RESUMO
The transmembrane serine protease 2 (TMPRSS2) activates the outer structural proteins of a number of respiratory viruses including influenza A virus (IAV), parainfluenza viruses, and various coronaviruses for membrane fusion. Previous studies showed that TMPRSS2 interacts with the carboxypeptidase angiotensin-converting enzyme 2 (ACE2), a cell surface protein that serves as an entry receptor for some coronaviruses. Here, by using protease activity assays, we determine that ACE2 increases the enzymatic activity of TMPRSS2 in a non-catalytic manner. Furthermore, we demonstrate that ACE2 knockdown inhibits TMPRSS2-mediated cleavage of IAV hemagglutinin (HA) in Calu-3 human airway cells and suppresses virus titers 100- to 1.000-fold. Transient expression of ACE2 in ACE2-deficient cells increased TMPRSS2-mediated HA cleavage and IAV replication. ACE2 knockdown also reduced titers of MERS-CoV and prevented S cleavage by TMPRSS2 in Calu-3 cells. By contrast, proteolytic activation and multicycle replication of IAV with multibasic HA cleavage site typically cleaved by furin were not affected by ACE2 knockdown. Co-immunoprecipitation analysis revealed that ACE2-TMPRSS2 interaction requires the enzymatic activity of TMPRSS2 and the carboxypeptidase domain of ACE2. Together, our data identify ACE2 as a new co-factor or stabilizer of TMPRSS2 activity and as a novel host cell factor involved in proteolytic activation and spread of IAV in human airway cells. Furthermore, our data indicate that ACE2 is involved in the TMPRSS2-catalyzed activation of additional respiratory viruses including MERS-CoV.IMPORTANCEProteolytic cleavage of viral envelope proteins by host cell proteases is essential for the infectivity of many viruses and relevant proteases provide promising drug targets. The transmembrane serine protease 2 (TMPRSS2) has been identified as a major activating protease of several respiratory viruses, including influenza A virus. TMPRSS2 was previously shown to interact with angiotensin-converting enzyme 2 (ACE2). Here, we report the mechanistic details of this interaction. We demonstrate that ACE2 increases or stabilizes the enzymatic activity of TMPRSS2. Furthermore, we describe ACE2 involvement in TMPRSS2-catalyzed cleavage of the influenza A virus hemagglutinin and MERS-CoV spike protein in human airway cells. These findings expand our knowledge of the activation of respiratory viruses by TMPRSS2 and the host cell factors involved. In addition, our results could help to elucidate a physiological role for TMPRSS2.
Assuntos
Enzima de Conversão de Angiotensina 2 , Vírus da Influenza A , Pulmão , Proteólise , Serina Endopeptidases , Animais , Cães , Humanos , Enzima de Conversão de Angiotensina 2/deficiência , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Biocatálise , Linhagem Celular , Furina/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/metabolismo , Pulmão/citologia , Pulmão/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Ligação Proteica , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus , Replicação ViralRESUMO
Science is humanity's best insurance against threats from nature, but it is a fragile enterprise that must be nourished and protected. The preponderance of scientific evidence indicates a natural origin for SARS-CoV-2. Yet, the theory that SARS-CoV-2 was engineered in and escaped from a lab dominates media attention, even in the absence of strong evidence. We discuss how the resulting anti-science movement puts the research community, scientific research, and pandemic preparedness at risk.
RESUMO
Type I interferons (IFN-I) are essential antiviral cytokines produced upon microbial infection. IFN-I elicits this activity through the upregulation of hundreds of IFN-I-stimulated genes (ISGs). The full breadth of ISG induction demands activation of a number of cellular factors including the IκB kinase epsilon (IKKε). However, the mechanism of IKKε activation upon IFN receptor signaling has remained elusive. Here we show that TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family of proteins, interacted with IKKε and promoted induction of IKKε-dependent ISGs. TRIM6 and the E2-ubiquitin conjugase UbE2K cooperated in the synthesis of unanchored K48-linked polyubiquitin chains, which activated IKKε for subsequent STAT1 phosphorylation. Our work attributes a previously unrecognized activating role of K48-linked unanchored polyubiquitin chains in kinase activation and identifies the UbE2K-TRIM6-ubiquitin axis as critical for IFN signaling and antiviral response.
