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UNLABELLED: Venezuelan equine encephalitis virus (VEEV) is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing (RNA-Seq) of poly(A) and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and 16 h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response factors, and components of the unfolded protein response (UPR) was observed. The protein kinase RNA-like endoplasmic reticulum kinase (PERK) arm of the UPR was activated, as the expression of both activating transcription factor 4 (ATF4) and CHOP (DDIT3), critical regulators of the pathway, was altered after infection. Expression of the transcription factor early growth response 1 (EGR1) was induced in a PERK-dependent manner. EGR1(-/-) mouse embryonic fibroblasts (MEFs) demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE: Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination of virus-host interactions. Currently there are no therapeutics for infections cause by encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus (VEEV) is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes. Our results indicated that the abundance of transcripts associated with the interferon and the unfolded protein response pathways was altered following infection and demonstrated that early growth response 1 (EGR1) contributed to VEEV-induced cell death.
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Apoptosis , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Virus de la Encefalitis Equina Venezolana/fisiología , Interacciones Huésped-Patógeno , Respuesta de Proteína Desplegada , Animales , Línea Celular , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones NoqueadosRESUMEN
Rift Valley fever virus (RVFV) is an arbovirus that is classified as a select agent, an emerging infectious virus, and an agricultural pathogen. Understanding RVFV-host interactions is imperative to the design of novel therapeutics. Here, we report that an infection by the MP-12 strain of RVFV induces phosphorylation of the p65 component of the NFκB cascade. We demonstrate that phosphorylation of p65 (serine 536) involves phosphorylation of IκBα and occurs through the classical NFκB cascade. A unique, low molecular weight complex of the IKK-ß subunit can be observed in MP-12-infected cells, which we have labeled IKK-ß2. The IKK-ß2 complex retains kinase activity and phosphorylates an IκBα substrate. Inhibition of the IKK complex using inhibitors impairs viral replication, thus alluding to the requirement of an active IKK complex to the viral life cycle. Curcumin strongly down-regulates levels of extracellular infectious virus. Our data demonstrated that curcumin binds to and inhibits kinase activity of the IKK-ß2 complex in infected cells. Curcumin partially exerts its inhibitory influence on RVFV replication by interfering with IKK-ß2-mediated phosphorylation of the viral protein NSs and by altering the cell cycle of treated cells. Curcumin also demonstrated efficacy against ZH501, the fully virulent version of RVFV. Curcumin treatment down-regulated viral replication in the liver of infected animals. Our data point to the possibility that RVFV infection may result in the generation of novel versions of host components (such as IKK-ß2) that, by virtue of altered protein interaction and function, qualify as unique therapeutic targets.
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Curcumina/farmacología , FN-kappa B/antagonistas & inhibidores , Virus de la Fiebre del Valle del Rift/metabolismo , Replicación Viral/efectos de los fármacos , Animales , Antivirales/farmacología , Línea Celular , Línea Celular Tumoral , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Regulación Viral de la Expresión Génica , Humanos , Quinasa I-kappa B/metabolismo , Ratones , Ratones Transgénicos , Fiebre del Valle del Rift/virología , Transcripción GenéticaRESUMEN
There are currently no therapeutics to treat infection with the alphavirus Venezuelan equine encephalitis virus (VEEV), which causes flu-like symptoms leading to neurological symptoms in up to 14% of cases. Large outbreaks of VEEV can result in 10,000 s of human cases and mass equine death. We previously showed that mifepristone (RU486) has anti-VEEV activity (EC50 = 20 µM) and only limited cytotoxicity (CC50 > 100 µM), but a limitation in its use is its abortifacient activity resulting from its ability to antagonize the progesterone receptor (PR). Here we generate a suite of new mifepristone analogues with enhanced antiviral properties, succeeding in achieving >11-fold improvement in anti-VEEV activity with no detectable increase in toxicity. Importantly, we were able to derive a lead compound with an EC50 of 7.2 µM and no detectable PR antagonism activity. Finally, based on our SAR analysis we propose avenues for the further development of these analogues as safe and effective anti-VEEV agents.
