RESUMEN
There is growing interest in evaluating the safety and therapeutic potential of existing treatments such as tocilizumab (TCZ), an IL-6 receptor antagonist used to treat inflammatory diseases. However, there have been reports of increased inflammation in patients with HTLV-1 uveitis after TCZ treatment, and its ocular safety in the HTLV-1 infected state remains unknown. This study focused on assessing the impact of TCZ on HTLV-1-infected ocular cells using an in vitro model in which retinal pigment epithelial cells were cocultured with irradiated HTLV-1-infected T-cell lines. TCZ did not significantly affect cellular viability, inflammatory markers, or HTLV-1 proviral loads at various concentrations (25/50/100 µg/ml), indicating no increased risk of HTLV-1 viral infection and no exacerbation of the inflammatory aspects of HTLV-1 infection in the ocular cells. These promising results support the potential of TCZ as a safe treatment option for HTLV-1-infected patients, particularly those with eye infections.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Infecciones por HTLV-I , Virus Linfotrópico T Tipo 1 Humano , Humanos , Infecciones por HTLV-I/tratamiento farmacológico , Infecciones por HTLV-I/inmunología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/efectos adversos , Línea Celular , Linfocitos T/inmunología , Linfocitos T/efectos de los fármacos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/virología , Epitelio Pigmentado de la Retina/inmunología , Técnicas de Cocultivo , Supervivencia Celular/efectos de los fármacos , Carga Viral/efectos de los fármacosRESUMEN
Although Herpes simplex virus type 1 (HSV-1) has been deeply studied, significant gaps remain in the fundamental understanding of HSV-host interactions: our work focused on studying the Infected Cell Protein 27 (ICP27) as an inhibitor of the Absent-in-melanoma-2 (AIM 2) inflammasome pathway, leading to reduced pro-inflammatory cytokines that influence the activation of a protective innate immune response to infection. To assess the inhibition of the inflammasome by the ICP27, hTert-immortalized Retinal Pigment Epithelial cells (hTert-RPE 1) infected with HSV-1 wild type were compared to HSV-1 lacking functional ICP27 (HSV-1∆ICP27) infected cells. The activation of the inflammasome by HSV-1∆ICP27 was demonstrated by quantifying the gene and protein expression of the inflammasome constituents using real-time PCR and Western blot. The detection of the cleavage of the pro-caspase-1 into the active form was performed by using a bioluminescent assay, while the quantification of interleukins 1ß (IL-1ß) and 18 (IL-18)released in the supernatant was quantified using an ELISA assay. The data showed that the presence of the ICP27 expressed by HSV-1 induces, in contrast to HSV-1∆ICP27 vector, a significant downregulation of AIM 2 inflammasome constituent proteins and, consequently, the release of pro-inflammatory interleukins into the extracellular environment reducing an effective response in counteracting infection.
Asunto(s)
Citocinas , Herpesvirus Humano 1 , Proteínas Inmediatas-Precoces , Inflamasomas , Epitelio Pigmentado de la Retina , Humanos , Línea Celular , Citocinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/virología , Herpes Simple/inmunología , Herpes Simple/metabolismo , Herpes Simple/virología , Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Inmediatas-Precoces/genética , Inflamasomas/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/virologíaRESUMEN
Recombinant adeno-associated viruses (rAAVs) have emerged as promising gene therapy vectors due to their proven efficacy and safety in clinical applications. In non-human primates (NHPs), rAAVs are administered via suprachoroidal injection at a higher dose. However, high doses of rAAVs tend to increase additional safety risks. Here, we present a novel AAV capsid (AAVv128), which exhibits significantly enhanced transduction efficiency for photoreceptors and retinal pigment epithelial (RPE) cells, along with a broader distribution across the layers of retinal tissues in different animal models (mice, rabbits, and NHPs) following intraocular injection. Notably, the suprachoroidal delivery of AAVv128-anti-VEGF vector completely suppresses the Grade IV lesions in a laser-induced choroidal neovascularization (CNV) NHP model for neovascular age-related macular degeneration (nAMD). Furthermore, cryo-EM analysis at 2.1 Å resolution reveals that the critical residues of AAVv128 exhibit a more robust advantage in AAV binding, the nuclear uptake and endosome escaping. Collectively, our findings highlight the potential of AAVv128 as a next generation ocular gene therapy vector, particularly using the suprachoroidal delivery route.