Assuntos
Quinase I-kappa B/imunologia , Interferon Tipo I/imunologia , Poliubiquitina/biossíntese , Ubiquitina-Proteína Ligases/imunologia , Animais , Antivirais , Células Cultivadas , Ativação Enzimática/imunologia , Humanos , Janus Quinase 1 , Camundongos , Fosforilação/imunologia , Interferência de RNA , RNA Interferente Pequeno , Fator de Transcrição STAT1/imunologia , Transdução de Sinais/imunologia , Proteínas com Motivo Tripartido , Enzimas de Conjugação de Ubiquitina/imunologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
Cleavage of the influenza A virus (IAV) hemagglutinin (HA) by host proteases is indispensable for virus replication. Most IAVs possess a monobasic HA cleavage site cleaved by trypsin-like proteases. Previously, the transmembrane protease TMPRSS2 was shown to be essential for proteolytic activation of IAV HA subtypes H1, H2, H7, and H10 in mice. In contrast, additional proteases are involved in activation of certain H3 IAVs, indicating that HAs with monobasic cleavage sites can differ in their sensitivity to host proteases. Here, we investigated the role of TMPRSS2 in proteolytic activation of avian HA subtypes H1 to H11 and H14 to H16 in human and mouse airway cell cultures. Using reassortant viruses carrying representative HAs, we analyzed HA cleavage and multicycle replication in (i) lung cells of TMPRSS2-deficient mice and (ii) Calu-3 cells and primary human bronchial cells subjected to morpholino oligomer-mediated knockdown of TMPRSS2 activity. TMPRSS2 was found to be crucial for activation of H1 to H11, H14, and H15 in airway cells of human and mouse. Only H9 with an R-S-S-R cleavage site and H16 were proteolytically activated in the absence of TMPRSS2 activity, albeit with reduced efficiency. Moreover, a TMPRSS2-orthologous protease from duck supported activation of H1 to H11, H15, and H16 in MDCK cells. Together, our data demonstrate that in human and murine respiratory cells, TMPRSS2 is the major activating protease of almost all IAV HA subtypes with monobasic cleavage sites. Furthermore, our results suggest that TMPRSS2 supports activation of IAV with a monobasic cleavage site in ducks. IMPORTANCE Human infections with avian influenza A viruses upon exposure to infected birds are frequently reported and have received attention as a potential pandemic threat. Cleavage of the envelope glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. In this study, we identify the transmembrane protease TMPRSS2 as the major activating protease of avian influenza virus HAs of subtypes H1 to H11, H14 and H15 in human and murine airway cells. Our data demonstrate that inhibition of TMPRSS2 activity may provide a useful approach for the treatment of human infections with avian influenza viruses that should be considered for pandemic preparedness as well. Additionally, we show that a TMPRSS2-orthologous protease from duck can activate avian influenza virus HAs with a monobasic cleavage site and, thus, represents a potential virus-activating protease in waterfowl, the primary reservoir for influenza A viruses.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/metabolismo , Serina Endopeptidases/metabolismo , Animais , Brônquios/citologia , Linhagem Celular , Cães , Feminino , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Vírus da Influenza A/imunologia , Vírus da Influenza A/patogenicidade , Pulmão/virologia , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeo Hidrolases/metabolismo , Proteólise , Mucosa Respiratória/metabolismo , Serina Endopeptidases/fisiologia , Replicação ViralRESUMO
BACKGROUND: As the causative agent of COVID-19, SARS-CoV-2 is a pathogen of immense importance to global public health. Development of innovative direct-acting antiviral agents is sorely needed to address this virus. Peptide-conjugated morpholino oligomers (PPMO) are antisense compounds composed of a phosphorodiamidate morpholino oligomer covalently conjugated to a cell-penetrating peptide. PPMO require no delivery assistance to enter cells and are able to reduce expression of targeted RNA through sequence-specific steric blocking. METHODS: Five PPMO designed against sequences of genomic RNA in the SARS-CoV-2 5'-untranslated region and a negative control PPMO of random sequence were synthesized. Each PPMO was evaluated for its effect on the viability of uninfected cells and its inhibitory effect on the replication of SARS-CoV-2 in Vero-E6 cell cultures. Cell viability was evaluated with an ATP-based method using a 48 h PPMO treatment time. Viral growth was measured with quantitative RT-PCR and TCID50 infectivity assays from experiments where cells received a 5 h PPMO treatment time. RESULTS: PPMO designed to base-pair with sequence in the 5' terminal region or the leader transcription regulatory sequence region of SARS-CoV-2 genomic RNA were highly efficacious, reducing viral titres by up to 4-6 log10 in cell cultures at 48-72 h post-infection, in a non-toxic and dose-responsive manner. CONCLUSIONS: The data indicate that PPMO have the ability to potently and specifically suppress SARS-CoV-2 growth and are promising candidates for further preclinical development.