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Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Mifepristona/análogos & derivados , Mifepristona/farmacología , Receptores de Progesterona/antagonistas & inhibidores , Transporte Activo de Núcleo Celular/efectos de los fármacos , Proteínas de la Cápside/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células HeLa , Humanos , Mifepristona/síntesis química , Mifepristona/química , Simulación del Acoplamiento Molecular , Unión Proteica/efectos de los fármacos , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/metabolismo , Relación Estructura-ActividadRESUMEN
The New World alphaviruses -Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV respectively) - cause a febrile disease that is often lethal in equines and children and leads to long-term neurological sequelae in survivors. Endemic to the Americas, epizootic outbreaks of the three viruses occur sporadically in the continental United States. All three viruses aerosolize readily, replicate to high titers in cell culture, and have low infectious doses. Additionally, there are no FDA-approved vaccines or therapeutics for human use. To address the therapeutic gap, a high throughput assay utilizing a luciferase reporter virus, TC83-luc, was performed to screen a library of commercially available, FDA-approved drugs for antiviral activity. From a group of twenty compounds found to significantly decrease luminescence, the carcinoma therapeutic sorafenib inhibited replication of VEEV-TC83 and TrD in vitro. Additionally, sorafenib inhibited replication of EEEV and two Old World alphaviruses, Sindbis virus and chikungunya virus, at 8 and 16â¯h post-infection. Sorafenib caused no toxicity in Vero cells, and coupled with a low EC50 value, yielded a selectivity index of >19. Mechanism of actions studies suggest that sorafenib inhibited viral translation through dephosphorylation of several key proteins, including eIF4E and p70S6K, leading to a reduction in viral protein production and overall viral replication.
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Alphavirus/efectos de los fármacos , Antineoplásicos/farmacología , Antivirales/farmacología , Reposicionamiento de Medicamentos , Sorafenib/farmacología , Replicación Viral/efectos de los fármacos , Alphavirus/crecimiento & desarrollo , Animales , Línea Celular , Evaluación Preclínica de Medicamentos/métodos , Genes Reporteros , Ensayos Analíticos de Alto Rendimiento , Luciferasas/análisis , Luciferasas/genética , Mediciones Luminiscentes , Genética InversaRESUMEN
Venezuelan equine encephalitis virus (VEEV) is a positive sense, single-stranded RNA virus and member of the New World alphaviruses. It causes a biphasic febrile illness that can be accompanied by central nervous system involvement and moderate morbidity in humans and severe mortality in equines. The virus has a history of weaponization, lacks FDA-approved therapeutics and vaccines in humans, and is considered a select agent. Like other RNA viruses, VEEV replicates in the cytoplasm of infected cells and eventually induces apoptosis. The capsid protein, which contains a nuclear localization and a nuclear export sequence, induces a shutdown of host transcription and nucleocytoplasmic trafficking. Here we show that infection with VEEV causes a dysregulation of cell cycling and a delay in the G0/G1 phase in Vero cells and U87MG astrocytes. Cells infected with VEEV encoding a capsid NLS mutant or treated with the capsid-importin α interaction inhibitor G281-1485 were partially rescued from this cell cycle dysregulation. Pathway analysis of previously published RNA-sequencing data from VEEV infected U87MG astrocytes identified alterations of canonical pathways involving cell cycle, checkpoint regulation, and proliferation. Multiple cyclins including cyclin D1, cyclin A2 and cyclin E2 and other regulators of the cell cycle were downregulated in infected cells in a capsid NLS dependent manner. Loss of Rb phosphorylation, which is a substrate for cyclin/cdk complexes was also observed. These data demonstrate the importance of capsid nuclear localization and/or importin α binding for inducing cell cycle arrest and transcriptional downregulation of key cell cycle regulators.
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Although the alphavirus Venezuelan equine encephalitis virus (VEEV) has been the cause of multiple outbreaks resulting in extensive human and equine mortality and morbidity, there are currently no anti-VEEV therapeutics available. VEEV pathogenicity is largely dependent on targeting of the viral capsid protein (CP) to the host cell nucleus through the nuclear transporting importin (Imp) α/ß1 heterodimer. Here we perform a high-throughput screen, combined with nested counterscreens to identify small molecules able to inhibit the Impα/ß1:CP interaction for the first time. Several compounds were able to significantly reduce viral replication in infected cells. Compound G281-1564 in particular could inhibit VEEV replication at low µM concentration, while showing minimal toxicity, with steady state and dynamic quantitative microscopic measurements confirming its ability to inhibit CP nuclear import. This study establishes the principle that inhibitors of CP nucleocytoplasmic trafficking can have potent antiviral activity against VEEV, and represents a platform for future development of safe anti-VEEV compounds with high efficacy and specificity.
