RESUMEN
This experimental laboratory-based study evaluated two disinfectants' efficacy against replication-competent severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) on three surfaces. Disinfectants were effictive at eliminating the presence, viability, and subsequent replication of SARS-CoV-2 on all surfaces. Although SARS-CoV-2 likely spreads primarily via airborne transmission, layered mitigation should include high-touch surface disinfection.
Asunto(s)
COVID-19 , Desinfectantes , Humanos , SARS-CoV-2 , Desinfectantes/farmacología , COVID-19/prevención & control , DesinfecciónRESUMEN
Bourbon virus (BRBV) is an emerging tick-borne orthomyxovirus that causes severe febrile illness in humans. There are no specific treatments for BRBV disease currently available. Here, we developed a highly accessible and robust, quantitative fluorescence-based BRBV minigenome (MG) system and applied it to high-throughput antiviral drug screening. We demonstrated that human dihydroorotate dehydrogenase (DHODH) inhibitors, hDHODH-IN-4 and brequinar, efficiently reduced BRBV RNA synthesis, and validated these findings using infectious BRBV in vitro. The DHODH inhibitors also exhibited high potency in inhibiting MG activities of other orthomyxoviruses with emerging zoonotic potential, including bat influenza A virus, swine influenza D virus, and Thogoto virus. Our newly developed MG system is a powerful platform for antiviral drug screening across the Orthomyxoviridae family, enabling rapid development and deployment of antivirals against future emerging orthomyxoviruses.
Asunto(s)
Thogotovirus , Garrapatas , Animales , Antivirales/farmacología , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Thogotovirus/genéticaRESUMEN
COVID-19 is a devastating respiratory and inflammatory illness caused by a new coronavirus that is rapidly spreading throughout the human population. Over the past 12 months, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, has already infected over 160 million (>20% located in United States) and killed more than 3.3 million people around the world (>20% deaths in USA). As we face one of the most challenging times in our recent history, there is an urgent need to identify drug candidates that can attack SARS-CoV-2 on multiple fronts. We have therefore initiated a computational dynamics drug pipeline using molecular modeling, structure simulation, docking and machine learning models to predict the inhibitory activity of several million compounds against two essential SARS-CoV-2 viral proteins and their host protein interactors-S/Ace2, Tmprss2, Cathepsins L and K, and Mpro-to prevent binding, membrane fusion and replication of the virus, respectively. All together, we generated an ensemble of structural conformations that increase high-quality docking outcomes to screen over >6 million compounds including all FDA-approved drugs, drugs under clinical trial (>3000) and an additional >30 million selected chemotypes from fragment libraries. Our results yielded an initial set of 350 high-value compounds from both new and FDA-approved compounds that can now be tested experimentally in appropriate biological model systems. We anticipate that our results will initiate screening campaigns and accelerate the discovery of COVID-19 treatments.
Asunto(s)
Antivirales/uso terapéutico , Tratamiento Farmacológico de COVID-19 , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Sitios de Unión , COVID-19/patología , COVID-19/virología , Descubrimiento de Drogas , Reposicionamiento de Medicamentos , Humanos , Aprendizaje Automático , Simulación del Acoplamiento Molecular , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Proteínas del Envoltorio Viral/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV) cause viral hemorrhagic fever-like illnesses in humans due to an aberrant host inflammatory response, which contributes to pathogenesis. Here, we established two separate minigenome (MG) systems based on the M-segment of SFTSV and HRTV. Following characterization of both systems for SFTSV and HRTV, we used them as a platform to screen potential compounds that inhibit viral RNA synthesis. We demonstrated that the NF-κB inhibitor, SC75741, reduces viral RNA synthesis of SFTSV and HRTV using our MG platform and validated these results using infectious SFTSV and HRTV. These results may lead to the use of MG systems as potential screening systems for the identification of antiviral compounds and yield novel insights into host-factors that could play role in bandavirus transcription and replication.