Assuntos
Antivirais/farmacologia , COVID-19/virologia , Peptídeos Penetradores de Células/farmacologia , Morfolinos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Chlorocebus aethiops , Efeito Citopatogênico Viral/efeitos dos fármacos , Morfolinos/química , SARS-CoV-2/genética , Células VeroRESUMO
Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is essential for virus infectivity and spread. We previously demonstrated in vitro that the transmembrane protease TMPRSS2 cleaves influenza A virus (IAV) and influenza B virus (IBV) HA possessing a monobasic cleavage site. Subsequent studies revealed that TMPRSS2 is crucial for the activation and pathogenesis of H1N1pdm and H7N9 IAV in mice. In contrast, activation of H3N2 IAV and IBV was found to be independent of TMPRSS2 expression and supported by an as-yet-undetermined protease(s). Here, we investigated the role of TMPRSS2 in proteolytic activation of IAV and IBV in three human airway cell culture systems: primary human bronchial epithelial cells (HBEC), primary type II alveolar epithelial cells (AECII), and Calu-3 cells. Knockdown of TMPRSS2 expression was performed using a previously described antisense peptide-conjugated phosphorodiamidate morpholino oligomer, T-ex5, that interferes with splicing of TMPRSS2 pre-mRNA, resulting in the expression of enzymatically inactive TMPRSS2. T-ex5 treatment produced efficient knockdown of active TMPRSS2 in all three airway cell culture models and prevented proteolytic activation and multiplication of H7N9 IAV in Calu-3 cells and H1N1pdm, H7N9, and H3N2 IAV in HBEC and AECII. T-ex5 treatment also inhibited the activation and spread of IBV in AECII but did not affect IBV activation in HBEC and Calu-3 cells. This study identifies TMPRSS2 as the major HA-activating protease of IAV in human airway cells and IBV in type II pneumocytes and as a potential target for the development of novel drugs to treat influenza infections.IMPORTANCE Influenza A viruses (IAV) and influenza B viruses (IBV) cause significant morbidity and mortality during seasonal outbreaks. Cleavage of the viral surface glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. Inhibition of relevant proteases provides a promising therapeutic approach that may avoid the development of drug resistance. HA of most influenza viruses is cleaved at a monobasic cleavage site, and a number of proteases have been shown to cleave HA in vitro This study demonstrates that the transmembrane protease TMPRSS2 is the major HA-activating protease of IAV in primary human bronchial cells and of both IAV and IBV in primary human type II pneumocytes. It further reveals that human and murine airway cells can differ in their HA-cleaving protease repertoires. Our data will help drive the development of potent and selective protease inhibitors as novel drugs for influenza treatment.
Assuntos
Vírus da Influenza A/fisiologia , Vírus da Influenza B/fisiologia , Influenza Humana/virologia , Serina Endopeptidases/metabolismo , Animais , Brônquios/citologia , Células Cultivadas , Células Epiteliais/virologia , Técnicas de Silenciamento de Genes , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Influenza Humana/enzimologia , Influenza Humana/metabolismo , Camundongos , Infecções por Orthomyxoviridae/enzimologia , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Alvéolos Pulmonares/citologia , Serina Endopeptidases/genética , Regulação para Cima , Replicação ViralRESUMO
BACKGROUND: Continuous administration of levodopa-carbidopa intestinal gel (carbidopa-levodopa enteral suspension) through a percutaneous endoscopic gastrojejunostomy is a treatment option for advanced Parkinson disease (PD) patients with motor fluctuations resistant to standard oral medications. Safety data from 4 prospective studies were integrated to assess the safety of this therapy. METHODS: Safety data from 4 studies were summarized using 2 overlapping data sets, permitting the separation of procedure/device-associated (n = 395) from non-procedure/device adverse events (n = 412). RESULTS: At the data cutoff, median exposure to levodopa-carbidopa intestinal gel was 911 days (range, 1-1980 days) with 963 total patient-years of exposure. Procedure/device adverse events occurred in 300 patients (76%), and serious adverse events occurred in 68 (17%); most frequently reported procedure/device adverse events and serious adverse events were complications of device insertion (41% and 8%, respectively) and abdominal pain (36% and 4%, respectively). Non-procedure/device adverse events occurred in 92% (379), with most frequently reported being insomnia (23%) and falls (23%); 42% (171) had non-procedure/device serious adverse events, with most frequently reported being pneumonia (5%) and PD symptoms (2%). Adverse events led to discontinuation in 17% (72), most frequently because of complication of device insertion (2.4%). There were 34 treatment-emergent deaths (8.3%) in the overlapping data sets, 2 of which (0.5%) were considered "possibly related" to the treatment system. CONCLUSION: In the largest collection of levodopa-carbidopa intestinal gel safety data from prospective clinical studies, procedure/device events were frequently reported and occasionally life threatening. Most non-procedure/device events were typical for levodopa treatment and an elderly population. These factors combined with high treatment efficacy led to a relatively low discontinuation rate in advanced PD patients.