Asunto(s)
Neovascularización Coroidal , Dependovirus , Terapia Genética , Vectores Genéticos , Epitelio Pigmentado de la Retina , Animales , Dependovirus/genética , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Terapia Genética/métodos , Ratones , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/virología , Neovascularización Coroidal/terapia , Neovascularización Coroidal/genética , Conejos , Humanos , Técnicas de Transferencia de Gen , Degeneración Macular/terapia , Degeneración Macular/genética , Degeneración Macular/patología , Modelos Animales de Enfermedad , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Transducción Genética , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ratones Endogámicos C57BL , Retina/metabolismo , Retina/virología , Masculino , Células HEK293RESUMEN
Ebola virus (EBOV) is a highly pathogenic virus that causes a severe illness called Ebola virus disease (EVD). EVD has a high mortality rate and remains a significant threat to public health. Research on EVD pathogenesis has traditionally focused on host transcriptional responses. Limited recent studies, however, have revealed some information on the significance of cellular microRNAs (miRNAs) in EBOV infection and pathogenic mechanisms, but further studies are needed. Thus, this study aimed to identify and validate additional known and novel human miRNAs in EBOV-infected adult retinal pigment epithelial (ARPE) cells and predict their potential roles in EBOV infection and pathogenic mechanisms. We analyzed previously available small RNA-Seq data obtained from ARPE cells and identified 23 upregulated and seven downregulated miRNAs in the EBOV-infected cells; these included two novel miRNAs and 17 additional known miRNAs not previously identified in ARPE cells. In addition to pathways previously identified by others, these miRNAs are associated with pathways and biological processes that include WNT, FoxO, and phosphatidylinositol signaling; these pathways were not identified in the original study. This study thus confirms and expands on the previous study using the same datasets and demonstrates further the importance of human miRNAs in the host response and EVD pathogenesis during infection.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , MicroARNs , Epitelio Pigmentado de la Retina , Humanos , MicroARNs/genética , Fiebre Hemorrágica Ebola/genética , Fiebre Hemorrágica Ebola/virología , Ebolavirus/genética , Ebolavirus/patogenicidad , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/virología , Epitelio Pigmentado de la Retina/patología , Línea CelularRESUMEN
Resveratrol (RES) is a polyphenol with increasing interest for its inhibitory effects on a wide variety of viruses. Zika virus (ZIKV) is an arbovirus which causes a broad spectrum of ophthalmological manifestations in humans. Currently there is no certified therapy or vaccine to treat it, thus it has become a major global health threat. Retinal pigment epithelium (RPE) is highly permissive and susceptible to ZIKV. This work explored the protective effects of RES on ZIKV-infected human RPE cells. RES treatment resulted in a significant reduction of infectious viral particles in infected male ARPE-19 and female hTERT-RPE1 cells. This protection was positively influenced by the action of RES on mitochondrial dynamics. Also, docking studies predicted that RES has a high affinity for two enzymes of the rate-limiting steps of pyrimidine and purine biosynthesis and viral polymerase. This evidence suggests that RES might be a potential antiviral agent to treat ZIKV-induced ocular abnormalities.
Asunto(s)
Antivirales/farmacología , Resveratrol/farmacología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/virología , Virus Zika/efectos de los fármacos , Antivirales/química , Sitios de Unión , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Desarrollo de Medicamentos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Humanos , Ligandos , Dinámicas Mitocondriales/efectos de los fármacos , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Resveratrol/química , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacos , Infección por el Virus Zika/tratamiento farmacológico , Infección por el Virus Zika/patología , Infección por el Virus Zika/virologíaRESUMEN
Several RNA viruses, including SARS-CoV-2, can infect or use the eye as an entry portal to cause ocular or systemic diseases. Povidone-Iodine (PVP-I) is routinely used during ocular surgeries and eye banking as a cost-effective disinfectant due to its broad-spectrum antimicrobial activity, including against viruses. However, whether PVP-I can exert antiviral activities in virus-infected cells remains elusive. In this study, using Zika (ZIKV) and Chikungunya (CHIKV) virus infection of human corneal and retinal pigment epithelial cells, we report antiviral mechanisms of PVP-I. Our data showed that PVP-I, even at the lowest concentration (0.01%), drastically reduced viral replication in corneal and retinal cells without causing cellular toxicity. Antiviral effects of PVP-I against ZIKV and CHIKV were mediated by direct viral inactivation, thus attenuating the ability of the virus to infect host cells. Moreover, one-minute PVP-I exposure of infected ocular cells drastically reduced viral replication and the production of infectious progeny virions. Furthermore, viral-induced (CHIKV) expression of inflammatory genes (TNF-α, IL-6, IL-8, and IL1ß) were markedly reduced in PVP-I treated corneal epithelial cells. Together, our results demonstrate potent antiviral effects of PVP-I against ZIKV and CHIKV infection of ocular cells. Thus, a low dose of PVP-I can be used during tissue harvesting for corneal transplants to prevent potential transmission of RNA viruses via infected cells.