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Antivirales/farmacología , Proteínas de la Cápside/metabolismo , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Encefalomielitis Equina Venezolana/virología , Carioferinas/antagonistas & inhibidores , Carioferinas/metabolismo , Replicación Viral/efectos de los fármacos , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Antivirales/química , Supervivencia Celular , Chlorocebus aethiops , Encefalomielitis Equina Venezolana/metabolismo , Ensayos Analíticos de Alto Rendimiento , Interacciones Huésped-Patógeno/efectos de los fármacos , Concentración 50 Inhibidora , Estructura Molecular , Unión Proteica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Células VeroRESUMEN
Viruses must parasitize host cell translational machinery in order to make proteins for viral progeny. In this study, we sought to use this signal transduction conduit against them by inhibiting multiple kinases that influence translation. Previous work indicated that several kinases involved in translation, including p70 S6K, p90RSK, ERK, and p38 MAPK, are phosphorylated following Rift Valley fever virus (RVFV) infection. Furthermore, inhibiting p70 S6K through treatment with the FDA approved drug rapamycin prevents RVFV pathogenesis in a mouse model of infection. We hypothesized that inhibiting either p70 S6K, p90RSK, or p90RSK’s upstream kinases, ERK and p38 MAPK, would decrease translation and subsequent viral replication. Treatment with the p70 S6K inhibitor PF-4708671 resulted in decreased phosphorylation of translational proteins and reduced RVFV titers. In contrast, treatment with the p90RSK inhibitor BI-D1870, p38MAPK inhibitor SB203580, or the ERK inhibitor PD0325901 alone had minimal influence on RVFV titers. The combination of PF-4708671 and BI-D1870 treatment resulted in robust inhibition of RVFV replication. Likewise, a synergistic inhibition of RVFV replication was observed with p38MAPK inhibitor SB203580 or the ERK inhibitor PD0325901 combined with rapamycin treatment. These findings serve as a proof of concept regarding combination kinase inhibitor treatment for RVFV infection.
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Antivirales/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Virus de la Fiebre del Valle del Rift/efectos de los fármacos , Virus de la Fiebre del Valle del Rift/fisiología , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , Ratones , Fosforilación , Biosíntesis de Proteínas/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Proteínas Ribosómicas/metabolismoRESUMEN
Venezuelan equine encephalitis virus (VEEV) is a New World alphavirus that is vectored by mosquitos and cycled in rodents. It can cause disease in equines and humans characterized by a febrile illness that may progress into encephalitis. Like the capsid protein of other viruses, VEEV capsid is an abundant structural protein that binds to the viral RNA and interacts with the membrane-bound glycoproteins. It also has protease activity, allowing cleavage of itself from the growing structural polypeptide during translation. However, VEEV capsid protein has additional nonstructural roles within the host cell functioning as the primary virulence factor for VEEV. VEEV capsid inhibits host transcription and blocks nuclear import in mammalian cells, at least partially due to its complexing with the host CRM1 and importin α/ß1 nuclear transport proteins. VEEV capsid also shuttles between the nucleus and cytoplasm and is susceptible to inhibitors of nuclear trafficking, making it a promising antiviral target. Herein, the role of VEEV capsid in viral replication and pathogenesis will be discussed including a comparison to proteins of other alphaviruses.
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Proteínas de la Cápside/metabolismo , Cápside/metabolismo , Virus de la Encefalitis Equina Venezolana/patogenicidad , ARN Viral/metabolismo , Replicación Viral , Transporte Activo de Núcleo Celular , Animales , Cápside/química , Proteínas de la Cápside/genética , Línea Celular , Virus de la Encefalitis Equina del Este , Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/metabolismo , Virus de la Encefalitis Equina del Oeste , Caballos , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Virulencia , Replicación Viral/genética , Proteína Exportina 1RESUMEN
Therapeutics are currently unavailable for Venezuelan equine encephalitis virus (VEEV), which elicits flu-like symptoms and encephalitis in humans, with an estimated 14% of cases resulting in neurological disease. Here we identify anti-VEEV agents using in silico structure-based-drug-design (SBDD) for the first time, characterising inhibitors that block recognition of VEEV capsid protein (C) by the host importin (IMP) α/ß1 nuclear transport proteins. From an initial screen of 1.5 million compounds, followed by in silico refinement and screening for biological activity in vitro, we identified 21 hit compounds which inhibited IMPα/ß1:C binding with IC50s as low as 5 µM. Four compounds were found to inhibit nuclear import of C in transfected cells, with one able to reduce VEEV replication at µM concentration, concomitant with reduced C nuclear accumulation in infected cells. Further, this compound was inactive against a mutant VEEV that lacks high affinity IMPα/ß1:C interaction, supporting the mode of its antiviral action to be through inhibiting C nuclear localization. This successful application of SBDD paves the way for lead optimization for VEEV antivirals, and is an exciting prospect to identify inhibitors for the many other viral pathogens of significance that require IMPα/ß1 in their infectious cycle.