Assuntos
Antiparkinsonianos/efeitos adversos , Carbidopa/efeitos adversos , Ensaios Clínicos Fase III como Assunto/estatística & dados numéricos , Derivação Gástrica/efeitos adversos , Infusões Parenterais/efeitos adversos , Levodopa/efeitos adversos , Avaliação de Resultados em Cuidados de Saúde/estatística & dados numéricos , Doença de Parkinson/tratamento farmacológico , Idoso , Antiparkinsonianos/administração & dosagem , Carbidopa/administração & dosagem , Combinação de Medicamentos , Feminino , Géis , Humanos , Levodopa/administração & dosagem , Masculino , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto/estatística & dados numéricos , Estudos ProspectivosRESUMO
UNLABELLED: Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions that cause significant morbidity and mortality worldwide. To date, no vaccines or antiviral therapeutics have been approved for combating DENV-associated disease. In this paper, we describe a class of tricyclic small-molecule compounds-dihydrodibenzothiepines (DHBTs), identified through high-throughput screening-with potent inhibitory activity against DENV serotype 2. SKI-417616, a highly active representative of this class, displayed activity against all four serotypes of DENV, as well as against a related flavivirus, West Nile virus (WNV), and an alphavirus, Sindbis virus (SINV). This compound was characterized to determine its mechanism of antiviral activity. Investigation of the stage of the viral life cycle affected revealed that an early event in the life cycle is inhibited. Due to the structural similarity of the DHBTs to known antagonists of the dopamine and serotonin receptors, we explored the roles of two of these receptors, serotonin receptor 2A (5HTR2A) and the D4 dopamine receptor (DRD4), in DENV infection. Antagonism of DRD4 and subsequent downstream phosphorylation of epidermal growth factor receptor (EGFR)-related kinase (ERK) were found to impact DENV infection negatively, and blockade of signaling through this network was confirmed as the mechanism of anti-DENV activity for this class of compounds. IMPORTANCE: The dengue viruses are mosquito-borne, reemerging human pathogens that are the etiological agents of a spectrum of febrile diseases. Currently, there are no approved therapeutic treatments for dengue-associated disease, nor is there a vaccine. This study identifies a small molecule, SKI-417616, with potent anti-dengue virus activity. Further analysis revealed that SKI-417616 acts through antagonism of the host cell dopamine D4 receptor and subsequent repression of the ERK phosphorylation pathway. These results suggest that SKI-417616, or other compounds targeting the same cellular pathways, may have therapeutic potential for the treatment of dengue virus infections.
Assuntos
Antivirais/metabolismo , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores de Dopamina D4/antagonistas & inibidores , Transdução de Sinais , Replicação Viral/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Sindbis virus/efeitos dos fármacos , Sindbis virus/fisiologia , Vírus do Nilo Ocidental/efeitos dos fármacos , Vírus do Nilo Ocidental/fisiologiaRESUMO
Dengue virus has emerged as a global health threat to over one-third of humankind. As a positive-strand RNA virus, dengue virus relies on the host cell metabolism for its translation, replication, and egress. Therefore, a better understanding of the host cell metabolic pathways required for dengue virus infection offers the opportunity to develop new approaches for therapeutic intervention. In a recently described screen of known drugs and bioactive molecules, we observed that methotrexate and floxuridine inhibited dengue virus infections at low micromolar concentrations. Here, we demonstrate that all serotypes of dengue virus, as well as West Nile virus, are highly sensitive to both methotrexate and floxuridine, whereas other RNA viruses (Sindbis virus and vesicular stomatitis virus) are not. Interestingly, flavivirus replication was restored by folinic acid, a thymidine precursor, in the presence of methotrexate and by thymidine in the presence of floxuridine, suggesting an unexpected role for thymidine in flavivirus replication. Since thymidine is not incorporated into RNA genomes, it is likely that increased thymidine production is indirectly involved in flavivirus replication. A possible mechanism is suggested by the finding that p53 inhibition restored dengue virus replication in the presence of floxuridine, consistent with thymidine-less stress triggering p53-mediated antiflavivirus effects in infected cells. Our data reveal thymidine synthesis pathways as new and unexpected therapeutic targets for antiflaviviral drug development.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/metabolismo , Flavivirus/efeitos dos fármacos , Flavivirus/metabolismo , Timidina/biossíntese , Animais , Linhagem Celular , Chlorocebus aethiops , Vírus de DNA/efeitos dos fármacos , Vírus da Dengue/fisiologia , Modelos Animais de Doenças , Flavivirus/fisiologia , Infecções por Flavivirus/tratamento farmacológico , Floxuridina/farmacologia , Células HEK293 , Células HeLa , Humanos , Leucovorina/farmacologia , Metotrexato/farmacologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Vírus de RNA/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos , Vírus do Nilo Ocidental/efeitos dos fármacos , Vírus do Nilo Ocidental/metabolismo , Vírus do Nilo Ocidental/fisiologiaRESUMO