Asunto(s)
Antivirales/farmacología , Povidona Yodada/farmacología , Virus ARN/fisiología , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , Virus Chikungunya/fisiología , Chlorocebus aethiops , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/virología , SARS-CoV-2/fisiología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Células Vero , Virus Zika/fisiologíaRESUMEN
PURPOSE: Epstein-Barr virus is a γ-herpes virus that infects primary B cells and can transform infected cells into immortalized lymphoblastoid cell lines (LCL). The role of EBV in malignancies such as Burkitt's lymphoma and nasopharyngeal carcinoma is well understood, however, its role in EBV-infected retinal cells remains poorly understood. Therefore, we investigated the effect of EBV on the growth of retinal cells. METHODS: Previously, we established and reported a cell line model to address the relationship between EBV infection and retinal cell proliferation that used adult retinal pigment epithelium (ARPE-19) and EBV infection. To determine the effect of EBV on ARPE-19 cells, cell death was measured by propidium iodine/annexin V staining and reactive oxygen species (ROS) were measured by FACS, and protein expression was evaluated using western blot analysis. Also, downregulation of LMP1 and NADPH oxidase 4 (NOX4) expression was accomplished using siRNA technology. RESULTS: We found that ROS were dramatically increased in EBV-infected ARPE19 cells (APRE19/EBV) relative to the parental cell line. Additionally, the expression level of NOX4, a main source of ROS, was upregulated by EBV infection. Interestingly, downregulation of LMP1, one of the EBV viral onco-proteins, completely decreased EBV-induced ROS accumulation and the upregulation of NOX4. Treatment with APX-115A, a pan-NOX inhibitor, induced apoptotic cell death of only the EBV-infected ARPE19 cells but not the parental cell line. Pretreatment with z-VAD, a pan-caspase inhibitor, inhibited NOX inhibitor-induced cell death in ARPE19/EBV cells. Furthermore, APX-115A-induced cell death mediated the activation of JNK and ERK. Finally, we confirmed the expression level of NOX4, and APX-115A induced cell death of EBV-infected human primary retina epithelial cells and the activation of JNK and ERK. CONCLUSION: Taken together, these our results suggest that APX-115A could be a therapeutic agent for treating EBV-infected retinal cells or diseases by inhibiting LMP1-NOX4-ROS signaling.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Herpesvirus Humano 4/fisiología , NADPH Oxidasa 4/metabolismo , Pirazoles/farmacología , Piridinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/patología , Western Blotting , Línea Celular , Proliferación Celular , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Humanos , NADPH Oxidasas/antagonistas & inhibidores , Epitelio Pigmentado de la Retina/enzimología , Epitelio Pigmentado de la Retina/virología , Transducción de Señal/efectos de los fármacosRESUMEN
Cellular microRNAs are known to modulate the life-cycle of different viruses. Surprisingly, very little data exists on AAV-induced changes to the cellular microRNAome in general and in hepatic and retinal cells, in particular. We reasoned that inducible microRNA in response to recombinant AAV infection may regulate immediate and long-lived cellular responses necessary for the cell's own survival as well as its ability to control several aspects of viral life-cycle. To study this, we performed a global small RNA sequencing analysis in Adeno-associated virus (AAV) serotypes 2 and 3 infected hepatic and retinal cell models. This screen identified multiple differentially expressed microRNAs, in AAV infected Huh-7 and ARPE-19 cells. Among these, one microRNA (miR-4488) was found to be significantly down regulated (-2.24 fold for AAV2 and -3.32 fold for ARPE-19) in AAV infected cells. An enrichment and pathway analysis of miR-4488 predicted its possible effects on gene targets involved in multiple biological processes including cell-cycle regulation, endoplasmic reticulum stress response and lipid-signalling pathways. Moreover, validation studies in miR-4488 mimic or sponge transfected cells revealed modulation of these target pathways in a cell-specific manner. Further studies demonstrated that overexpression of miR-4488, modestly increased gene expression (126-128%) from AAV2 and AAV3 vectors in Huh-7 cells whereas miR-4488 inhibition in ARPE-19 cells had a similar increase (142-158%) on AAV2 or AAV3 transduction. Our results highlight that recombinant AAV mediated microRNA expression is cell-type and serotype-specific and can target specific host cellular biological pathways.
