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1.
Sci Rep ; 12(1): 7348, 2022 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-35513694

RESUMO

Zika virus (ZIKV) infection at the maternal-placental interface is associated with adverse pregnancy outcomes including fetal demise and pregnancy loss. To determine how infection impacts placental trophoblasts, we utilized rhesus macaque trophoblast stem cells (TSC) that can be differentiated into early gestation syncytiotrophoblasts (ST) and extravillous trophoblasts (EVT). TSCs and STs, but not EVTs, were highly permissive to productive infection with ZIKV strain DAK AR 41524. The impact of ZIKV on the cellular transcriptome showed that infection of TSCs and STs increased expression of immune related genes, including those involved in type I and type III interferon responses. ZIKV exposure altered extracellular vesicle (EV) mRNA, miRNA and protein cargo, including ZIKV proteins, regardless of productive infection. These findings suggest that early gestation macaque TSCs and STs are permissive to ZIKV infection, and that EV analysis may provide a foundation for identifying non-invasive biomarkers of placental infection in a highly translational model.


Assuntos
Vesículas Extracelulares , Complicações Infecciosas na Gravidez , Infecção por Zika virus , Zika virus , Animais , Vesículas Extracelulares/genética , Feminino , Expressão Gênica , Humanos , Macaca mulatta , Placenta/metabolismo , Gravidez , Trofoblastos/metabolismo , Zika virus/fisiologia
2.
PLoS Negl Trop Dis ; 16(5): e0010366, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35522620

RESUMO

Zika virus (ZIKV) is a mosquito-borne flavivirus and can cause neurodevelopmental disorders in fetus. As a neurotropic virus, ZIKV persistently infects neural tissues during pregnancy but the viral pathogenesis remains largely unknown. ZIKV has a positive-sense and single-stranded RNA genome, which encodes 7 non-structural (NS) proteins, participating in viral replication and dysregulation of host immunity. Like those in many other viruses, NS proteins are considered to be products evolutionarily beneficiary to viruses and some are virulence factors. However, we found that some NS proteins encoded by ZIKV genome appeared to function against the viral replication. In this report we showed that exogenously expressed ZIKV NS2A and NS4A inhibited ZIKV infection by inhibiting viral RNA replication in microglial cells and astrocytes. To understand how viral NS proteins suppressed viral replication, we analyzed the transcriptome of the microglial cells and astrocytes and found that expression of NS4A induced the upregulation of ISGs, including MX1/2, OAS1/2/3, IFITM1, IFIT1, IFI6, IFI27, ISG15 or BST2 through activating the ISGF3 signaling pathway. Upregulation of these ISGs seemed to be related to the inhibition of ZIKV replication, since the anti-ZIKV function of NS4A was partially attenuated when the cells were treated with Abrocitinib, an inhibitor of the ISGF3 signaling pathway, or were knocked down with STAT2. Aborting the protein expression of NS4A, but not its nucleic acid, eliminated the antiviral activity of NS4A effectively. Dynamic expression of viral NS proteins was examined in ZIKV-infected microglial cells and astrocytes, which showed comparatively NS4A occurred later than other NS proteins during the infection. We hypothesize that NS4A may possess intrinsic features to serve as a unique type of pathogen associated molecular pattern (PAMP), detectable by the cells to induce an innate immune response, or function with other mechanisms, to restrict the viral replication to a certain level as a negative feedback, which may help ZIKV maintain its persistent infection in fetal neural tissues.


Assuntos
Infecção por Zika virus , Zika virus , Animais , RNA Viral/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Zika virus/fisiologia
3.
Acta Neuropathol Commun ; 10(1): 46, 2022 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-35379362

RESUMO

The mechanisms by which vertically transmitted Zika virus (ZIKV) causes postnatal brain development abnormalities and congenital disease remain poorly understood. Here, we optimized the established anti-IFNAR1 treated, Rag1-/- (AIR) mouse model of ZIKV infection to examine the consequence of vertical transmission on neonate survival and postnatal brain development. We found that modulating the infectious dose and the frequency of anti-IFNAR1 treatment of pregnant mice (termed AIRlow mice) prolonged neonatal survival allowing for pathogenesis studies of brain tissues at critical postnatal time points. Postnatal AIRlow mice all had chronic ZIKV infection in the brain that was associated with decreased cortical thickness and cerebellar volume, increased gliosis, and higher levels of cell death in many brain areas including cortex, hippocampus and cerebellum when compared to controls. Interestingly, despite active infection and brain abnormalities, the neurodevelopmental program remained active in AIRlow mice as indicated by elevated mRNA expression of critical neurodevelopmental genes in the brain and enlargement of neural-progenitor rich regions of the cerebellum at a developmental time point analogous to birth in humans. Nevertheless, around the developmental time point when the brain is fully populated by neurons, AIRlow mice developed neurologic disease associated with persistent ZIKV infection in the brain, gliosis, and increased cell death. Together, these data show that vertically transmitted ZIKV infection in the brain of postnatal AIRlow mice strongly influences brain development resulting in structural abnormalities and cell death in multiple regions of the brain.