Influenza A viruses constitute a major and ongoing global public health concern. Current antiviral strategies target viral gene products; however, the emergence of drug-resistant viruses highlights the need for novel antiviral approaches. Cleavage of the influenza virus hemagglutinin (HA) by host cell proteases is crucial for viral infectivity and therefore presents a potential drug target. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded-DNA-like antisense agents that readily enter cells and can act as antisense agents by sterically blocking cRNA. Here, we evaluated the effect of PPMO targeted to regions of the pre-mRNA or mRNA of the HA-cleaving protease TMPRSS2 on proteolytic activation and spread of influenza viruses in human Calu-3 airway epithelial cells. We found that treatment of cells with a PPMO (T-ex5) designed to interfere with TMPRSS2 pre-mRNA splicing resulted in TMPRSS2 mRNA lacking exon 5 and consequently the expression of a truncated and enzymatically inactive form of TMPRSS2. Altered splicing of TMPRSS2 mRNA by the T-ex5 PPMO prevented HA cleavage in different human seasonal and pandemic influenza A viruses and suppressed viral titers by 2 to 3 log(10) units, strongly suggesting that TMPRSS2 is responsible for HA cleavage in Calu-3 airway cells. The data indicate that PPMO provide a useful reagent for investigating HA-activating proteases and may represent a promising strategy for the development of novel therapeutics to address influenza infections.
Assuntos
Células Epiteliais/virologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/patogenicidade , Metaloendopeptidases/metabolismo , Morfolinas/farmacologia , Serina Endopeptidases/metabolismo , Animais , Brônquios/citologia , Linhagem Celular , Células Cultivadas , Embrião de Galinha , Cães , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/enzimologia , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/enzimologia , Vírus da Influenza A Subtipo H7N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H7N1/enzimologia , Vírus da Influenza A/enzimologia , Morfolinos , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The dengue viruses (DENVs) exist as numerous genetic strains that are grouped into four antigenically distinct serotypes. DENV strains from each serotype can cause severe disease and threaten public health in tropical and subtropical regions worldwide. No licensed antiviral agent to treat DENV infections is currently available, and there is an acute need for the development of novel therapeutics. We found that a synthetic small interfering RNA (siRNA) (DC-3) targeting the highly conserved 5' cyclization sequence (5'CS) region of the DENV genome reduced, by more than 100-fold, the titers of representative strains from each DENV serotype in vitro. To determine if DC-3 siRNA could inhibit DENV in vivo, an "in vivo-ready" version of DC-3 was synthesized and tested against DENV-2 by using a mouse model of antibody-dependent enhancement of infection (ADE)-induced disease. Compared with the rapid weight loss and 5-day average survival time of the control groups, mice receiving the DC-3 siRNA had an average survival time of 15 days and showed little weight loss for approximately 12 days. DC-3-treated mice also contained significantly less virus than control groups in several tissues at various time points postinfection. These results suggest that exogenously introduced siRNA combined with the endogenous RNA interference processing machinery has the capacity to prevent severe dengue disease. Overall, the data indicate that DC-3 siRNA represents a useful research reagent and has potential as a novel approach to therapeutic intervention against the genetically diverse dengue viruses.
Assuntos
Antivirais/administração & dosagem , Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Dengue/tratamento farmacológico , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacologia , Animais , Anticorpos Facilitadores , Produtos Biológicos/administração & dosagem , Produtos Biológicos/farmacologia , Peso Corporal , Técnicas de Cultura de Células , Chlorocebus aethiops , Sequência Conservada , Dengue/patologia , Dengue/virologia , Vírus da Dengue/genética , Modelos Animais de Doenças , Humanos , Camundongos , RNA Interferente Pequeno/genética , Doenças dos Roedores/tratamento farmacológico , Doenças dos Roedores/patologia , Doenças dos Roedores/virologia , Análise de SobrevidaRESUMO
Molecular responses to influenza A virus (IAV) infections vary between mammalian species. To identify conserved and species-specific molecular responses, we perform a comparative study of transcriptomic data derived from blood cells, primary epithelial cells, and lung tissues collected from IAV-infected humans, ferrets, and mice. The molecular responses in the human host have unique functions such as antigen processing that are not observed in mice or ferrets. Highly conserved gene coexpression modules across the three species are enriched for IAV infection-induced pathways including cell cycle and interferon (IFN) signaling. TDRD7 is predicted as an IFN-inducible host factor that is up-regulated upon IAV infection in the three species. TDRD7 is required for antiviral IFN response, potentially modulating IFN signaling via the JAK/STAT/IRF9 pathway. Identification of the common and species-specific molecular signatures, networks, and regulators of IAV infection provides insights into host-defense mechanisms and will facilitate the development of novel therapeutic interventions against IAV infection.