Asunto(s)
Dependovirus/genética , MicroARNs/genética , Infecciones por Parvoviridae/genética , Epitelio Pigmentado de la Retina/virología , Transducción Genética/métodos , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Ciclo Celular/genética , Línea Celular , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/genética , Perfilación de la Expresión Génica , Humanos , Metabolismo de los Lípidos/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Parvovirinae/genética , Reproducibilidad de los Resultados , Epitelio Pigmentado de la Retina/citología , TransgenesRESUMEN
We report the analysis of a complex enveloped human virus, herpes simplex virus (HSV), assembled after in vivo incorporation of bio-orthogonal methionine analogues homopropargylglycine (HPG) or azidohomoalanine (AHA). We optimised protocols for the production of virions incorporating AHA (termed HSVAHA), identifying conditions which resulted in normal yields of HSV and normal particle/pfu ratios. Moreover we show that essentially every single HSVAHA capsid-containing particle was detectable at the individual particle level by chemical ligation of azide-linked fluorochromes to AHA-containing structural proteins. This was a completely specific chemical ligation, with no capsids assembled under normal methionine-containing conditions detected in parallel. We demonstrate by quantitative mass spectrometric analysis that HSVAHA virions exhibit no qualitative or quantitative differences in the repertoires of structural proteins compared to virions assembled under normal conditions. Individual proteins and AHA incorporation sites were identified in capsid, tegument and envelope compartments, including major essential structural proteins. Finally we reveal novel aspects of entry pathways using HSVAHA and chemical fluorochrome ligation that were not apparent from conventional immunofluorescence. Since ligation targets total AHA-containing protein and peptides, our results demonstrate the presence of abundant AHA-labelled products in cytoplasmic macrodomains and tubules which no longer contain intact particles detectable by immunofluorescence. Although these do not co-localise with lysosomal markers, we propose they may represent sites of proteolytic virion processing. Analysis of HSVAHA also enabled the discrimination from primary entering from secondary assembling virions, demonstrating assembly and second round infection within 6 hrs of initial infection and dual infections of primary and secondary virus in spatially restricted cytoplasmic areas of the same cell. Together with other demonstrated applications e.g., in genome biology, lipid and protein trafficking, this work further exemplifies the utility and potential of bio-orthogonal chemistry for studies in many aspects of virus-host interactions.
Asunto(s)
Aminoácidos/metabolismo , Herpes Simple/virología , Herpesvirus Humano 1/fisiología , Epitelio Pigmentado de la Retina/virología , Proteínas Estructurales Virales/metabolismo , Ensamble de Virus , Internalización del Virus , Proliferación Celular , Células Cultivadas , Herpes Simple/metabolismo , Humanos , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Human retinal pigment epithelial (hRPE) cells are important for the establishment and maintenance of the immune privilege of the eye. They function as target cells for human cytomegalovirus (hCMV), but are able to restrict viral replication. hCMV causes opportunistic posterior uveitis such as retinitis and chorioretinitis. Both mainly occur in severely immunocompromised patients and rarely manifest in immunocompetent individuals. In this study, hRPE cells were infected with hCMV in vitro and activated with proinflammatory cytokines. The enzymatic activities of indoleamine 2,3-dioxygenase-1 (IDO1) and inducible nitric oxide synthase (iNOS) were determined. The antimicrobial capacity of both molecules was analyzed in co-infection experiments using Staphylococcus aureus (S. aureus) and Toxoplasma gondii (T. gondii), causing uveitis in patients. We show that an hCMV infection of hRPE cells blocks IDO1 and iNOS mediated antimicrobial defense mechanisms necessary for the control of S. aureus and T. gondii. hCMV also inhibits immune suppressive effector mechanisms in hRPE. The interferon gamma-induced IDO1 dependent immune regulation was severely blocked, as detected by the loss of T cell inhibition. We conclude that an active hCMV infection in the eye might favor the replication of pathogens causing co-infections in immunosuppressed individuals. An hCMV caused blockade of IDO1 might weaken the eye's immune privilege and favor the development of post-infectious autoimmune uveitis.
