 well microplate format. The screen identified 56 hits that inhibited EBOV MGA activity by more than 70% while exhibiting less than 20% cell cytotoxicity. Inhibitory chemical scaffolds included angelicin derivatives, derivatives of the antiviral compound GSK983 and benzoquinolines. Structure-activity relationship (SAR) studies of the benzoquinoline scaffold produced â¼50 analogs and led to identification of an optimized compound, SW456, with a submicromolar IC50 in the EBOV MGA and antiviral activity against infectious EBOV in cell culture. The compound was also active against a MGA for another deadly filovirus, Marburg virus. It also exhibited antiviral activity towards a negative-sense RNA virus from the rhabdovirus family, vesicular stomatitis virus, and a positive-sense RNA virus, Zika virus. Overall, these data demonstrate the potential of the EBOV MGA to identify anti-EBOV compounds and identifies the benzoquinoline series as a broad-spectrum antiviral lead.