Assuntos
Doenças Transmissíveis , Vírus da Influenza A , Influenza Humana , Infecções por Orthomyxoviridae , Animais , Antivirais , Furões/metabolismo , Humanos , Vírus da Influenza A/fisiologia , Influenza Humana/genética , Interferons/metabolismo , Camundongos , Infecções por Orthomyxoviridae/genética , RibonucleoproteínasRESUMO
Members of the Arenaviridae family are a threat to public health and can cause meningitis and hemorrhagic fever, and yet treatment options remain limited by a lack of effective antivirals. In this study, we found that peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) complementary to viral genomic RNA were effective in reducing arenavirus replication in cell cultures and in vivo. PPMO complementary to the Junín virus genome were designed to interfere with viral RNA synthesis or translation or both. However, only PPMO designed to potentially interfere with translation were effective in reducing virus replication. PPMO complementary to sequences that are highly conserved across the arenaviruses and located at the 5' termini of both genomic segments were effective against Junín virus, Tacaribe virus, Pichinde virus, and lymphocytic choriomeningitis virus (LCMV)-infected cell cultures and suppressed viral titers in the livers of LCMV-infected mice. These results suggest that arenavirus 5' genomic termini represent promising targets for pan-arenavirus antiviral therapeutic development.
Assuntos
Antivirais/farmacologia , Arenavirus/efeitos dos fármacos , Morfolinos/farmacologia , Peptídeos/farmacologia , Animais , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/virologia , Arenavirus/genética , Arenavirus/crescimento & desenvolvimento , Arenavirus do Novo Mundo/efeitos dos fármacos , Linhagem Celular , Chlorocebus aethiops , Genoma Viral , Vírus Junin/efeitos dos fármacos , Vírus da Coriomeningite Linfocítica/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Vírus Pichinde/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , RNA Viral/genética , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Cardiomyocyte apoptosis is a hallmark of coxsackievirus B3 (CVB3)-induced myocarditis. We used cardiomyocytes and HeLa cells to explore the cellular response to CVB3 infection, with a focus on pathways leading to apoptosis. CVB3 infection triggered endoplasmic reticulum (ER) stress and differentially regulated the three arms of the unfolded protein response (UPR) initiated by the proximal ER stress sensors ATF6a (activating transcription factor 6a), IRE1-XBP1 (X box binding protein 1), and PERK (PKR-like ER protein kinase). Upon CVB3 infection, glucose-regulated protein 78 expression was upregulated, and in turn ATF6a and XBP1 were activated via protein cleavage and mRNA splicing, respectively. UPR activity was further confirmed by the enhanced expression of UPR target genes ERdj4 and EDEM1. Surprisingly, another UPR-associated gene, p58(IPK), which often is upregulated during infections with other types of viruses, was downregulated at both mRNA and protein levels after CVB3 infection. These findings were observed similarly for uninfected Tet-On HeLa cells induced to overexpress ATF6a or XBP1. In exploring potential connections between the three UPR pathways, we found that the ATF6a-induced downregulation of p58(IPK) was associated with the activation of PKR (PERK) and the phosphorylation of eIF2alpha, suggesting that p58(IPK), a negative regulator of PERK and PKR, mediates cross-talk between the ATF6a/IRE1-XBP1 and PERK arms. Finally, we found that CVB3 infection eventually produced the induction of the proapoptoic transcription factor CHOP and the activation of SREBP1 and caspase-12. Taken together, these data suggest that CVB3 infection activates UPR pathways and induces ER stress-mediated apoptosis through the suppression of P58(IPK) and induction/activation of CHOP, SREBP1, and caspase-12.