Asunto(s)
Ojo/inmunología , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Epitelio Pigmentado de la Retina/inmunología , Uveítis/inmunología , Proliferación Celular/genética , Coinfección/inmunología , Coinfección/microbiología , Coinfección/virología , Citomegalovirus/genética , Citomegalovirus/inmunología , Ojo/microbiología , Ojo/virología , Citometría de Flujo , Humanos , Privilegio Inmunológico/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Interferón gamma/inmunología , Óxido Nítrico Sintasa de Tipo II/genética , Epitelio Pigmentado de la Retina/microbiología , Epitelio Pigmentado de la Retina/virología , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/patogenicidad , Linfocitos T/inmunología , Linfocitos T/microbiología , Linfocitos T/virología , Toxoplasma/crecimiento & desarrollo , Toxoplasma/patogenicidad , Uveítis/microbiología , Uveítis/virologíaRESUMEN
BACKGROUND: Zika virus (ZIKV) has recently re-emerged as a pathogenic agent with epidemic capacities as was well illustrated in South America. Because of the extent of this health crisis, a number of more serious symptoms have become associated with ZIKV infection than what was initially described. In particular, neuronal and ocular disorders have been characterized, both in infants and in adults. Notably, the macula and the retina can be strongly affected by ZIKV, possibly by a direct effect of the virus. This is supported by the detection of replicative and infectious virus in lachrimal fluid in human patients and mouse models. METHODS: Here, we used an innovative, state-of-the-art iPSC-derived human retinal pigment epithelium (RPE) model to study ZIKV retinal impairment. FINDINGS: We showed that the human RPE is highly susceptible to ZIKV infection and that a ZIKV African strain was more virulent and led to a more potent epithelium disruption and stronger anti-viral response than an Asian strain, suggesting lineage differences. Moreover, ZIKV infection led to impaired membrane dynamics involved in endocytosis, organelle biogenesis and potentially secretion, key mechanisms of RPE homeostasis and function. INTERPRETATION: Taken together, our results suggest that ZIKV has a highly efficient ocular tropism, which creates a strong inflammatory environment that could have acute or chronic adverse effects. FUND: This work was funded by Retina France, REACTing and La Région Languedoc-Roussillon.
Asunto(s)
Interferones/metabolismo , Epitelio Pigmentado de la Retina/virología , Infección por el Virus Zika/inmunología , Virus Zika/patogenicidad , Células Cultivadas , Homeostasis , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/virología , Interferones/genética , Modelos Biológicos , Fagocitosis , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/inmunología , Tropismo Viral , Replicación Viral , Virus Zika/clasificación , Virus Zika/fisiología , Infección por el Virus Zika/genética , Infección por el Virus Zika/virologíaRESUMEN
A fundamental signal-processing problem is how biological systems maintain phenotypic states (i.e., canalization) long after degradation of initial catalyst signals. For example, to efficiently replicate, herpesviruses (e.g., human cytomegalovirus, HCMV) rapidly counteract cell-mediated silencing using transactivators packaged in the tegument of the infecting virion particle. However, the activity of these tegument transactivators is inherently transient-they undergo immediate proteolysis but delayed synthesis-and how transient activation sustains lytic viral gene expression despite cell-mediated silencing is unclear. By constructing a two-color, conditional-feedback HCMV mutant, we find that positive feedback in HCMV's immediate-early 1 (IE1) protein is of sufficient strength to sustain HCMV lytic expression. Single-cell time-lapse imaging and mathematical modeling show that IE1 positive feedback converts transient transactivation signals from tegument pp71 proteins into sustained lytic expression, which is obligate for efficient viral replication, whereas attenuating feedback decreases fitness by promoting a reversible silenced state. Together, these results identify a regulatory mechanism enabling herpesviruses to sustain expression despite transient activation signals-akin to early electronic transistors-and expose a potential target for therapeutic intervention.
Asunto(s)
Citomegalovirus/genética , Retroalimentación Fisiológica , Regulación Viral de la Expresión Génica , Replicación Viral/genética , Línea Celular , Células Cultivadas , Citomegalovirus/fisiología , Interacciones Huésped-Patógeno , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Microscopía Fluorescente , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/virología , Imagen de Lapso de Tiempo/métodosRESUMEN
Previously, we reported that Zika virus (ZIKV) causes ocular complications such as chorioretinal atrophy, by infecting cells lining the blood-retinal barrier, including the retinal pigment epithelium (RPE). To understand the molecular basis of ZIKV-induced retinal pathology, we performed a meta-analysis of transcriptome profiles of ZIKV-infected human primary RPE and other cell types infected with either ZIKV or other related flaviviruses (Japanese encephalitis, West Nile, and Dengue). This led to identification of a unique ZIKV infection signature comprising 43 genes (35 upregulated and 8 downregulated). The major biological processes perturbed include SH3/SH2 adaptor activity, lipid and ceramide metabolism, and embryonic organ development. Further, a comparative analysis of some differentially regulated genes (ABCG1, SH2B3, SIX4, and TNFSF13B) revealed that ZIKV induced their expression relatively more than dengue virus did in RPE. Importantly, the pharmacological inhibition of ABCG1, a membrane transporter of cholesterol, resulted in reduced ZIKV infectivity. Interestingly, the ZIKV infection signature revealed the downregulation of ALDH5A1 and CHML, genes implicated in neurological (cognitive impairment, expressive language deficit, and mild ataxia) and ophthalmic (choroideremia) disorders, respectively. Collectively, our study revealed that ZIKV induces differential gene expression in RPE cells, and the identified genes/pathways (e.g., ABCG1) could potentially contribute to ZIKV-associated ocular pathologies.