Assuntos
Encefalopatias , Infecção por Zika virus , Zika virus , Animais , Encéfalo/patologia , Encefalopatias/complicações , Feminino , Proteínas de Homeodomínio , Interferons , Camundongos , Gravidez , Receptor de Interferon alfa e beta , Zika virus/fisiologia , Infecção por Zika virus/complicações , Infecção por Zika virus/genética
4.
Front Immunol ; 13: 773191, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35371036

RESUMO

Zika virus (ZIKV), despite being discovered six decades earlier, became a major health concern only after an epidemic in French Polynesia and an increase in the number of microcephaly cases in Brazil. Substantial evidence has been found to support the link between ZIKV and neurological complications in infants. The virus targets various cells in the brain, including radial glial cells, neural progenitor cells (NPCs), astrocytes, microglial and glioblastoma stem cells. It affects the brain cells by exploiting different mechanisms, mainly through apoptosis and cell cycle dysregulation. The modulation of host immune response and the inflammatory process has also been demonstrated to play a critical role in ZIKV induced neurological complications. In addition to that, different ZIKV strains have exhibited specific neurotropism and unique molecular mechanisms. This review provides a comprehensive and up-to-date overview of ZIKV-induced neuroimmunopathogenesis by dissecting its main target cells in the brain, and the underlying cellular and molecular mechanisms. We highlighted the roles of the different ZIKV host factors and how they exploit specific host factors through various mechanisms. Overall, it covers key components for understanding the crosstalk between ZIKV and the brain.


Assuntos
Microcefalia , Doenças do Sistema Nervoso , Células-Tronco Neurais , Infecção por Zika virus , Zika virus , Encéfalo/patologia , Humanos , Microcefalia/patologia , Doenças do Sistema Nervoso/patologia , Células-Tronco Neurais/patologia , Zika virus/fisiologia
5.
Elife ; 112022 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-35261339

RESUMO

We evaluated neuropathological consequences of fetal ZIKV exposure in rhesus monkeys, a translatable animal model for human neural development, by carrying out quantitative neuroanatomical analyses of the nearly full-term brains of fetuses infected with ZIKV and procedure-matched controls. For each animal, a complete cerebral hemisphere was evaluated using immunohistochemical (IHC) and neuroanatomical techniques to detect virus, identify affected cell types, and evaluate gross neuroanatomical abnormalities. IHC staining revealed the presence of ZIKV in the frontal lobe, which contained activated microglia and showed increased apoptosis of immature neurons. ZIKV-infected animals exhibited macrostructural changes within the visual pathway. Regional differences tracked with the developmental timing of the brain, suggesting inflammatory processes related to viral infiltration swept through the cortex, followed by a wave of cell death resulting in morphological changes. These findings may help explain why some infants born with normal sized heads during the ZIKV epidemic manifest developmental challenges as they age.


Assuntos
Infecção por Zika virus , Zika virus , Animais , Encéfalo/patologia , Feto , Humanos , Macaca mulatta , Zika virus/fisiologia
6.
Commun Biol ; 5(1): 243, 2022 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-35304593

RESUMO

Zika virus infection can result in devastating pregnancy outcomes when it crosses the placental barrier. For human pregnancies, the mechanisms of vertical transmission remain enigmatic. Utilizing a human placenta-cotyledon perfusion model, we examined Zika virus exposure in the absence of maternal factors. To distinguish responses related to viral infection vs. recognition, we evaluated cotyledons perfused with either active or inactivated Zika virus. Active Zika virus exposure resulted in infection, cell death and syncytium injury. Pathology corresponded with transcriptional changes related to inflammation and innate immunity. Inactive Zika virus exposure also led to syncytium injury and related changes in gene expression but not cell death. Our observations reveal pathologies and innate immune responses that are dependent on infection or virus placenta interactions independent of productive infection. Importantly, our findings indicate that Zika virus can infect and compromise placentas in the absence of maternal humoral factors that may be protective.