Assuntos
Apoptose , Infecções por Coxsackievirus/metabolismo , Regulação para Baixo , Enterovirus/fisiologia , Proteínas de Choque Térmico HSP40/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Fator de Transcrição CHOP/metabolismo , Resposta a Proteínas não Dobradas , Animais , Caspase 12/genética , Caspase 12/metabolismo , Infecções por Coxsackievirus/genética , Infecções por Coxsackievirus/fisiopatologia , Infecções por Coxsackievirus/virologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Choque Térmico HSP40/genética , Células HeLa , Humanos , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/virologia , Fosforilação , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Fator de Transcrição CHOP/genéticaRESUMO
The fate of influenza A virus (IAV) infection in the host cell depends on the balance between cellular defence mechanisms and viral evasion strategies. To illuminate the landscape of IAV cellular restriction, we generated and integrated global genetic loss-of-function screens with transcriptomics and proteomics data. Our multi-omics analysis revealed a subset of both IFN-dependent and independent cellular defence mechanisms that inhibit IAV replication. Amongst these, the autophagy regulator TBC1 domain family member 5 (TBC1D5), which binds Rab7 to enable fusion of autophagosomes and lysosomes, was found to control IAV replication in vitro and in vivo and to promote lysosomal targeting of IAV M2 protein. Notably, IAV M2 was observed to abrogate TBC1D5-Rab7 binding through a physical interaction with TBC1D5 via its cytoplasmic tail. Our results provide evidence for the molecular mechanism utilised by IAV M2 protein to escape lysosomal degradation and traffic to the cell membrane, where it supports IAV budding and growth.
Assuntos
Autofagia , Evasão da Resposta Imune , Vírus da Influenza A/fisiologia , Antivirais/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/patogenicidade , Lisossomos/metabolismo , Ligação Proteica , Proteínas da Matriz Viral/metabolismo , Replicação Viral , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
BACKGROUND: Japanese encephalitis virus (JEV) has a significant impact on public health throughout Asia, and there is a pressing need for development of new therapeutics against it. METHODS: Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are antisense agents that enter cells readily and interfere with gene expression. Four PPMOs, targeting various locations in the JEV genome, were evaluated for antiviral activity against JEV in cultured cells and the mouse model of JEV infection. RESULTS: A PPMO (P10882) targeting the JEV 3' cyclization sequence (3'CSI) had significant antiviral activity in Vero (epithelial), Neuro2A (neuronal) and J774E (macrophage) cells at concentrations that were not cytotoxic. P10882 added before infection suppressed JEV replication to an undetectable level in Vero cells and produced a 93% and 66% reduction in titre in J774E and Neuro2A cells, respectively, when measured at 24 h post-infection. In uninfected cells, fluorescein-labelled PPMOs entered J774E cells most efficiently, followed by Vero and Neuro2A cells. The antiviral effect of P10882 was also demonstrated in vivo, where 60%-80% of 1-week-old mice treated intracerebrally with a 20 mg/kg dose of P10882 every 12 h for 5 days were protected from a lethal dose of JEV and showed an undetectable level of virus in brain tissue at 2 days post-infection. CONCLUSIONS: P10882, which targets sequence that is highly conserved across members of the JEV serocomplex, was previously shown to be effective in a mouse model of West Nile disease, and represents a candidate antiviral agent against members of the JEV serocomplex.
Assuntos
Antivirais/farmacologia , Vírus da Encefalite Japonesa (Espécie)/efeitos dos fármacos , Oligonucleotídeos/farmacologia , Peptídeos/farmacologia , Animais , Antivirais/uso terapêutico , Linhagem Celular , Chlorocebus aethiops , Encefalite Japonesa/tratamento farmacológico , Células Epiteliais/virologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/virologia , Oligonucleotídeos/uso terapêutico , Peptídeos/uso terapêutico , Análise de SobrevidaRESUMO
In this study, infection of 293/ACE2 cells with severe acute respiratory syndrome coronavirus (SARS-CoV) activated several apoptosis-associated events, namely, cleavage of caspase-3, caspase-8, and poly(ADP-ribose) polymerase 1 (PARP), and chromatin condensation and the phosphorylation and hence inactivation of the eukaryotic translation initiation factor 2alpha (eIF2alpha). In addition, two of the three cellular eIF2alpha kinases known to be virus induced, protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK), were activated by SARS-CoV. The third kinase, general control nonderepressible-2 kinase (GCN2), was not activated, but late in infection the level of GCN2 protein was significantly reduced. Reverse transcription-PCR analyses revealed that the reduction of GCN2 protein was not due to decreased transcription or stability of GCN2 mRNA. The specific reduction of PKR protein expression by antisense peptide-conjugated phosphorodiamidate morpholino oligomers strongly reduced cleavage of PARP in infected cells. Surprisingly, the knockdown of PKR neither enhanced SARS-CoV replication nor abrogated SARS-CoV-induced eIF2alpha phosphorylation. Pretreatment of cells with beta interferon prior to SARS-CoV infection led to a significant decrease in PERK activation, eIF2alpha phosphorylation, and SARS-CoV replication. The various effects of beta interferon treatment were found to function independently on the expression of PKR. Our results show that SARS-CoV infection activates PKR and PERK, leading to sustained eIF2alpha phosphorylation. However, virus replication was not impaired by these events, suggesting that SARS-CoV possesses a mechanism to overcome the inhibitory effects of phosphorylated eIF2alpha on viral mRNA translation. Furthermore, our data suggest that viral activation of PKR can lead to apoptosis via a pathway that is independent of eIF2alpha phosphorylation.
