RESUMEN
Chikungunya virus (CHIKV) is an emerging/re-emerging mosquito-borne pathogen responsible for explosive epidemics of febrile illness characterized by debilitating polyarthralgia and the risk of lethal infection among the most severe cases. Despite the public health risk posed by CHIKV, no vaccine is currently available. Using a site-directed hydrogen peroxide-based inactivation approach, we developed a new CHIKV vaccine, HydroVax-CHIKV. This vaccine technology was compared to other common virus inactivation approaches including ß-propiolactone (BPL), formaldehyde, heat, and ultraviolet (UV) irradiation. Heat, UV, and BPL were efficient at inactivating CHIKV-181/25 but caused substantial damage to neutralizing epitopes and failed to induce high-titer neutralizing antibodies in vaccinated mice. HydroVax-CHIKV and formaldehyde-inactivated CHIKV retained intact neutralizing epitopes similar to live virus controls but the HydroVax-CHIKV approach demonstrated a more rapid rate of virus inactivation. HydroVax-CHIKV vaccination induced high neutralizing responses to homologous and heterologous CHIKV clades as well as to other alphaviruses including Mayaro virus, O'nyong'nyong virus, and Una virus. Following heterologous infection with CHIKV-SL15649, HydroVax-CHIKV-immunized mice were protected against viremia, CHIKV-associated arthritic disease, and lethal CHIKV infection by an antibody-dependent mechanism. In contrast, animals vaccinated with Heat- or UV-inactivated virus showed no protection against viremia in addition to demonstrating significantly exacerbated CD4+ T cell-mediated footpad swelling after CHIKV infection. Together, these results demonstrate the risks associated with using suboptimal inactivation methods that fail to elicit protective neutralizing antibody responses and show that HydroVax-CHIKV represents a promising new vaccine candidate for prevention of CHIKV-associated disease.
Asunto(s)
Fiebre Chikungunya , Virus Chikungunya , Vacunas Virales , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Fiebre Chikungunya/prevención & control , Epítopos , Formaldehído , Ratones , ViremiaRESUMEN
Human cytomegalovirus (HCMV) microRNAs (miRNAs) significantly rewire host signaling pathways to support the viral lifecycle and regulate host cell responses. Here we show that SMAD3 expression is regulated by HCMV miR-UL22A and contributes to the IRF7-mediated induction of type I IFNs and IFN-stimulated genes (ISGs) in human fibroblasts. Addition of exogenous TGFß interferes with the replication of a miR-UL22A mutant virus in a SMAD3-dependent manner in wild type fibroblasts, but not in cells lacking IRF7, indicating that downregulation of SMAD3 expression to limit IFN induction is important for efficient lytic replication. These findings uncover a novel interplay between SMAD3 and innate immunity during HCMV infection and highlight the role of viral miRNAs in modulating these responses.
Asunto(s)
Infecciones por Citomegalovirus/microbiología , Citomegalovirus/fisiología , Fibroblastos/microbiología , Inmunidad Innata/inmunología , Interferón Tipo I/metabolismo , MicroARNs/genética , Factor de Crecimiento Transformador beta/metabolismo , Fibroblastos/inmunología , Fibroblastos/patología , Interacciones Huésped-Patógeno , Humanos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Interferón Tipo I/genética , Proteína smad3/genética , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/genética , Fenómenos Fisiológicos de los VirusRESUMEN
Chikungunya is a clinically and economically important arbovirus that has spread globally in the twenty-first century. While uncommonly fatal, infection with the virus can lead to incapacitating arthralgia that can persist for months to years. The adverse impacts of viral spread are most severe in developing low- and middle-income countries in which medical infrastructure is insufficient and manual labor is an economic driver. Unfortunately, no prophylactic or therapeutic treatments are approved for human use to combat the virus. Historically, vaccination has proven to be the most efficient and successful strategy for protecting populations and eradicating infectious disease. A large and diverse range of promising vaccination approaches for use against Chikungunya has emerged in recent years and been shown to safely elicit protective immune responses in animal models and humans. Importantly, many of these are based on technologies that have been clinically approved for use against other pathogens. Furthermore, clinical trials are currently ongoing for a subset of these. The purpose of this review is to provide a description of the relevant immunobiology of Chikungunya infection, to present immune-stimulating technologies that have been successfully employed to protect against infection, and discuss priorities and challenges regarding the future development of a vaccine for clinical use.