Asunto(s)
Epitelio Pigmentado de la Retina/metabolismo , Transcriptoma/genética , Infección por el Virus Zika/genética , Virus Zika/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/genética , Proteínas Adaptadoras Transductoras de Señales , Factor Activador de Células B/genética , Dengue/genética , Dengue/patología , Dengue/virología , Virus del Dengue/patogenicidad , Virus de la Encefalitis Japonesa (Subgrupo)/patogenicidad , Infecciones por Flavivirus/genética , Infecciones por Flavivirus/patología , Infecciones por Flavivirus/virología , Regulación de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Cultivo Primario de Células , Proteínas/genética , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/virología , Transactivadores/genética , Replicación Viral/genética , Fiebre del Nilo Occidental/genética , Fiebre del Nilo Occidental/patología , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/patogenicidad , Virus Zika/patogenicidad , Infección por el Virus Zika/patología , Infección por el Virus Zika/virologíaRESUMEN
Purpose: The purpose of this study was to determine whether the blood-retina barrier is compromised by choroidal murine cytomegalovirus (MCMV) infection, using electron microscopy. Methods: BALB/c mice were immunosuppressed with methylprednisolone and monoclonal antibodies to CD4 and CD8. At several time points post-MCMV intraperitoneal inoculation, the eyes were removed and analyzed with western blotting and immunoelectron microscopy for the presence of MCMV early antigen (EA) and the host protein RIP3. Posterior eyecups from RIP3-/- and RIP3+/+ mice were cultured and inoculated with MCMV. At days 4, 7, and 11 post-infection, cultures were collected and analyzed with plaque assay, immunohistochemical staining, and real-time PCR (RT-PCR). Results: MCMV EA was observed in the nuclei of vascular endothelial cells and pericytes in the choriocapillaris. Disruption of Bruch's membrane was observed, especially at sites adjacent to activated platelets, and a few RPE cells containing some enlarged vesicles were found directly beneath disrupted Bruch's membrane. Some virus particles were also observed in the enlarged vesicles of RPE cells. Levels of the RIP3 protein, which was observed mainly in the RPE cells and the basement membrane of the choriocapillaris, were greatly increased following MCMV infection, while depletion of RIP3 resulted in greatly decreased inflammasome formation, as well as expression of downstream inflammation factors. Conclusions: The results suggest that systemic MCMV spreads to the choroid and replicates in vascular endothelia and pericytes of the choriocapillaris during immunosuppression. Choroidal MCMV infection is associated with in situ inflammation and subsequent disruption of Bruch's membrane and the outer blood-retina barrier.
Asunto(s)
Coroides/inmunología , Infecciones por Citomegalovirus/inmunología , Infecciones Virales del Ojo/inmunología , Huésped Inmunocomprometido , Retina/inmunología , Retinitis/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Antígenos Virales/genética , Plaquetas/inmunología , Plaquetas/patología , Plaquetas/virología , Barrera Hematorretinal/inmunología , Barrera Hematorretinal/patología , Barrera Hematorretinal/virología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Coroides/irrigación sanguínea , Coroides/patología , Coroides/virología , Infecciones por Citomegalovirus/patología , Infecciones por Citomegalovirus/virología , Células Endoteliales , Infecciones Virales del Ojo/patología , Infecciones Virales del Ojo/virología , Femenino , Proteínas Inmediatas-Precoces/genética , Inflamasomas/inmunología , Metilprednisolona/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Muromegalovirus/crecimiento & desarrollo , Muromegalovirus/patogenicidad , Pericitos/inmunología , Pericitos/patología , Pericitos/virología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Retina/patología , Retina/virología , Epitelio Pigmentado de la Retina/inmunología , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/virología , Retinitis/patología , Retinitis/virologíaRESUMEN
Purpose: The purpose of this study was to determine if the receptor-interacting protein kinase 3 (RIP3) plays a significant role in innate immune responses and death of bystander retinal neurons during murine cytomegalovirus (MCMV) retinal infection, by comparing the innate immune response and cell death in RIP3-depleted mice (Rip3-/-) and Rip3+/+ control mice. Methods: Rip3-/- and Rip3+/+ mice were immunosuppressed (IS) and inoculated with MCMV via the supraciliary route. Virus-injected and mock-injected control eyes were removed at days 4, 7, and 10 post infection (p.i.) and markers of innate immunity and cell death were analyzed. Results: Compared to Rip3+/+ mice, significantly more MCMV was recovered and more MCMV-infected RPE cells were observed in injected eyes of Rip3-/- mice at days 4 and 7 p.i. In contrast, fewer TUNEL-stained photoreceptors were observed in Rip3-/- eyes than in Rip3+/+ eyes at these times. Electron microscopy showed