Asunto(s)
Antivirales/aislamiento & purificación , Antivirales/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Phlebovirus/efectos de los fármacos , Garrapatas/virología , Animales , Chlorocebus aethiops , Descubrimiento de Drogas , Genoma Viral , Células HEK293 , Humanos , Concentración 50 Inhibidora , Phlebovirus/clasificación , Phlebovirus/genética , Phlebovirus/patogenicidad , Células THP-1 , Células VeroRESUMEN
Cells require ribonucleotide reductase (RNR) activity for DNA replication. In bacteria, electrons can flow from NADPH to RNR by either a thioredoxin-reductase- or a glutathione-reductase-dependent route. Yeast and plants artificially lacking thioredoxin reductases exhibit a slow-growth phenotype, suggesting glutathione-reductase-dependent routes are poor at supporting DNA replication in these organisms. We have studied proliferation of thioredoxin-reductase-1 (Txnrd1)-deficient hepatocytes in mice. During development and regeneration, normal mice and mice having Txnrd1-deficient hepatocytes exhibited similar liver growth rates. Proportions of hepatocytes that immunostained for PCNA, phosphohistone H3 or incorporated BrdU were also similar, indicating livers of either genotype had similar levels of proliferative, S and M phase hepatocytes, respectively. Replication was blocked by hydroxyurea, confirming that RNR activity was required by Txnrd1-deficient hepatocytes. Regenerative thymidine incorporation was similar in normal and Txnrd1-deficient livers, further indicating that DNA synthesis was unaffected. Using genetic chimeras in which a fluorescently marked subset of hepatocytes was Txnrd1-deficient while others were not, we found that the multigenerational contributions of both hepatocyte types to development and to liver regeneration were indistinguishable. We conclude that, in mouse hepatocytes, a Txnrd1-independent route for the supply of electrons to RNR can fully support DNA replication and normal proliferative growth.
Asunto(s)
Hepatocitos/metabolismo , Hígado/metabolismo , Tiorredoxina Reductasa 1/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , Hepatectomía , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Hidroxiurea/farmacología , Hígado/embriología , Hígado/patología , Hígado/cirugía , Regeneración Hepática , Ratones , Ratones Endogámicos C57BL , Organogénesis/genética , Receptores Nucleares Huérfanos/metabolismo , Eliminación de Secuencia/genética , Tiorredoxina Reductasa 1/genéticaRESUMEN
ICP0 is a regulatory protein that plays a critical role in the replication-latency balance of herpes simplex virus (HSV). Absence of ICP0 renders HSV prone to establish quiescent infections, and thus cellular repressor(s) are believed to silence HSV mRNA synthesis when ICP0 fails to accumulate. To date, an ICP0-antagonized repressor has not been identified that restricts HSV mRNA synthesis by more than 2-fold. We report the unexpected discovery that HSV's major transcriptional regulator, ICP4, meets the criteria of a bona fide ICP0-antagonized repressor of viral mRNA synthesis. Our study began when we noted a repressive activity that restricted ICP0 mRNA synthesis by up to 30-fold in the absence of ICP0. When ICP0 accumulated, the repressor only restricted ICP0 mRNA synthesis by 3-fold. ICP4 proved to be necessary and sufficient to repress ICP0 mRNA synthesis, and did so in an ICP4-binding-site-dependent manner. ICP4 co-immunoprecipitated with FLAG-tagged ICP0; thus, a physical interaction likely explains how ICP0 antagonizes ICP4's capacity to silence the ICP0 gene. These findings suggest that ICP0 mRNA synthesis is differentially regulated in HSV-infected cells by the virus-encoded repressor activity embedded in ICP4, and a virus-encoded antirepressor, ICP0. Bacteriophage lambda relies on a similar repression-antirepression regulatory scheme to "decide" whether a given infection will be productive or silent. Therefore, our findings appear to add to the growing list of inexplicable similarities that point to a common evolutionary ancestry between the herpesviruses and tailed bacteriophage.
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Silenciador del Gen , Herpesvirus Humano 1/genética , Proteínas Inmediatas-Precoces/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Animales , Chlorocebus aethiops , Proteínas Inmediatas-Precoces/genética , ARN Mensajero/genética , Ubiquitina-Proteína Ligasas/genética , Células VeroRESUMEN
The albCre transgene, having Cre recombinase driven by the serum albumin (alb) gene promoter, is commonly used to generate adult mice having reliable hepatocyte-specific recombination of loxP-flanked ("floxed") alleles. Based on previous studies, it has been unclear whether albCre transgenes are also reliable in fetal and juvenile mice. Perinatal liver undergoes a dynamic transition from being predominantly hematopoietic to predominantly hepatic. We evaluated Cre activity during this transition in albCre mice using a sensitive two-color fluorescent reporter system. From fetal through adult stages, in situ patterns of Cre-dependent recombination of the reporter closely matched expression of endogenous Alb mRNA or protein, indicating most or all hepatocytes, including those in fetal and juvenile livers, had expressed Cre and recombined the reporter. Our results indicate the albCre transgene is effective in converting simple floxed alleles in fetal and neonatal mice and is an appropriate tool for studies on hepatocyte development.