Asunto(s)
Fiebre Chikungunya , Virus Chikungunya , Vacunas Virales , Animales , Fiebre Chikungunya/prevención & control , Humanos , VacunaciónRESUMEN
Venezuelan equine encephalitis virus (VEEV) is a reemerging alphavirus that can cause encephalitis resulting in severe human morbidity and mortality. Using a high-throughput cell-based screen, we identified a quinolinone compound that protected against VEEV-induced cytopathic effects. Analysis of viral replication in cells identified several quinolinone compounds with potent inhibitory activity against vaccine and virulent strains of VEEV. These quinolinones also displayed inhibitory activity against additional alphaviruses, such as Mayaro virus and Ross River virus, although the potency was greatly reduced. Time-of-addition studies indicated that these compounds inhibit the early-to-mid stage of viral replication. Deep sequencing and reverse genetics studies identified two unique resistance mutations in the nsP2 gene (Y102S/C; stalk domain) that conferred VEEV resistance on this chemical series. Moreover, introduction of a K102Y mutation into the nsP2 gene enhanced the sensitivity of chikungunya virus (CHIKV) to this chemical series. Computational modeling of CHIKV and VEEV nsP2 identified a highly probable docking alignment for the quinolinone compounds that require a tyrosine residue at position 102 within the helicase stalk domain. These studies identified a class of compounds with antiviral activity against VEEV and other alphaviruses and provide further evidence that therapeutics targeting nsP2 may be useful against alphavirus infection.
Asunto(s)
Virus Chikungunya , Virus de la Encefalitis Equina Venezolana , Quinolonas , Animales , Antivirales/farmacología , Virus de la Encefalitis Equina Venezolana/genética , Caballos , Humanos , Quinolonas/farmacología , Replicación ViralRESUMEN
Alphaviruses are arthropod-transmitted RNA viruses that can cause arthralgia, myalgia, and encephalitis in humans. Since the role of cellular kinases in alphavirus replication is unknown, we profiled kinetic changes in host kinase abundance and phosphorylation following chikungunya virus (CHIKV) infection of fibroblasts. Based upon the results of this study, we treated CHIKV-infected cells with kinase inhibitors targeting the Src family kinase (SFK)-phosphatidylinositol 3-kinase (PI3K)-AKT-mTORC signaling pathways. Treatment of cells with SFK inhibitors blocked the replication of CHIKV as well as multiple other alphaviruses, including Mayaro virus, O'nyong-nyong virus, Ross River virus, and Venezuelan equine encephalitis virus. Dissecting the effect of SFK inhibition on alphavirus replication, we found that viral structural protein levels were significantly reduced, but synthesis of viral genomic and subgenomic RNAs was unaffected. By measuring the association of viral RNA with polyribosomes, we found that the SFK inhibitor dasatinib blocks alphavirus subgenomic RNA translation. Our results demonstrate a role for SFK signaling in alphavirus subgenomic RNA translation and replication. Targeting host factors involved in alphavirus replication represents an innovative, perhaps paradigm-shifting, strategy for exploring the replication of CHIKV and other alphaviruses while promoting antiviral therapeutic development.
Asunto(s)
Infecciones por Alphavirus/tratamiento farmacológico , Alphavirus/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , Familia-src Quinasas/genética , Alphavirus/genética , Infecciones por Alphavirus/virología , Animales , Antivirales/farmacología , Línea Celular , Chlorocebus aethiops , Genoma Viral/efectos de los fármacos , Genoma Viral/genética , Humanos , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , ARN Viral/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Células Vero , Proteínas Virales/genética , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
The type I interferon (IFN) system represents an essential innate immune response that renders cells resistant to virus growth via the molecular actions of IFN-induced effector proteins. IFN-mediated cellular states inhibit growth of numerous and diverse virus types, including those of known pathogenicity as well as potentially emerging agents. As such, targeted pharmacologic activation of the IFN response may represent a novel therapeutic strategy to prevent infection or spread of clinically impactful viruses. In light of this, we employed a high-throughput screen to identify small molecules capable of permeating the cell and of activating IFN-dependent signaling processes. Here we report the identification and characterization of N-(methylcarbamoyl)-2-{[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-2-phenylacetamide (referred to as C11), a novel compound capable of inducing IFN secretion from human cells. Using reverse genetics-based loss-of-function assays, we show that C11 activates the type I IFN response in a manner that requires the adaptor protein STING but not the alternative adaptors MAVS and TRIF. Importantly, treatment of cells with C11 generated a cellular state that potently blocked replication of multiple emerging alphavirus types, including chikungunya, Ross River, Venezuelan equine encephalitis, Mayaro, and O'nyong-nyong viruses. The antiviral effects of C11 were subsequently abrogated in cells lacking STING or the type I IFN receptor, indicating that they are mediated, at least predominantly, by way of STING-mediated IFN secretion and subsequent autocrine/paracrine signaling. This work also allowed characterization of differential antiviral roles of innate immune signaling adaptors and IFN-mediated responses and identified MAVS as being crucial to cellular resistance to alphavirus infection.IMPORTANCE Due to the increase in emerging arthropod-borne viruses, such as chikungunya virus, that lack FDA-approved therapeutics and vaccines, it is important to better understand the signaling pathways that lead to clearance of virus. Here we show that C11 treatment makes human cells refractory to replication of a number of these viruses, which supports its value in increasing our understanding of the immune response and viral pathogenesis required to establish host infection. We also show that C11 depends on signaling through STING to produce antiviral type I interferon, which further supports its potential as a therapeutic drug or research tool.