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Proteínas de la Cápside/efectos de los fármacos , Descubrimiento de Drogas/métodos , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Antivirales/farmacología , Cápside , Proteínas de la Cápside/metabolismo , Núcleo Celular/metabolismo , Chlorocebus aethiops , Simulación por Computador , Diseño de Fármacos , Virus de la Encefalitis Equina Venezolana/patogenicidad , Humanos , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Células Vero , Replicación Viral/efectos de los fármacos , alfa Carioferinas/antagonistas & inhibidores , alfa Carioferinas/metabolismo , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/metabolismoRESUMEN
Despite over 60 years of research on antiviral drugs, very few are FDA approved to treat acute viral infections. Rift Valley fever virus (RVFV), an arthropod borne virus that causes hemorrhagic fever in severe cases, currently lacks effective treatments. Existing as obligate intracellular parasites, viruses have evolved to manipulate host cell signaling pathways to meet their replication needs. Specifically, translation modulation is often necessary for viruses to establish infection in their host. Here we demonstrated phosphorylation of p70 S6 kinase, S6 ribosomal protein, and eIF4G following RVFV infection in vitro through western blot analysis and in a mouse model of infection through reverse phase protein microarrays (RPPA). Inhibition of p70 S6 kinase through rapamycin treatment reduced viral titers in vitro and increased survival and mitigated clinical disease in RVFV challenged mice. Additionally, the phosphorylation of p70 S6 kinase was decreased following rapamycin treatment in vivo. Collectively these data demonstrate modulating p70 S6 kinase can be an effective antiviral strategy.
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Proteínas Quinasas S6 Ribosómicas 70-kDa/efectos de los fármacos , Virus de la Fiebre del Valle del Rift/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sirolimus/antagonistas & inhibidores , Animales , Antivirales/farmacología , Apoptosis/efectos de los fármacos , Línea Celular , Chlorocebus aethiops , Replicación del ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Factor 4G Eucariótico de Iniciación/metabolismo , Femenino , Inmunohistoquímica , Hígado/patología , Ratones , Ratones Endogámicos BALB C , Fosforilación/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Fiebre del Valle del Rift/tratamiento farmacológico , Fiebre del Valle del Rift/patología , Fiebre del Valle del Rift/virología , Virus de la Fiebre del Valle del Rift/genética , Virus de la Fiebre del Valle del Rift/crecimiento & desarrollo , Virus de la Fiebre del Valle del Rift/patogenicidad , Sirolimus/metabolismo , Sirolimus/uso terapéutico , Análisis de Supervivencia , Células Vero , Carga Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Rift Valley fever (RVF) is a zoonotic disease that can cause severe illness in humans and livestock, triggering spontaneous abortion in almost 100% of pregnant ruminants. In this study, we demonstrate that signal transducer and activator of transcription 3 (STAT3) is phosphorylated on its conserved tyrosine residue (Y705) following RVFV infection. This phosphorylation was dependent on a major virulence factor, the viral nonstructural protein NSs. Loss of STAT3 had little effect on viral replication, but rather resulted in cells being more susceptible to RVFV-induced cell death. Phosphorylated STAT3 translocated to the nucleus, coinciding with inhibition of fos, jun, and nr4a2 gene expression, and the presence of STAT3 and NSs at the nr4a2 promoter. NSs was found predominantly in the cytoplasm of STAT3 null cells, indicating that STAT3 influences NSs nuclear localization. Collectively, these data demonstrate that STAT3 functions in a pro-survival capacity through modulation of NSs localization.
