RESUMEN
Crimean-Congo hemorrhagic fever virus (CCHFV) is an important human pathogen. In cell culture, CCHFV is sensed by the cytoplasmic RNA sensor retinoic acid-inducible gene I (RIG-I) molecule and its adaptor molecule mitochondrial antiviral signaling (MAVS) protein. MAVS initiates both type I interferon (IFN-I) and proinflammatory responses. Here, we studied the role MAVS plays in CCHFV infection in mice in both the presence and absence of IFN-I activity. MAVS-deficient mice were not susceptible to CCHFV infection when IFN-I signaling was active and showed no signs of disease. When IFN-I signaling was blocked by antibody, MAVS-deficient mice lost significant weight, but were uniformly protected from lethal disease, whereas all control mice succumbed to infection. Cytokine activity in the infected MAVS-deficient mice was markedly blunted. Subsequent investigation revealed that CCHFV infected mice lacking TNF-α receptor signaling (TNFA-R-deficient), but not IL-6 or IL-1 activity, had more limited liver injury and were largely protected from lethal outcomes. Treatment of mice with an anti-TNF-α neutralizing antibody also conferred partial protection in a post-virus exposure setting. Additionally, we found that a disease causing, but non-lethal strain of CCHFV produced more blunted inflammatory cytokine responses compared to a lethal strain in mice. Our work reveals that MAVS activation and cytokine production both contribute to CCHFV pathogenesis, potentially identifying new therapeutic targets to treat this disease.
Asunto(s)
Virus de la Fiebre Hemorrágica de Crimea-Congo , Fiebre Hemorrágica de Crimea , Animales , Citocinas , Modelos Animales de Enfermedad , Virus de la Fiebre Hemorrágica de Crimea-Congo/genética , Ratones , Ratones Noqueados , Índice de Severidad de la Enfermedad , Inhibidores del Factor de Necrosis TumoralRESUMEN
Protein phosphorylation plays an important role during the life cycle of many viruses. Venezuelan equine encephalitis virus (VEEV) capsid protein has recently been shown to be phosphorylated at four residues. Here those studies are extended to determine the kinase responsible for phosphorylation and the importance of capsid phosphorylation during the viral life cycle. Phosphorylation site prediction software suggests that Protein Kinase C (PKC) is responsible for phosphorylation of VEEV capsid. VEEV capsid co-immunoprecipitated with PKCδ, but not other PKC isoforms and siRNA knockdown of PKCδ caused a decrease in viral replication. Furthermore, knockdown of PKCδ by siRNA decreased capsid phosphorylation. A virus with capsid phosphorylation sites mutated to alanine (VEEV CPD) displayed a lower genomic copy to pfu ratio than the parental virus; suggesting more efficient viral assembly and more infectious particles being released. RNA:capsid binding was significantly increased in the mutant virus, confirming these results. Finally, VEEV CPD is attenuated in a mouse model of infection, with mice showing increased survival and decreased clinical signs as compared to mice infected with the parental virus. Collectively our data support a model in which PKCδ mediated capsid phosphorylation regulates viral RNA binding and assembly, significantly impacting viral pathogenesis.
Asunto(s)
Proteínas de la Cápside/metabolismo , Virus de la Encefalitis Equina Venezolana/metabolismo , Encefalomielitis Equina Venezolana/enzimología , Proteína Quinasa C-delta/metabolismo , ARN Viral/metabolismo , Animales , Cápside/metabolismo , Proteínas de la Cápside/genética , Virus de la Encefalitis Equina Venezolana/genética , Encefalomielitis Equina Venezolana/genética , Encefalomielitis Equina Venezolana/virología , Femenino , Caballos , Interacciones Huésped-Patógeno , Ratones , Ratones Endogámicos C3H , Fosforilación , Unión Proteica , Proteína Quinasa C-delta/genética , ARN Viral/genéticaRESUMEN
Protein phosphatase 1 (PP1) is a serine/threonine phosphatase which has been implicated in the regulation of a number of viruses, including HIV-1, Ebolavirus, and Rift Valley fever virus. Catalytic subunits of PP1 (PP1α, PP1ß, and PP1γ) interact with a host of regulatory subunits and target a wide variety of cellular substrates through a combination of short binding motifs, including an RVxF motif present in the majority of PP1 regulatory subunits. Targeting the RVxF-interacting site on PP1 with the small molecule 1E7-03 inhibits HIV-1, Ebolavirus, and Rift Valley fever virus replication. In this study, we determined the effect of PP1 on Venezuelan equine encephalitis virus (VEEV) replication. Treatment of VEEV-infected cells with 1E7-03 decreased viral replication by more than 2 logs (50% effective concentration [EC50] = 0.6 µM). 1E7-03 treatment reduced viral titers starting at 8 h postinfection. Viral replication was also decreased after treatment with PP1α-targeting small interfering RNA (siRNA). Confocal microscopy demonstrated that PP1α shuttles toward the cytosol during infection with VEEV and that PP1α colocalizes with VEEV capsid. Coimmunoprecipitation experiments confirmed VEEV capsid interaction with PP1α. Furthermore, immunoprecipitation and mass spectrometry data showed that VEEV capsid is phosphorylated and that phosphorylation is moderated by PP1α. Finally, less viral RNA is associated with capsid after treatment with 1E7-03. Coupled with data showing that 1E7-03 inhibits several alphaviruses, this study indicates that inhibition of the PP1α RVxF binding pocket is a promising therapeutic target and provides novel evidence that PP1α modulation of VEEV capsid phosphorylation influences viral replication.IMPORTANCE Venezuelan equine encephalitis virus (VEEV) causes moderate flu-like symptoms and can lead to severe encephalitic disease and potentially death. There are currently no FDA-approved therapeutics or vaccines for human use, and understanding the molecular underpinning of host-virus interactions can aid in the rational design of intervention strategies. The significance of our research is in identifying the interaction between protein phosphatase 1 (PP1) and the viral capsid protein. This interaction is important for viral replication, as inhibition of PP1 results in decrease viral replication. Inhibition of PP1 also inhibited multiple biomedically important alphaviruses, indicating that PP1 may be a potential therapeutic target for alphavirus-induced disease.