Asunto(s)
Alphavirus/metabolismo , Antivirales/farmacología , Fibroblastos/metabolismo , Proteínas de la Membrana/agonistas , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Comunicación Autocrina/efectos de los fármacos , Comunicación Autocrina/genética , Fibroblastos/patología , Fibroblastos/virología , Humanos , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Comunicación Paracrina/efectos de los fármacos , Comunicación Paracrina/genética , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Transducción de Señal/genéticaRESUMEN
Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1-7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.
Asunto(s)
Infección por el Virus Zika/patología , Infección por el Virus Zika/virología , Animales , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Hibridación in Situ , Macaca mulatta , Masculino , Pruebas de Neutralización , Reacción en Cadena de la Polimerasa , Viremia/virología , Virus ZikaRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1006219.].
RESUMEN
Chikungunya virus (CHIKV) is a re-emerging global pathogen with pandemic potential, which causes fever, rash and debilitating arthralgia. Older adults over 65 years are particularly susceptible to severe and chronic CHIKV disease (CHIKVD), accounting for >90% of all CHIKV-related deaths. There are currently no approved vaccines or antiviral treatments available to limit chronic CHIKVD. Here we show that in old mice excessive, dysregulated TGFß production during acute infection leads to a reduced immune response and subsequent chronic disease. Humans suffering from CHIKV infection also exhibited high TGFß levels and a pronounced age-related defect in neutralizing anti-CHIKV antibody production. In vivo reduction of TGFß levels minimized acute joint swelling, restored neutralizing antibody production and diminished chronic joint pathology in old mice. This study identifies increased and dysregulated TGFß secretion as one key mechanism contributing to the age-related loss of protective anti-CHIKV-immunity leading to chronic CHIKVD.
Asunto(s)
Envejecimiento/inmunología , Fiebre Chikungunya/inmunología , Factor de Crecimiento Transformador beta/inmunología , Adulto , Anciano , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Virus Chikungunya , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
Human Cytomegalovirus (HCMV) encodes multiple microRNAs (miRNAs) whose functions are just beginning to be uncovered. Using in silico approaches, we identified the Toll-Like Receptor (TLR) innate immunity pathway as a possible target of HCMV miRNAs. Luciferase reporter assay screens further identified TLR2 as a target of HCMV miR-UL112-3p. TLR2 plays a major role in innate immune response by detecting both bacterial and viral ligands, including HCMV envelope proteins gB and gH. TLR2 activates a variety of signal transduction routes including the NFκB pathway. Furthermore, TLR2 plays an important role in controlling CMV infection both in humans and in mice. Immunoblot analysis of cells transfected with a miR-UL112-3p mimic revealed that endogenous TLR2 is down-regulated by miR-UL112-3p with similar efficiency as a TLR2-targeting siRNA (siTLR2). We next found that TLR2 protein level decreases at late times during HCMV infection and correlates with miR-UL112-3p accumulation in fibroblasts and monocytic THP1 cells. Confirming direct miR-UL112-3p targeting, down-regulation of endogenous TLR2 was not observed in cells infected with HCMV mutants deficient in miR-UL112-3p expression, but transfection of miR-UL112-3p in these cells restored TLR2 down-regulation. Using a NFκB reporter cell line, we found that miR-UL112-3p transfection significantly inhibited NFκB-dependent luciferase activity with similar efficiency as siTLR2. Consistent with this observation, miR-UL112-3p transfection significantly reduced the expression of multiple cytokines (IL-1ß, IL-6 and IL-8) upon stimulation with a TLR2 agonist. Finally, miR-UL112-3p transfection significantly inhibited the TLR2-induced post-translational activation of IRAK1, a kinase located in the upstream section of the TLR2/NFκB signaling axis. To our knowledge, this is the first identified mechanism of TLR2 modulation by HCMV and is the first report of functional targeting of TLR2 by a viral miRNA. These results provide a novel mechanism through which a HCMV miRNA regulates the innate immune response by down-regulating TLR-2 expression.