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Fiebre del Valle del Rift/metabolismo , Fiebre del Valle del Rift/virología , Virus de la Fiebre del Valle del Rift/fisiología , Factor de Transcripción STAT3/metabolismo , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Humanos , Fosforilación , Regiones Promotoras Genéticas , Unión Proteica , Transporte de Proteínas , Fiebre del Valle del Rift/genética , Virus de la Fiebre del Valle del Rift/efectos de los fármacos , Tirosina/metabolismo , Células Vero , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
The capsid structural protein of the New World alphavirus, Venezuelan equine encephalitis virus (VEEV), interacts with the host nuclear transport proteins importin α/ß1 and CRM1. Novel selective inhibitor of nuclear export (SINE) compounds, KPT-185, KPT-335 (verdinexor), and KPT-350, target the host's primary nuclear export protein, CRM1, in a manner similar to the archetypical inhibitor Leptomycin B. One major limitation of Leptomycin B is its irreversible binding to CRM1; which SINE compounds alleviate because they are slowly reversible. Chemically inhibiting CRM1 with these compounds enhanced capsid localization to the nucleus compared to the inactive compound KPT-301, as indicated by immunofluorescent confocal microscopy. Differences in extracellular versus intracellular viral RNA, as well as decreased capsid in cell free supernatants, indicated the inhibitors affected viral assembly, which led to a decrease in viral titers. The decrease in viral replication was confirmed using a luciferase-tagged virus and through plaque assays. SINE compounds had no effect on VEEV TC83_Cm, which encodes a mutated form of capsid that is unable to enter the nucleus. Serially passaging VEEV in the presence of KPT-185 resulted in mutations within the nuclear localization and nuclear export signals of capsid. Finally, SINE compound treatment also reduced the viral titers of the related eastern and western equine encephalitis viruses, suggesting that CRM1 maintains a common interaction with capsid proteins across the New World alphavirus genus.
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Infecciones por Alphavirus/virología , Alphavirus/efectos de los fármacos , Antivirales/farmacología , Proteínas de la Cápside/metabolismo , Replicación Viral/efectos de los fármacos , Transporte Activo de Núcleo Celular/efectos de los fármacos , Alphavirus/genética , Alphavirus/fisiología , Animales , Proteínas de la Cápside/genética , Núcleo Celular/virología , Humanos , Carioferinas/antagonistas & inhibidores , Carioferinas/genética , Carioferinas/metabolismo , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Ensamble de Virus/efectos de los fármacos , Proteína Exportina 1RESUMEN
BACKGROUND: Rift Valley fever virus (RVFV) is a highly pathogenic arthropod-borne virus that has a detrimental effect on both livestock and human populations. While there are several diagnostic methodologies available for RVFV detection, many are not sensitive enough to diagnose early infections. Furthermore, detection may be hindered by high abundant proteins such as albumin. Previous findings have shown that Nanotrap particles can be used to significantly enhance detection of various small analytes of low abundance. We have expanded upon this repertoire to show that this simple and efficient sample preparation technology can drastically improve the detection of the RVFV nucleoprotein (NP), the most abundant and widely used viral protein for RVFV diagnostics. RESULTS: After screening multiple Nanotrap particle architectures, we found that one particle, NT45, was optimal for RVFV NP capture, as demonstrated by western blotting. NT45 significantly enhanced detection of the NP at levels undetectable without the technology. Importantly, we demonstrated that Nanotrap particles are capable of concentrating NP in a number of matrices, including infected cell lysates, viral supernatants, and animal sera. Specifically, NT45 enhanced detection of NP at various viral titers, multiplicity of infections, and time points. Our most dramatic results were observed in spiked serum samples, where high abundance serum proteins hindered detection of NP without Nanotrap particles. Nanotrap particles allowed for sample cleanup and subsequent detection of RVFV NP. Finally, we demonstrated that incubation of our samples with Nanotrap particles protects the NP from degradation over extended periods of time (up to 120 hours) and at elevated temperatures (at 37ºC). CONCLUSION: This study demonstrates that Nanotrap particles are capable of drastically lowering the limit of detection for RVFV NP by capturing, concentrating, and preserving RVFV NP in clinically relevant matrices. These studies can be extended to a wide range of pathogens and their analytes of diagnostic interest.