Assuntos
Apoptose , Infecções por Coronavirus/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , eIF-2 Quinase/metabolismo , Animais , Chlorocebus aethiops , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Interferon beta/farmacologia , Morfolinas/farmacologia , Morfolinos , Fosforilação , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Células Vero , eIF-2 Quinase/genéticaRESUMO
BACKGROUND: SARS-CoV-2 is the causative agent of COVID-19 and a pathogen of immense global public health importance. Development of innovative direct-acting antiviral agents is sorely needed to address this virus. Peptide-conjugated morpholino oligomers (PPMO) are antisense agents composed of a phosphordiamidate morpholino oligomer covalently conjugated to a cell-penetrating peptide. PPMO require no delivery assistance to enter cells and are able to reduce expression of targeted RNA through sequence-specific steric blocking. OBJECTIVES AND METHODS: Five PPMO designed against sequences of genomic RNA in the SARS-CoV-2 5'-untranslated region and a negative control PPMO of random sequence were synthesized. Each PPMO was evaluated for its effect on the viability of uninfected cells and its inhibitory effect on the replication of SARS-CoV-2 in Vero-E6 cell cultures. Cell viability was evaluated with an ATP-based method and viral growth was measured with quantitative RT-PCR and TCID 50 infectivity assays. RESULTS: PPMO designed to base-pair with sequence in the 5'-terminal region or the leader transcription regulatory sequence-region of SARS-CoV-2 genomic RNA were highly efficacious, reducing viral titers by up to 4-6 log10 in cell cultures at 48-72 hours post-infection, in a non-toxic and dose-responsive manner. CONCLUSION: The data indicate that PPMO have the ability to potently and specifically suppress SARS-CoV-2 growth and are promising candidates for further pre-clinical development.
RESUMO
The novel emerged SARS-CoV-2 has rapidly spread around the world causing acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. For SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study, we show that S can be cleaved by the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2' site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 human airway epithelial cells through antisense-mediated knockdown of TMPRSS2 expression. Furthermore, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851 in human airway cells. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Combining various TMPRSS2 inhibitors with furin inhibitor MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. Therefore, this approach has considerable therapeutic potential for treatment of COVID-19.
Assuntos
Células Epiteliais Alveolares/virologia , Betacoronavirus/fisiologia , Furina/genética , Serina Endopeptidases/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Epiteliais Alveolares/citologia , Animais , Sítios de Ligação , Linhagem Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Proteólise , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Células Vero , Internalização do Vírus , Replicação ViralRESUMO
BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) causes extensive economic losses in the swine industry. Current strategies and vaccines to control the disease are inadequate. We previously demonstrated that peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) could potently inhibit PRRS virus (PRRSV) replication in cell cultures. PPMOs are single-stranded DNA analogues containing a modified backbone and cell-penetrating peptide. PPMOs are nuclease-resistant, water-soluble, can enter cells readily and exhibit highly specific binding to complementary RNA. In this study, we examined PPMO-mediated inhibition of PRRSV replication in a primary culture of porcine pulmonary alveolar macrophages (PAMs). METHODS: PAMs were collected from piglets, pre-incubated in culture and infected with PRRSV. Viability, cytopathic effects, virus yield and apoptosis of PAMs in the presence or absence of a PPMO (5UP2) were examined. The 5UP2 PPMO is complementary to a conserved sequence in the 5'-terminal region of the PRRSV genome. The level of several interferon-associated gene products and activity of caspases were monitored. RESULTS: PRRSV infection induced the activity of caspases-3/7, -8 and -9 significantly. Treatment of PAMs with 5UP2 resulted in protection of the cells from PRRSV-induced cell death for at least 7 days and avoided the activation of the caspases evaluated. 5UP2 treatment of PRRSV-infected PAMs also prevented the vigorous induction of interferon-beta and chemokines observed in infected and mock-treated PAMs. CONCLUSIONS: PPMO-mediated suppression of PRRSV replication in PAMs was associated with a reduction of apoptotic and inflammatory responses. These results provide further rationale for the development of PPMO 5UP2 as an antiviral to control PRRSV infection.