Asunto(s)
Citomegalovirus/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , MicroARNs/metabolismo , Interferencia de ARN , ARN Viral/metabolismo , Transducción de Señal , Receptor Toll-Like 2/antagonistas & inhibidores , Regiones no Traducidas 3' , Células Cultivadas , Fibroblastos/citología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Genes Reporteros , Células HEK293 , Humanos , Inmunidad Innata , Quinasas Asociadas a Receptores de Interleucina-1/genética , Ligandos , MicroARNs/genética , Monocitos/citología , Monocitos/inmunología , Monocitos/metabolismo , Mutación , FN-kappa B/genética , FN-kappa B/metabolismo , ARN Interferente Pequeño , ARN Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismoRESUMEN
Varicella zoster virus (VZV) causes chickenpox in humans and, subsequently, establishes latency in the sensory ganglia from where it reactivates to cause herpes zoster. Infection of rhesus macaques with simian varicella virus (SVV) recapitulates VZV pathogenesis in humans thus representing a suitable animal model for VZV infection. While the type I interferon (IFN) response has been shown to affect VZV replication, the virus employs counter mechanisms to prevent the induction of anti-viral IFN stimulated genes (ISG). Here, we demonstrate that SVV inhibits type I IFN-activated signal transduction via the JAK-STAT pathway. SVV-infected rhesus fibroblasts were refractory to IFN stimulation displaying reduced protein levels of IRF9 and lacking STAT2 phosphorylation. Since previous work implicated involvement of the VZV immediate early gene product ORF63 in preventing ISG-induction we studied the role of SVV ORF63 in generating resistance to IFN treatment. Interestingly, SVV ORF63 did not affect STAT2 phosphorylation but caused IRF9 degradation in a proteasome-dependent manner, suggesting that SVV employs multiple mechanisms to counteract the effect of IFN. Control of SVV ORF63 protein levels via fusion to a dihydrofolate reductase (DHFR)-degradation domain additionally confirmed its requirement for viral replication. Our results also show a prominent reduction of IRF9 and inhibition of STAT2 phosphorylation in VZV-infected cells. In addition, cells expressing VZV ORF63 blocked IFN-stimulation and displayed reduced levels of the IRF9 protein. Taken together, our data suggest that varicella ORF63 prevents ISG-induction both directly via IRF9 degradation and indirectly via transcriptional control of viral proteins that interfere with STAT2 phosphorylation. SVV and VZV thus encode multiple viral gene products that tightly control IFN-induced anti-viral responses.
Asunto(s)
Infecciones por Herpesviridae/metabolismo , Interacciones Huésped-Patógeno , Interferón Tipo I/metabolismo , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Varicellovirus/fisiología , Animales , Línea Celular , Cercopithecinae , Varicela/inmunología , Varicela/metabolismo , Varicela/patología , Varicela/virología , ADN Recombinante/metabolismo , Regulación Viral de la Expresión Génica , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Herpesvirus Humano 3/inmunología , Herpesvirus Humano 3/fisiología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Inmunidad Innata , Interferón Tipo I/antagonistas & inhibidores , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/antagonistas & inhibidores , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/metabolismo , Fosforilación , Complejo de la Endopetidasa Proteasomal , Procesamiento Proteico-Postraduccional , Proteolisis , Proteínas Recombinantes/metabolismo , Factores de Transcripción STAT/genética , Varicellovirus/inmunologíaRESUMEN
Pharmacologic stimulation of innate immune processes represents an attractive strategy to achieve multiple therapeutic outcomes including inhibition of virus replication, boosting antitumor immunity, and enhancing vaccine immunogenicity. In light of this we sought to identify small molecules capable of activating the type I interferon (IFN) response by way of the transcription factor IFN regulatory factor 3 (IRF3). A high throughput in vitro screen yielded 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (referred to herein as G10), which was found to trigger IRF3/IFN-associated transcription in human fibroblasts. Further examination of the cellular response to this molecule revealed expression of multiple IRF3-dependent antiviral effector genes as well as type I and III IFN subtypes. This led to the establishment of a cellular state that prevented replication of emerging Alphavirus species including Chikungunya virus, Venezuelan Equine Encephalitis virus, and Sindbis virus. To define cellular proteins essential to elicitation of the antiviral activity by the compound we employed a reverse genetics approach that utilized genome editing via CRISPR/Cas9 technology. This allowed the identification of IRF3, the IRF3-activating adaptor molecule STING, and the IFN-associated transcription factor STAT1 as required for observed gene induction and antiviral effects. Biochemical analysis indicates that G10 does not bind to STING directly, however. Thus the compound may represent the first synthetic small molecule characterized as an indirect activator of human STING-dependent phenotypes. In vivo stimulation of STING-dependent activity by an unrelated small molecule in a mouse model of Chikungunya virus infection blocked viremia demonstrating that pharmacologic activation of this signaling pathway may represent a feasible strategy for combating emerging Alphaviruses.