Asunto(s)
Proteínas de la Cápside/metabolismo , Cápside/metabolismo , Virus de la Encefalitis Equina Venezolana/fisiología , Proteína Fosfatasa 1/metabolismo , Replicación Viral/fisiología , Animales , Proteínas de la Cápside/genética , Chlorocebus aethiops , Fosforilación/genética , Proteína Fosfatasa 1/genética , Células VeroRESUMEN
BACKGROUND: Syrian hamsters infected with Andes virus (ANDV) develop a disease that recapitulates many of the salient features of human hantavirus pulmonary syndrome (HPS), including lethality. Infection of hamsters with Hantaan virus (HTNV) results in an asymptomatic, disseminated infection. In order to explore this dichotomy, we examined the transcriptome of ANDV- and HTNV-infected hamsters. RESULTS: Using NanoString technology, we examined kinetic transcriptional responses in whole blood collected from ANDV- and HTNV-infected hamsters. Of the 770 genes analyzed, key differences were noted in the kinetics of type I interferon sensing and signaling responses, complement activation, and apoptosis pathways between ANDV- and HTNV-infected hamsters. CONCLUSIONS: Delayed activation of type I interferon responses in ANDV-infected hamsters represents a potential mechanism that ANDV uses to subvert host immune responses and enhance disease. This is the first genome-wide analysis of hantavirus-infected hamsters and provides insight into potential avenues for therapeutics to hantavirus disease.
Asunto(s)
Infecciones por Hantavirus/patología , Síndrome Pulmonar por Hantavirus/patología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Orthohantavirus/genética , Orthohantavirus/patogenicidad , Animales , Chlorocebus aethiops , Cricetinae , Femenino , Orthohantavirus/aislamiento & purificación , Mesocricetus , Células VeroRESUMEN
Nanotrap® (NT) particles are hydrogel microspheres developed for target analyte separation and discovery applications. NT particles consist of cross-linked N-isopropylacrylamide (NIPAm) copolymers that are functionalized with a variety of chemical affinity baits to enable broad-spectrum collection and retention of target proteins, nucleic acids, and pathogens. NT particles have been previously shown to capture and enrich arboviruses including Rift Valley fever and Venezuelan equine encephalitis viruses. Yet, there is still a need to enhance the detection ability for other re-emerging viruses such as Zika (ZIKV), chikungunya (CHIKV), and dengue (DENV) viruses. In this study, we exploited NT particles with different affinity baits, including cibacron blue, acrylic acid, and reactive red 120, to evaluate their capturing and enrichment capability for ZIKV, DENV and CHIKV in human fluids. Our results demonstrate that CN1030, a NT particle conjugated with reactive red 120, can recover between 8-16-fold greater genomic copies of ZIKV, CHIKV and DENV in virus spiked urine samples via RT-qPCR, superior to the other chemical baits. Also, we observed that CN1030 simultaneously enriched ZIKV, CHIKV and DENV in co-infection-based settings and could stabilize ZIKV, but not CHIKV infectivity in saliva spiked samples. CN1030 enriched viral detection at various viral concentrations, with significant enhancement observed at viral titers as low as 100 PFU/mL for ZIKV and 10 PFU/mL for CHIKV. The detection of ZIKV was further enhanced with NT particles by processing of larger volume urine samples. Furthermore, we developed a magnetic NT particle, CN3080, based on the same backbone of CN1030, and demonstrated that CN3080 could also capture and enrich ZIKV and CHIKV in a dose-dependent manner. Finally, in silico docking predictions support that the affinity between reactive red 120 and ZIKV or CHIKV envelope proteins appeared to be greater than acrylic acid. Overall, our data show that NT particles along with reactive red 120 can be utilized as a pre-processing technology for enhancement of detecting febrile-illness causing viruses.