Assuntos
Complicações Infecciosas na Gravidez , Infecção por Zika virus , Zika virus , Feminino , Humanos , Transmissão Vertical de Doenças Infecciosas , Placenta , Gravidez , Complicações Infecciosas na Gravidez/patologia , Zika virus/fisiologia
7.
Proc Natl Acad Sci U S A ; 119(8)2022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-35193960

RESUMO

Emerging microbe infections, such as Zika virus (ZIKV), pose an increasing threat to human health. Investigations on ZIKV replication have revealed the construction of replication complexes (RCs), but the role of cytoskeleton in this process is largely unknown. Here, we investigated the function of cytoskeletal intermediate filament protein vimentin in the life cycle of ZIKV infection. Using advanced imaging techniques, we uncovered that vimentin filaments undergo drastic reorganization upon viral protein synthesis to form a perinuclear cage-like structure that embraces and concentrates RCs. Genetic removal of vimentin markedly disrupted the integrity of RCs and resulted in fragmented subcellular dispersion of viral proteins. This led to reduced viral genome replication, viral protein production, and release of infectious virions, without interrupting viral binding and entry. Furthermore, mass spectrometry and RNA-sequencing screens identified interactions and interplay between vimentin and hundreds of endoplasmic reticulum (ER)-resident RNA-binding proteins. Among them, the cytoplasmic-region of ribosome receptor binding protein 1, an ER transmembrane protein that directly binds viral RNA, interacted with and was regulated by vimentin, resulting in modulation of ZIKV replication. Together, the data in our work reveal a dual role for vimentin as a structural element for RC integrity and as an RNA-binding-regulating hub during ZIKV infection, thus unveiling a layer of interplay between Zika virus and host cell.


Assuntos
Vimentina/metabolismo , Infecção por Zika virus/metabolismo , Animais , Linhagem Celular , China , Citoesqueleto/metabolismo , Retículo Endoplasmático/metabolismo , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Filamentos Intermediários/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Vimentina/fisiologia , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Zika virus/metabolismo , Zika virus/patogenicidade , Zika virus/fisiologia , Infecção por Zika virus/virologia
8.
Int J Mol Sci ; 23(3)2022 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-35163212

RESUMO

Cell death by apoptosis is a major cellular response in the control of tissue homeostasis and as a defense mechanism in the case of cellular aggression such as an infection. Cell self-destruction is part of antiviral responses, aimed at limiting the spread of a virus. Although it may contribute to the deleterious effects in infectious pathology, apoptosis remains a key mechanism for viral clearance and the resolution of infection. The control mechanisms of cell death processes by viruses have been extensively studied. Apoptosis can be triggered by different viral determinants through different pathways as a result of virally induced cell stresses and innate immune responses. Zika virus (ZIKV) induces Zika disease in humans, which has caused severe neurological forms, birth defects, and microcephaly in newborns during the last epidemics. ZIKV also surprised by revealing an ability to persist in the genital tract and in semen, thus being sexually transmitted. Mechanisms of diverting antiviral responses such as the interferon response, the role of cytopathic effects and apoptosis in the etiology of the disease have been widely studied and debated. In this review, we examined the interplay between ZIKV infection of different cell types and apoptosis and how the virus deals with this cellular response. We illustrate a duality in the effects of ZIKV-controlled apoptosis, depending on whether it occurs too early or too late, respectively, in neuropathogenesis, or in long-term viral persistence. We further discuss a prospective role for apoptosis in ZIKV-related therapies, and the use of ZIKV as an oncolytic agent.


Assuntos
Apoptose/fisiologia , Infecção por Zika virus/metabolismo , Zika virus/fisiologia , Animais , Antivirais/uso terapêutico , Morte Celular/fisiologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/imunologia , Interferons/uso terapêutico , Microcefalia/virologia , Fenômenos Fisiológicos Virais/imunologia , Replicação Viral/fisiologia , Zika virus/genética , Zika virus/patogenicidade , Infecção por Zika virus/virologia
9.
Viruses ; 14(2)2022 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-35215846

RESUMO

Flaviviruses such as dengue virus (DENV) and Zika virus (ZIKV) have evolved sophisticated mechanisms to suppress the host immune system. For instance, flavivirus infections were found to sabotage peroxisomes, organelles with an important role in innate immunity. The current model suggests that the capsid (C) proteins of DENV and ZIKV downregulate peroxisomes, ultimately resulting in reduced production of interferons by interacting with the host protein PEX19, a crucial chaperone in peroxisomal biogenesis. Here, we aimed to explore the importance of peroxisomes and the role of C interaction with PEX19 in the flavivirus life cycle. By infecting cells lacking peroxisomes we show that this organelle is required for optimal DENV replication. Moreover, we demonstrate that DENV and ZIKV C bind PEX19 through a conserved PEX19-binding motif, which is also commonly found in cellular peroxisomal membrane proteins (PMPs). However, in contrast to PMPs, this interaction does not result in the targeting of C to peroxisomes. Furthermore, we show that the presence of C results in peroxisome loss due to impaired peroxisomal biogenesis, which appears to occur by a PEX19-independent mechanism. Hence, these findings challenge the current model of how flavivirus C might downregulate peroxisomal abundance and suggest a yet unknown role of peroxisomes in flavivirus biology.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Vírus da Dengue/fisiologia , Proteínas de Membrana/metabolismo , Domínios e Motivos de Interação entre Proteínas , Zika virus/fisiologia , Animais , Linhagem Celular , Vírus da Dengue/química , Humanos , Biogênese de Organelas , Peroxissomos/fisiologia , Replicação Viral , Zika virus/química
10.
Viruses ; 14(2)2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35215908