Asunto(s)
Antivirales/farmacología , Fiebre Chikungunya/inmunología , Proteínas de la Membrana/agonistas , Transducción de Señal/inmunología , Tiazinas/farmacología , Alphavirus/inmunología , Infecciones por Alphavirus/inmunología , Animales , Células Cultivadas , Virus Chikungunya/inmunología , Ensayos Analíticos de Alto Rendimiento , Humanos , Immunoblotting , Factor 3 Regulador del Interferón/inmunología , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
UNLABELLED: Varicella-zoster virus (VZV) causes chickenpox upon primary infection and establishes latency in ganglia. Reactivation from latency causes herpes zoster, which may be complicated by postherpetic neuralgia. Innate immunity mediated by interferon and proinflammatory cytokines represents the first line of immune defense upon infection and reactivation. VZV is known to interfere with multiple innate immune signaling pathways, including the central transcription factor NF-κB. However, the role of these inhibitory mechanisms in vivo is unknown. Simian varicella virus (SVV) infection of rhesus macaques recapitulates key aspects of VZV pathogenesis, and this model thus permits examination of the role of immune evasion mechanisms in vivo. Here, we compare SVV and VZV with respect to interference with NF-κB activation. We demonstrate that both viruses prevent ubiquitination of the NF-κB inhibitor IκBα, whereas SVV additionally prevents IκBα phosphorylation. We show that the ORF61 proteins of VZV and SVV are sufficient to prevent IκBα ubiquitination upon ectopic expression. We further demonstrate that SVV ORF61 interacts with ß-TrCP, a subunit of the SCF ubiquitin ligase complex that mediates the degradation of IκBα. This interaction seems to inactivate SCF-mediated protein degradation in general, since the unrelated ß-TrCP target Snail is also stabilized by ORF61. In addition to ORF61, SVV seems to encode additional inhibitors of the NF-κB pathway, since SVV with ORF61 deleted still prevented IκBα phosphorylation and degradation. Taken together, our data demonstrate that SVV interferes with tumor necrosis factor alpha (TNF-α)-induced NF-κB activation at multiple levels, which is consistent with the importance of these countermechanisms for varicella virus infection. IMPORTANCE: The role of innate immunity during the establishment of primary infection, latency, and reactivation by varicella-zoster virus (VZV) is incompletely understood. Since infection of rhesus macaques by simian varicella virus (SVV) is used as an animal model of VZV infection, we characterized the molecular mechanism by which SVV interferes with innate immune activation. Specifically, we studied how SVV prevents activation of the transcription factor NF-κB, a central factor in eliciting proinflammatory responses. The identification of molecular mechanisms that counteract innate immunity might ultimately lead to better vaccines and treatments for VZV, since overcoming these mechanisms, either by small-molecule inhibition or by genetic modification of vaccine strains, is expected to reduce the pathogenic potential of VZV. Moreover, using SVV infection of rhesus macaques, it will be possible to study how increasing the vulnerability of varicella viruses to innate immunity will impact viral pathogenesis.