that significantly more apoptotic photoreceptor cells were present in Rip3+/+ mice than in Rip3-/- mice. Immunohistochemistry showed that the majority of TUNEL-stained photoreceptors died via mitochondrial flavoprotein apoptosis-inducing factor (AIF)-mediated, caspase 3-independent apoptosis. The majority of RIP3-expressing cells in infected eyes were RPE cells, microglia/macrophages, and glia, whereas retinal neurons contained much lower amounts of RIP3. Western blots showed significantly higher levels of activated nuclear factor-κB and caspase 1 were present in Rip3+/+ eyes compared to Rip3-/- eyes. Conclusions: Our results suggest that RIP3 enhances innate immune responses against ocular MCMV infection via activation of the inflammasome and nuclear factor-κB, which also leads to inflammation and death of bystander cells by multiple pathways including apoptosis and necroptosis.
Asunto(s)
Apoptosis , Infecciones Virales del Ojo/patología , Infecciones por Herpesviridae/patología , Muromegalovirus/aislamiento & purificación , Células Fotorreceptoras de Vertebrados/patología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/fisiología , Enfermedades de la Retina/patología , Animales , Biomarcadores/metabolismo , Western Blotting , Supervivencia Celular/fisiología , Infecciones Virales del Ojo/metabolismo , Infecciones Virales del Ojo/virología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Infecciones por Herpesviridae/metabolismo , Infecciones por Herpesviridae/virología , Inmunidad Innata/fisiología , Etiquetado Corte-Fin in Situ , Inflamasomas/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Electrónica , FN-kappa B/metabolismo , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/virología , Epitelio Pigmentado de la Retina/virologíaRESUMEN
The present study aimed to explore the effects of a stabilizing ligand, Shield1, on the replication of recombinant varicellazoster virus (VZV) containing FK506 binding protein (FKPB) tags in essential open reading frames (ORF) 4 and 48. A specific galactokinase (galK) selection method was conducted, following the addition of galK labels to VZV ORF4 and 48, using a SW102 VZV bacterial artificial chromosome (BAC) system. Subsequently, recombinant VZV containing FKPB tags in ORF4 and 48 was constructed by counterselection and homologous recombination. Recombinant viral plasmids containing FKPBtagged VZV ORF4 and 48 were extracted and transfected into human acute retinal pigment epithelial ARPE19 cells. The results demonstrated that the FKPBtagged viral protein was rapidly degraded by proteases in recombinant virusinfected ARPE19 cells. In addition, the recombinant VZVORF4FKBPORF48FKBP virus could not grow if a synthetic ligand of FKBP, Shield1, was not added to the ARPE19 cell culture medium; however, the degradation of FKPBtagged viral protein was prevented if Shield1 was added to the ARPE19 cell culture medium, thereby allowing viral replication in ARPE19 cells. These results indicated that Shield1 may regulate replication of recombinant VZVORF4FKBPORF48FKBP following transfection into human epithelial cells.
Asunto(s)
Herpesvirus Humano 3/genética , Proteínas Inmediatas-Precoces/genética , Proteínas de Unión a Tacrolimus/genética , Replicación Viral/genética , Línea Celular , Cromosomas Artificiales Bacterianos/genética , Medios de Cultivo/farmacología , Herpesvirus Humano 3/crecimiento & desarrollo , Herpesvirus Humano 3/patogenicidad , Humanos , Ligandos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/virología , Transfección , Replicación Viral/efectos de los fármacosRESUMEN
Purpose: Emerging evidence has shown that both congenital and adult Zika virus (ZIKV) infection can cause eye diseases. The goals of the current study were to explore mechanisms and pathophysiology of ZIKV-induced eye defects. Methods: Wild-type or A129 interferon type I receptor-deficient mice were infected by either FSS13025 or Mex1-7 strain of ZIKV. Retinal histopathology was measured at different time points after infection. The presence of viral RNA and protein in the retina was determined by in situ hybridization and immunofluorescence staining, respectively. Growth curves of ZIKV in permissive retinal cells were assessed in cultured retinal pigment epithelial (RPE) and Müller glial cells. Results: ZIKV-infected mice developed a spectrum of ocular pathologies that affected multiple layers of the retina. A primary target of ZIKV in the eye was Müller glial cells, which displayed decreased neurotrophic function and increased expression of proinflammatory cytokines after infection. ZIKV also infected RPE; and both the RPE and Müller cells expressed viral entry receptors TYRO3 and AXL. Retinitis, focal retinal degeneration, and ganglion cell loss were observed after the clearance of viral particles. Conclusions: Our data suggest that ZIKV can infect infant eyes with immature blood-retinal barrier and cause structural damages to the retina. The ocular findings in microcephalic infants may not be solely caused by ZIKV-induced impairment of neurodevelopment.