Asunto(s)
Infecciones por Arbovirus/orina , Virus Chikungunya/aislamiento & purificación , Virus del Dengue/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , Nanopartículas/química , Virus Zika/aislamiento & purificación , Infecciones por Arbovirus/diagnóstico , Infecciones por Arbovirus/virología , Virus Chikungunya/genética , Virus Chikungunya/patogenicidad , Colorantes/química , Virus del Dengue/genética , Virus del Dengue/patogenicidad , Humanos , Hidrogeles/química , Nanopartículas/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Unión Proteica , Saliva/virología , Orina/virología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Virus Zika/genética , Virus Zika/patogenicidadRESUMEN
Venezuelan equine encephalitis virus (VEEV) is a neurotropic virus that causes significant disease in both humans and equines. Here we characterized the impact of VEEV on signaling pathways regulating cell death in human primary astrocytes. VEEV productively infected primary astrocytes and caused an upregulation of early growth response 1 (EGR1) gene expression at 9 and 18â¯h post infection. EGR1 induction was dependent on extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), but not on p38 mitogen activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling. Knockdown of EGR1 significantly reduced VEEV-induced apoptosis and impacted viral replication. Knockdown of ERK1/2 or PERK significantly reduced EGR1 gene expression, dramatically reduced viral replication, and increased cell survival as well as rescued cells from VEEV-induced apoptosis. These data indicate that EGR1 activation and subsequent cell death are regulated through ERK and PERK pathways in VEEV infected primary astrocytes.
Asunto(s)
Muerte Celular , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Virus de la Encefalitis Equina Venezolana/fisiología , Encefalomielitis Equina Venezolana/virología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , eIF-2 Quinasa/metabolismo , Apoptosis , Astrocitos/metabolismo , Astrocitos/patología , Astrocitos/virología , Supervivencia Celular , Células Cultivadas , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Virus de la Encefalitis Equina Venezolana/patogenicidad , Encefalomielitis Equina Venezolana/metabolismo , Encefalomielitis Equina Venezolana/patología , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Transducción de Señal , Replicación Viral , eIF-2 Quinasa/genéticaRESUMEN
The emergence of SARS-CoV-2 has created an international health crisis, and small animal models mirroring SARS-CoV-2 human disease are essential for medical countermeasure (MCM) development. Mice are refractory to SARS-CoV-2 infection owing to low-affinity binding to the murine angiotensin-converting enzyme 2 (ACE2) protein. Here, we evaluated the pathogenesis of SARS-CoV-2 in male and female mice expressing the human ACE2 gene under the control of the keratin 18 promoter (K18). In contrast to nontransgenic mice, intranasal exposure of K18-hACE2 animals to 2 different doses of SARS-CoV-2 resulted in acute disease, including weight loss, lung injury, brain infection, and lethality. Vasculitis was the most prominent finding in the lungs of infected mice. Transcriptomic analysis from lungs of infected animals showed increases in transcripts involved in lung injury and inflammatory cytokines. In the low-dose challenge groups, there was a survival advantage in the female mice, with 60% surviving infection, whereas all male mice succumbed to disease. Male mice that succumbed to disease had higher levels of inflammatory transcripts compared with female mice. To our knowledge, this is the first highly lethal murine infection model for SARS-CoV-2 and should be valuable for the study of SARS-CoV-2 pathogenesis and for the assessment of MCMs.
Asunto(s)
Causas de Muerte , Infecciones por Coronavirus/patología , Progresión de la Enfermedad , Peptidil-Dipeptidasa A/genética , Neumonía Viral/patología , Síndrome Respiratorio Agudo Grave/patología , Enzima Convertidora de Angiotensina 2 , Animales , COVID-19 , Infecciones por Coronavirus/fisiopatología , Modelos Animales de Enfermedad , Femenino , Humanos , Pulmón/patología , Masculino , Ratones , Ratones Transgénicos , Pandemias , Neumonía Viral/fisiopatología , Síndrome Respiratorio Agudo Grave/fisiopatología , Índice de Severidad de la Enfermedad , Tasa de Supervivencia , Replicación Viral/genéticaRESUMEN
The New World alphaviruses, Venezuelan, eastern and western equine encephalitis viruses (VEEV, EEEV, and WEEV), are important human pathogens due to their ability to cause varying levels of morbidity and mortality in humans. There is also concern about VEEV and EEEV being used as bioweapons. Currently, a FDA-approved antiviral is lacking for New World alphaviruses. In this review, the function of each viral protein is discussed with an emphasis on how these functions can be targeted by therapeutics. Both direct acting antivirals as well as inhibitors that impact host protein interactions with viral proteins are described. Non-structural protein 3 (nsP3), capsid, and E2 proteins have garnered attention in recent years, whereas little is known regarding host protein interactions of the other viral proteins and is an important avenue for future study.
Asunto(s)
Infecciones por Alphavirus/tratamiento farmacológico , Alphavirus/efectos de los fármacos , Antivirales/uso terapéutico , Proteínas Virales/química , Alphavirus/fisiología , Animales , Antivirales/farmacología , Línea Celular , Ensayos Clínicos como Asunto , Interacciones Microbiota-Huesped/efectos de los fármacos , Humanos , Ratones , Replicación Viral/efectos de los fármacosRESUMEN
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.
RESUMEN
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.