Asunto(s)
Herpesvirus Humano 3/genética , Proteínas I-kappa B/metabolismo , FN-kappa B/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Varicela/inmunología , Varicela/virología , Modelos Animales de Enfermedad , Activación Enzimática , Eliminación de Gen , Células HEK293 , Herpesvirus Humano 3/inmunología , Humanos , Evasión Inmune/inmunología , Inmunidad Innata/inmunología , Macaca mulatta , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Fosforilación , Proteínas Ligasas SKP Cullina F-box/metabolismo , Factores de Transcripción de la Familia Snail , Factores de Transcripción/metabolismo , Ubiquitinación , Carga Viral , Proteínas Virales/genética , Activación Viral/inmunología , Proteínas con Repetición de beta-Transducina/metabolismoRESUMEN
UNLABELLED: The cytosolic RIG-I (retinoic acid-inducible gene I) receptor plays a pivotal role in the initiation of the immune response against RNA virus infection by recognizing short 5'-triphosphate (5'ppp)-containing viral RNA and activating the host antiviral innate response. In the present study, we generated novel 5'ppp RIG-I agonists of varieous lengths, structures, and sequences and evaluated the generation of the antiviral and inflammatory responses in human epithelial A549 cells, human innate immune primary cells, and murine models of influenza and chikungunya viral pathogenesis. A 99-nucleotide, uridine-rich hairpin 5'pppRNA termed M8 stimulated an extensive and robust interferon response compared to other modified 5'pppRNA structures, RIG-I aptamers, or poly(I·C). Interestingly, manipulation of the primary RNA sequence alone was sufficient to modulate antiviral activity and inflammatory response, in a manner dependent exclusively on RIG-I and independent of MDA5 and TLR3. Both prophylactic and therapeutic administration of M8 effectively inhibited influenza virus and dengue virus replication in vitro. Furthermore, multiple strains of influenza virus that were resistant to oseltamivir, an FDA-approved therapeutic treatment for influenza, were highly sensitive to inhibition by M8. Finally, prophylactic M8 treatment in vivo prolonged survival and reduced lung viral titers of mice challenged with influenza virus, as well as reducing chikungunya virus-associated foot swelling and viral load. Altogether, these results demonstrate that 5'pppRNA can be rationally designed to achieve a maximal RIG-I-mediated protective antiviral response against human-pathogenic RNA viruses. IMPORTANCE: The development of novel therapeutics to treat human-pathogenic RNA viral infections is an important goal to reduce spread of infection and to improve human health and safety. This study investigated the design of an RNA agonist with enhanced antiviral and inflammatory properties against influenza, dengue, and chikungunya viruses. A novel, sequence-dependent, uridine-rich RIG-I agonist generated a protective antiviral response in vitro and in vivo and was effective at concentrations 100-fold lower than prototype sequences or other RNA agonists, highlighting the robust activity and potential clinical use of the 5'pppRNA against RNA virus infection. Altogether, the results identify a novel, sequence-specific RIG-I agonist as an attractive therapeutic candidate for the treatment of a broad range of RNA viruses, a pressing issue in which a need for new and more effective options persists.
Asunto(s)
Virus Chikungunya/inmunología , ARN Helicasas DEAD-box/inmunología , Virus del Dengue/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , ARN Viral/agonistas , ARN Viral/inmunología , Virosis/inmunología , Animales , Línea Celular , Virus Chikungunya/química , Virus Chikungunya/genética , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , Virus del Dengue/química , Virus del Dengue/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/química , Subtipo H1N1 del Virus de la Influenza A/genética , Ratones , Ratones Endogámicos BALB C , Conformación de Ácido Nucleico , ARN Viral/genética , Receptores Inmunológicos , Virosis/genética , Virosis/virologíaRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related gamma-2 herpesvirus rhesus macaque (RM) rhadinovirus (RRV) are the only known viruses to encode viral homologues of the cellular interferon (IFN) regulatory factors (IRFs). Recent characterization of a viral IRF (vIRF) deletion clone of RRV (vIRF-knockout RRV [vIRF-ko RRV]) demonstrated that vIRFs inhibit induction of type I and type II IFNs during RRV infection of peripheral blood mononuclear cells. Because the IFN response is a key component to a host's antiviral defenses, this study has investigated the role of vIRFs in viral replication and the development of the immune response during in vivo infection in RMs, the natural host of RRV. Experimental infection of RMs with vIRF-ko RRV resulted in decreased viral loads and diminished B cell hyperplasia, a characteristic pathology during acute RRV infection that often develops into more severe lymphoproliferative disorders in immune-compromised animals, similar to pathologies in KSHV-infected individuals. Moreover, in vivo infection with vIRF-ko RRV resulted in earlier and sustained production of proinflammatory cytokines and earlier induction of an anti-RRV T cell response compared to wild-type RRV infection. These findings reveal the broad impact that vIRFs have on pathogenesis and the immune response in vivo and are the first to validate the importance of vIRFs during de novo infection in the host.