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Hepatocitos/metabolismo , Integrasas/metabolismo , Regiones Promotoras Genéticas/genética , Albúmina Sérica/genética , Animales , Animales Recién Nacidos , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hepatocitos/citología , Hibridación in Situ , Integrasas/genética , Hígado/citología , Hígado/embriología , Hígado/metabolismo , Masculino , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Transgenes/genéticaRESUMEN
BACKGROUND: Metabolically active cells require robust mechanisms to combat oxidative stress. The cytoplasmic thioredoxin reductase/thioredoxin (Txnrd1/Txn1) system maintains reduced protein dithiols and provides electrons to some cellular reductases, including peroxiredoxins. PRINCIPAL FINDINGS: Here we generated mice in which the txnrd1 gene, encoding Txnrd1, was specifically disrupted in all parenchymal hepatocytes. Txnrd1-deficient livers exhibited a transcriptome response in which 56 mRNAs were induced and 12 were repressed. Based on the global hybridization profile, this represented only 0.3% of the liver transcriptome. Since most liver mRNAs were unaffected, compensatory responses were evidently effective. Nuclear pre-mRNA levels indicated the response was transcriptional. Twenty-one of the induced genes contained known antioxidant response elements (AREs), which are binding sites for the oxidative and chemical stress-induced transcription factor Nrf2. Txnrd1-deficient livers showed increased accumulation of nuclear Nrf2 protein and chromatin immunoprecipitation on the endogenous nqo1 and aox1 promoters in fibroblasts indicated that Txnrd1 ablation triggered in vivo assembly of Nrf2 on each. CONCLUSIONS: Chronic deletion of Txnrd1 results in induction of the Nrf2 pathway, which contributes to an effective compensatory response.
Asunto(s)
Hepatocitos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Tiorredoxina Reductasa 1/fisiología , Animales , Células Cultivadas , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Inmunohistoquímica , Ratones , Oxidación-Reducción , ARN Mensajero/genética , Tiorredoxina Reductasa 1/genéticaRESUMEN
Rotavirus mRNAs are transcribed from 11 genomic dsRNA segments within a subviral particle. The mRNAs are extruded into the cytoplasm where they serve as mRNA for protein synthesis and as templates for packaging and replication into dsRNA. The molecular steps in the replication pathway that regulate the levels of viral gene expression are not well defined. We have investigated potential mechanisms of regulation of rotavirus gene expression by functional evaluation of two differentially expressed viral mRNAs. NSP1 (gene 5) and VP6 (gene 6) are expressed early in infection, and VP6 is expressed in excess over NSP1. We formulated the hypothesis that the amounts of NSP1 and VP6 were regulated by the translational efficiencies of the respective mRNAs. We measured the levels of gene 5 and gene 6 mRNA and showed that they were not significantly different, and protein analysis indicated no difference in stability of NSP1 compared with VP6. Polyribosome analysis showed that the majority of gene 6 mRNA was present on large polysomes. In contrast, sedimentation of more than half of the gene 5 mRNA was subpolysomal. The change in distribution of gene 5 mRNA in polyribosome gradients in response to treatment with low concentrations of cycloheximide suggested that gene 5 is a poor translation initiation template compared with gene 6 mRNA. These data define a regulatory mechanism for the difference in amounts of VP6 and NSP1 and provide evidence for post-transcriptional control of rotavirus gene expression mediated by the translational efficiency of individual viral mRNAs.
Asunto(s)
Antígenos Virales , Proteínas de la Cápside/metabolismo , Regulación Viral de la Expresión Génica , Biosíntesis de Proteínas , Rotavirus/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Proteínas de la Cápside/genética , Bovinos , Línea Celular , Datos de Secuencia Molecular , Polirribosomas/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rotavirus/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
The rotavirus nonstructural protein NSP1 is the least conserved protein in the rotavirus genome, and its function in the replication cycle is not known. We employed NSP1 as bait in the yeast two-hybrid interaction trap to identify candidate cellular partners of NSP1 that may provide clues to its function. Interferon regulatory factor 3 (IRF-3) was identified as an NSP1 interactor. NSP1 synthesized in rotavirus-infected cells bound IRF-3 in a glutathione S-transferase pull-down assay, indicating that the interaction was not unique to the two-hybrid system. NSP1 of murine rotavirus strain EW also interacted with IRF-3. NSP1 deletion and point mutants were constructed to map domains important in the interaction between NSP1 and IRF-3. The data suggest that a binding domain resides in the C terminus of NSP1 and that the N-terminal conserved zinc finger is important but not sufficient to mediate binding to IRF-3. We predict that a role for NSP1 in rotavirus-infected cells is to inhibit activation of IRF-3 and diminish the cellular interferon response.