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Nanopartículas/química , Nucleoproteínas/química , Virus de la Fiebre del Valle del Rift/química , Proteínas Virales/química , Animales , Chlorocebus aethiops , Humanos , Nanopartículas/metabolismo , Nucleoproteínas/metabolismo , Fiebre del Valle del Rift/diagnóstico , Fiebre del Valle del Rift/metabolismo , Virus de la Fiebre del Valle del Rift/metabolismo , Células Vero , Proteínas Virales/metabolismoRESUMEN
There are currently no FDA-approved therapeutics available to treat Rift Valley fever virus (RVFV) infection. In an effort to repurpose drugs for RVFV treatment, a library of FDA-approved drugs was screened to determine their ability to inhibit RVFV. Several drugs from varying compound classes, including inhibitors of growth factor receptors, microtubule assembly/disassembly, and DNA synthesis, were found to reduce RVFV replication. The hepatocellular and renal cell carcinoma drug, sorafenib, was the most effective inhibitor, being non-toxic and demonstrating inhibition of RVFV in a cell-type and virus strain independent manner. Mechanism of action studies indicated that sorafenib targets at least two stages in the virus infectious cycle, RNA synthesis and viral egress. Computational modeling studies also support this conclusion. siRNA knockdown of Raf proteins indicated that non-classical targets of sorafenib are likely important for the replication of RVFV.
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Detection of early infectious disease may be challenging due to the low copy number of organisms present. To overcome this limitation and rapidly measure low concentrations of the pathogen, we developed a novel technology: Nanotrap particles, which are designed to capture, concentrate, and protect biomarkers from complex biofluids. Nanotrap particles are thermoresponsive hydrogels that are capable of antigen capture through the coupling of affinity baits to the particles. Here, we describe recent findings demonstrating that Nanotrap particles are able to capture live infectious virus, viral RNA, and viral proteins. Capture is possible even in complex mixtures such as serum and allows the concentration and protection of these analytes, providing increased performance of downstream assays. The Nanotrap particles are a versatile sample preparation technology that has far reaching implications for biomarker discovery and diagnostic assays.
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Armas Biológicas , Enfermedades Transmisibles Emergentes/diagnóstico , Técnicas Microbiológicas/métodos , Animales , Antígenos Virales/aislamiento & purificación , Líquidos Corporales/virología , Diagnóstico Precoz , Humanos , ARN Viral/aislamiento & purificación , Virosis/diagnóstico , Virus/aislamiento & purificaciónRESUMEN
Venezuelan equine encephalitis virus (VEEV) is classified as a Category B Select Agent and potential bioterror weapon for its severe disease course in humans and equines and its potential for aerosol transmission. There are no current FDA licensed vaccines or specific therapies against VEEV, making identification of potential therapeutic targets a priority. With this aim, our research focuses on the interactions of VEEV with host microRNA (miRNA) machinery. miRNAs are small non-coding RNAs that act as master regulators of gene expression by downregulating or degrading messenger RNA, thus suppressing production of the resultant proteins. Recent publications implicate miRNA interactions in the pathogenesis of various viral diseases. To test the importance of miRNA processing for VEEV replication, cells deficient in Ago2, an important component of the RNA-induced silencing complex (RISC), and cells treated with known Ago2 inhibitors, notably acriflavine (ACF), were utilized. Both conditions caused decreased viral replication and capsid expression. ACF treatment promoted increased survival of neuronal cells over a non-treated, infected control and reduced viral titers of fully virulent VEEV as well as Eastern and Western Equine Encephalitis Viruses and West Nile Virus, but not Vesicular Stomatitis Virus. ACF treatment of VEEV TC-83 infected mice resulted in increased in vivo survival, but did not affect survival or viral loads when mice were challenged with fully virulent VEEV TrD. These results suggest that inhibition of Ago2 results in decreased replication of encephalitic alphaviruses in vitro and this pathway may be an avenue to explore for future therapeutic development.