Asunto(s)
Modelos Animales de Enfermedad , Infecciones por Herpesviridae/inmunología , Factores Reguladores del Interferón/inmunología , Macaca mulatta , Rhadinovirus/inmunología , Proteínas Virales/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/virología , Infecciones por Herpesviridae/virología , Humanos , Factores Reguladores del Interferón/genética , Interferones/inmunología , Rhadinovirus/genética , Proteínas Virales/genéticaRESUMEN
Human cytomegalovirus (HCMV) is linked to the acceleration of vascular diseases such as atherosclerosis and transplant vasculopathy. One of the hallmarks of these diseases is angiogenesis (AG) and neovessel formation. Endothelial cells (ECs) are an integral part of AG and are sites of HCMV persistence. AG requires multiple synchronous processes that include EC proliferation, migration, and vessel stabilization. Virus-free supernatant (secretome) from HCMV-infected ECs induces AG. To identify factor(s) involved in this process, we performed a human cytokine array. Several cytokines were significantly induced in the HCMV secretomes including interleukin-6 (IL-6), granulocyte macrophage colony-stimulating factor, and IL-8/CXCL8. Using in vitro AG assays, neutralization of IL-6 significantly reduced neovessel formation. Addition of the HCMV secretome to preformed vessels extended neovessel survival, but this effect was blocked by neutralization of IL-6. In these cells, IL-6 prevented apoptosis by blocking caspase-3 and -7 activation through the induction of survivin. Neutralization of IL-6 receptor on ECs abolished the ability of HCMV secretome to increase survivin expression and activated effector caspases. Moreover, survivin shRNA expression induced rapid regression of tubule capillary networks in ECs stimulated with HCMV secretome and activated effector caspases. These observations may explain how CMV accelerates vascular disease despite limited infection in tissues.
Asunto(s)
Inductores de la Angiogénesis/metabolismo , Citomegalovirus/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Interleucina-6/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Apoptosis , Western Blotting , Caspasa 3/metabolismo , Adhesión Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Infecciones por Citomegalovirus/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis , Interleucina-8/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Receptores de Interleucina-6/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Survivin , Venas Umbilicales/citología , Venas Umbilicales/metabolismoRESUMEN
Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes debilitating and persistent arthritogenic disease. While MAYV was previously reported to infect non-human primates (NHP), characterization of MAYV pathogenesis is currently lacking. Therefore, in this study we characterized MAYV infection and immunity in rhesus macaques. To inform the selection of a viral strain for NHP experiments, we evaluated five MAYV strains in C57BL/6 mice and showed that MAYV strain BeAr505411 induced robust tissue dissemination and disease. Three male rhesus macaques were subcutaneously challenged with 105 plaque-forming units of this strain into the arms. Peak plasma viremia occurred at 2 days post-infection (dpi). NHPs were taken to necropsy at 10 dpi to assess viral dissemination, which included the muscles and joints, lymphoid tissues, major organs, male reproductive tissues, as well as peripheral and central nervous system tissues. Histological examination demonstrated that MAYV infection was associated with appendicular joint and muscle inflammation as well as presence of perivascular inflammation in a wide variety of tissues. One animal developed a maculopapular rash and two NHP had viral RNA detected in upper torso skin samples, which was associated with the presence of perivascular and perifollicular lymphocytic aggregation. Analysis of longitudinal peripheral blood samples indicated a robust innate and adaptive immune activation, including the presence of anti-MAYV neutralizing antibodies with activity against related Una virus and chikungunya virus. Inflammatory cytokines and monocyte activation also peaked coincident with viremia, which was well supported by our transcriptomic analysis highlighting enrichment of interferon signaling and other antiviral processes at 2 days post MAYV infection. The rhesus macaque model of MAYV infection recapitulates many of the aspects of human infection and is poised to facilitate the evaluation of novel therapies and vaccines targeting this re-emerging virus.
Asunto(s)
Infecciones por Alphavirus , Alphavirus , Virus Chikungunya , Animales , Ratones , Masculino , Macaca mulatta , Viremia , Ratones Endogámicos C57BL , Anticuerpos AntiviralesRESUMEN
Although the lineages of human papillomavirus type 31 (HPV31) variants are recognized, their clinical relevance is unknown. The purpose of our study was to examine risk of cervical intraepithelial neoplasia Grades 2-3 (CIN2/3) by HPV31 variants. Study subjects were women who participated in the atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesion Triage Study and who had HPV31 infections detected at one or more visits. They were followed semi-annually over 2 years for detection of HPV DNA and cervical lesion. HPV31 isolates were characterized by DNA sequencing and assigned into 1 of 3 variant lineages. CIN2/3 was histologically confirmed in 127 (27.0%) of the 470 HPV31-positive women, 83 diagnosed at the first HPV31-positive visit and 44 thereafter. The odds ratio for the association of 2-year cumulative risk of CIN2/3 was 1.7 (95% CI: 1.0-2.9) for infections with A variants and 2.2 (95% CI: 1.2-3.9) for infections with B variants as compared to those with C variants. Among women without CIN2/3 at the first HPV31-positive visit, the risk of subsequent CIN2/3 was 2.2-fold greater for those with A variants (95% CI: 1.0-4.8) and 2.0-fold greater for those with B variants (95% CI: 0.9-4.9) as compared to those with C variants. Similar associations were observed when CIN3 was used as the endpoint. The findings from our study help to tag HPV31 variants that differ in risk of CIN2/3 and to explain in part why some HPV31 infections regress spontaneously and others lead to disease progression.