Asunto(s)
Modelos Animales de Enfermedad , Células Ependimogliales/virología , Infecciones Virales del Ojo/virología , Enfermedades de la Retina/virología , Epitelio Pigmentado de la Retina/virología , Infección por el Virus Zika/virología , Animales , Barrera Hematorretinal , Células Cultivadas , Células Ependimogliales/patología , Infecciones Virales del Ojo/patología , Citometría de Flujo , Hibridación Fluorescente in Situ , Ratones , Ratones Endogámicos C57BL , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Enfermedades de la Retina/patología , Epitelio Pigmentado de la Retina/patología , Uveítis Posterior/patología , Uveítis Posterior/virología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiología , Virus Zika/fisiología , Infección por el Virus Zika/patologíaRESUMEN
Zika virus (ZIKV) is an important pathogen that causes not only neurologic, but also ocular, abnormalities. Thus, it is imperative that models to study ZIKV pathogenesis in the eye are developed to identify potential targets for interventions. Here, we studied ZIKV interactions with human retinal cells and evaluated ZIKV's pathobiology in mouse eyes. We showed that cells lining the blood-retinal barrier (BRB), the retinal endothelium, and retinal pigment epithelium (RPE) were highly permissive and susceptible to ZIKV-induced cell death. Direct inoculation of ZIKV in eyes of adult C57BL/6 and IFN-stimulated gene 15 (ISG15) KO mice caused chorioretinal atrophy with RPE mottling, a common ocular manifestation of congenital ZIKV infection in humans. This response was associated with induced expression of multiple inflammatory and antiviral (IFNs) response genes in the infected mouse retina. Interestingly, ISG15 KO eyes exhibited severe chorioretinitis, which coincided with increased retinal cell death and higher ZIKV replication. Collectively, our study provides the first evidence to our knowledge that ZIKV causes retinal lesions and infects the cells lining the BRB and that ISG15 plays a role in retinal innate defense against ZIKV infection. Our mouse model can be used to study mechanisms underlying ZIKV-induced chorioretinitis and to gauge ocular antiviral therapies.
Asunto(s)
Barrera Hematorretinal/virología , Coriorretinitis/virología , Coroides/virología , Endotelio/virología , Epitelio Pigmentado de la Retina/virología , Infección por el Virus Zika/patología , Virus Zika , Animales , Atrofia , Barrera Hematorretinal/citología , Muerte Celular , Línea Celular , Coriorretinitis/patología , Coroides/patología , Citocinas/genética , Modelos Animales de Enfermedad , Endotelio/citología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Retina/patología , Retina/virología , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/patología , Ubiquitinas/genética , Replicación ViralRESUMEN
An epidemic of Zika virus (ZIKV) infection associated with congenital abnormalities such as microcephaly, is ongoing in the Americas and the Pacific. Currently there are no approved therapies to treat this emerging viral disease. Here, we tested three cell-directed broad-spectrum antiviral compounds against ZIKV replication using human retinal pigment epithelial (RPE) cells and a low-passage ZIKV strain isolated from fetal brain. We found that obatoclax, SaliPhe, and gemcitabine inhibited ZIKV infections at noncytotoxic concentrations. Moreover, all three compounds prevented production of viral RNA and proteins as well as activation of cellular caspase 8, 3 and 7. However, these compounds differentially affected ZIKV-mediated transcription, translation and posttranslational modifications of cellular factors as well as metabolic pathways indicating that these agents possess different mechanisms of action. Interestingly, combination of obatoclax and SaliPhe at nanomolar concentrations had a synergistic effect against ZIKV infection. Thus, our results provided the foundation for development of broad-spectrum cell-directed antivirals or their combinations for treatment of ZIKV and other emerging viral diseases.