Asunto(s)
Antivirales/farmacología , Proteínas Argonautas/antagonistas & inhibidores , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Virus de la Encefalitis Equina Venezolana/fisiología , Inhibidores Enzimáticos/farmacología , Replicación Viral/efectos de los fármacos , Acriflavina/farmacología , Acriflavina/uso terapéutico , Animales , Antivirales/uso terapéutico , Proteínas de la Cápside/biosíntesis , Supervivencia Celular , Modelos Animales de Enfermedad , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Encefalomielitis Equina Venezolana/virología , Inhibidores Enzimáticos/uso terapéutico , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Neuronas/fisiología , Neuronas/virología , Análisis de Supervivencia , Resultado del Tratamiento , Carga ViralRESUMEN
Targeting host responses to invading viruses has been the focus of recent antiviral research. Venezuelan Equine Encephalitis Virus (VEEV) is able to modulate host transcription and block nuclear trafficking at least partially due to its capsid protein forming a complex with the host proteins importin α/ß1 and CRM1. We hypothesized that disrupting the interaction of capsid with importin α/ß1 or the interaction of capsid with CRM1 would alter capsid localization, thereby lowering viral titers in vitro. siRNA mediated knockdown of importin α, importin ß1, and CRM1 altered capsid localization, confirming their role in modulating capsid trafficking. Mifepristone and ivermectin, inhibitors of importin α/ß-mediated import, were able to reduce nuclear-associated capsid, while leptomycin B, a potent CRM1 inhibitor, confined capsid to the nucleus. In addition to altering the level and distribution of capsid, the three inhibitors were able to reduce viral titers in a relevant mammalian cell line with varying degrees of efficacy. The inhibitors were also able to reduce the cytopathic effects associated with VEEV infection, hinting that nuclear import inhibitors may be protecting cells from apoptosis in addition to disrupting the function of an essential viral protein. Our results confirm that VEEV uses host importins and exportins during part of its life cycle. Further, it suggests that temporarily targeting host proteins that are hijacked for use by viruses is a viable antiviral therapy.
Asunto(s)
Transporte Activo de Núcleo Celular/efectos de los fármacos , Cápside/metabolismo , Núcleo Celular/metabolismo , Virus de la Encefalitis Equina Venezolana/efectos de los fármacos , Ivermectina/farmacología , Mifepristona/farmacología , Replicación Viral/efectos de los fármacos , Animales , Astrocitoma/patología , Compartimento Celular , Línea Celular Tumoral , Chlorocebus aethiops , Efecto Citopatogénico Viral , Virus de la Encefalitis Equina Venezolana/fisiología , Ácidos Grasos Insaturados/farmacología , Humanos , Carioferinas/antagonistas & inhibidores , Sustancias Macromoleculares , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Células Vero , Cultivo de Virus , alfa Carioferinas/antagonistas & inhibidores , alfa Carioferinas/genética , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/genética , Proteína Exportina 1RESUMEN
In most bacteria, the nonmevalonate pathway is used to synthesize isoprene units. Dxr, the second step in the pathway, catalyzes the NADPH-dependent reductive isomerization of 1-deoxy-D-xylulose-5-phosphate (DXP) to 2-C-methyl-D-erythritol-4-phosphate (MEP). Dxr is inhibited by natural products fosmidomycin and FR900098, which bind in the DXP binding site. These compounds, while potent inhibitors of Dxr, lack whole cell activity against Mycobacterium tuberculosis (Mtb) due to their polarity. Our goal was to use the Mtb Dxr-fosmidomycin co-crystal structure to design bisubstrate ligands to bind to both the DXP and NADPH sites. Such compounds would be expected to demonstrate improved whole cell activity due to increased lipophilicity. Two series of compounds were designed and synthesized. Compounds from both series inhibited Mtb Dxr. The most potent compound (8) has an IC50 of 17.8 µM. Analysis shows 8 binds to Mtb Dxr via a novel, non-bisubstrate mechanism. Further, the diethyl ester of 8 inhibits Mtb growth making this class of compounds interesting lead molecules in the search for new antitubercular agents.
RESUMEN
Rift Valley fever virus (RVFV) is an emerging viral zoonosis that is responsible for devastating outbreaks among livestock and is capable of causing potentially fatal disease in humans. Studies have shown that upon infection, certain viruses have the capability of utilizing particular cellular signaling pathways to propagate viral infection. Activation of p53 is important for the DNA damage signaling cascade, initiation of apoptosis, cell cycle arrest and transcriptional regulation of multiple genes. The current study focuses on the role of p53 signaling in RVFV infection and viral replication. These results show an up-regulation of p53 phosphorylation at several serine sites after RVFV MP-12 infection that is highly dependent on the viral protein NSs. qRT-PCR data showed a transcriptional up-regulation of several p53 targeted genes involved in cell cycle and apoptosis regulation following RVFV infection. Cell viability assays demonstrate that loss of p53 results in less RVFV induced cell death. Furthermore, decreased viral titers in p53 null cells indicate that RVFV utilizes p53 to enhance viral production. Collectively, these experiments indicate that the p53 signaling pathway is utilized during RVFV infection to induce cell death and increase viral production.