Asunto(s)
Papillomavirus Humano 31/genética , Displasia del Cuello del Útero/patología , Displasia del Cuello del Útero/virología , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virología , Adolescente , Adulto , Femenino , Papillomavirus Humano 31/clasificación , Humanos , Estimación de Kaplan-Meier , Clasificación del Tumor , Infecciones por Papillomavirus , Riesgo , Neoplasias del Cuello Uterino/mortalidad , Frotis Vaginal , Adulto Joven , Displasia del Cuello del Útero/mortalidadRESUMEN
Chikungunya virus (CHIKV) is an arthritogenic mosquito-transmitted alphavirus that is undergoing reemergence in areas around the Indian Ocean. Despite the current and potential danger posed by this virus, we know surprisingly little about the induction and evasion of CHIKV-associated antiviral immune responses. With this in mind we investigated innate immune reactions to CHIKV in human fibroblasts, a demonstrable in vivo target of virus replication and spread. We show that CHIKV infection leads to activation of the transcription factor interferon regulatory factor 3 (IRF3) and subsequent transcription of IRF3-dependent antiviral genes, including beta interferon (IFN-ß). IRF3 activation occurs by way of a virus-induced innate immune signaling pathway that includes the adaptor molecule interferon promoter stimulator 1 (IPS-1). Despite strong transcriptional upregulation of these genes, however, translation of the corresponding proteins is not observed. We further demonstrate that translation of cellular (but not viral) genes is blocked during infection and that although CHIKV is found to trigger inactivation of the translational molecule eukaryotic initiation factor subunit 2α by way of the double-stranded RNA sensor protein kinase R, this response is not required for the block to protein synthesis. Furthermore, overall diminution of cellular RNA synthesis is also observed in the presence of CHIKV and transcription of IRF3-dependent antiviral genes appears specifically blocked late in infection. We hypothesize that the observed absence of IFN-ß and antiviral proteins during infection results from an evasion mechanism exhibited by CHIKV that is dependent on widespread shutoff of cellular protein synthesis and a targeted block to late synthesis of antiviral mRNA transcripts.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Virus Chikungunya/patogenicidad , Regulación de la Expresión Génica , Evasión Inmune/inmunología , Inmunidad Innata/inmunología , Biosíntesis de Proteínas/inmunología , eIF-2 Quinasa/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular , Células Cultivadas , Virus Chikungunya/inmunología , Cricetinae , Fibroblastos/inmunología , Fibroblastos/virología , Humanos , Interferón beta , Proteínas/genética , Proteínas/metabolismoRESUMEN
We sought to develop a small-molecule activator of interferon regulatory factor 3 (IRF3), an essential innate immune transcription factor, which could potentially be used therapeutically in multiple disease settings. Using a high-throughput screen, we identified small-molecule entities that activate a type I interferon response, with minimal off-target NFκB activation. We identified 399 compounds at a hit rate of 0.24% from singlicate primary screening. Secondary screening included the primary hits and additional compounds with similar chemical structures obtained from other library sources and resulted in 142 candidate compounds. The hit compounds were sorted and ranked to identify compound groups with activity in both human and mouse backgrounds to facilitate animal model engagement for translational development. Chemical modifications within two groups of small molecules produced leads with improved activity over original hits. Furthermore, these leads demonstrated activity in ex vivo cytokine release assays from human blood- and mouse bone marrow-derived macrophages. Dependence on IRF3 was demonstrated using bone marrow-derived macrophages from IRF3-deficient mice, which were not responsive to the molecules. To identify the upstream pathway leading to IRF3 activation, we used a library of CRISPR knockout cell lines to test the key innate immune adaptor and receptor molecules. These studies indicated a surprising toll-interleukin-1 receptor-domain-containing-adapter-inducing interferon-ß-dependent but TLR3/4-independent mechanism of IRF3 activation.