RESUMO

The transcription factors IRF3 and NF-κB are crucial in innate immune signalling in response to many viral and bacterial pathogens. However, mechanisms leading to their activation remain incompletely understood. Viral RNA can be detected by RLR receptors, such as RIG-I and MDA5, and the dsRNA receptor TLR3. Alternatively, the DExD-Box RNA helicases DDX1-DDX21-DHX36 activate IRF3/NF-κB in a TRIF-dependent manner independent of RIG-I, MDA5, or TLR3. Here, we describe DDX50, which shares 55.6% amino acid identity with DDX21, as a non-redundant factor that promotes activation of the IRF3 signalling pathway following its stimulation with viral RNA or infection with RNA and DNA viruses. Deletion of DDX50 in mouse and human cells impaired IRF3 phosphorylation and IRF3-dependent endogenous gene expression and cytokine/chemokine production in response to cytoplasmic dsRNA (polyIC transfection), and infection by RNA and DNA viruses. Mechanistically, whilst DDX50 co-immunoprecipitated TRIF, it acted independently to the previously described TRIF-dependent RNA sensor DDX1. Indeed, shRNA-mediated depletion of DDX1 showed DDX1 was dispensable for signalling in response to RNA virus infection. Importantly, loss of DDX50 resulted in a significant increase in replication and dissemination of virus following infection with vaccinia virus, herpes simplex virus, or Zika virus, highlighting its important role as a broad-ranging viral restriction factor.


Assuntos
RNA Helicases DEAD-box/metabolismo , Herpes Simples/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Simplexvirus/fisiologia , Vírus Vaccinia/fisiologia , Vaccinia/metabolismo , Infecção por Zika virus/metabolismo , Zika virus/fisiologia , Animais , RNA Helicases DEAD-box/genética , Herpes Simples/genética , Herpes Simples/virologia , Interações Hospedeiro-Patógeno , Humanos , Fator Regulador 3 de Interferon/genética , Camundongos , Fosforilação , Transdução de Sinais , Simplexvirus/genética , Vaccinia/genética , Vaccinia/virologia , Vírus Vaccinia/genética , Zika virus/genética , Infecção por Zika virus/genética , Infecção por Zika virus/virologia
11.
Viruses ; 14(2)2022 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-35215978

RESUMO

Zika virus (ZIKV) infection during pregnancy can result in a significant impact on the brain and eye of the developing fetus, termed congenital zika syndrome (CZS). At a morphological level, the main serious presentations of CZS are microcephaly and retinal scarring. At a cellular level, many cell types of the brain may be involved, but primarily neuronal progenitor cells (NPC) and developing neurons. Vav proteins have guanine exchange activity in converting GDP to GTP on proteins such as Rac1, Cdc42 and RhoA to stimulate intracellular signaling pathways. These signaling pathways are known to play important roles in maintaining the polarity and self-renewal of NPC pools by coordinating the formation of adherens junctions with cytoskeletal rearrangements. In developing neurons, these same pathways are adopted to control the formation and growth of neurites and mediate axonal guidance and targeting in the brain and retina. This review describes the role of Vavs in these processes and highlights the points of potential ZIKV interaction, such as (i) the binding and entry of ZIKV in cells via TAM receptors, which may activate Vav/Rac/RhoA signaling; (ii) the functional convergence of ZIKV NS2A with Vav in modulating adherens junctions; (iii) ZIKV NS4A/4B protein effects on PI3K/AKT in a regulatory loop via PPI3 to influence Vav/Rac1 signaling in neurite outgrowth; and (iv) the induction of SOCS1 and USP9X following ZIKV infection to regulate Vav protein degradation or activation, respectively, and impact Vav/Rac/RhoA signaling in NPC and neurons. Experiments to define these interactions will further our understanding of the molecular basis of CZS and potentially other developmental disorders stemming from in utero infections. Additionally, Vav/Rac/RhoA signaling pathways may present tractable targets for therapeutic intervention or molecular rationale for disease severity in CZS.


Assuntos
Encéfalo/patologia , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Infecção por Zika virus/patologia , Zika virus/fisiologia , Encéfalo/embriologia , Encéfalo/virologia , Proteínas de Ciclo Celular/metabolismo , Feminino , Humanos , Microcefalia/patologia , Microcefalia/virologia , Neurônios/patologia , Neurônios/virologia , Fosfatidilinositol 3-Quinases/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-vav/metabolismo , Infecção por Zika virus/genética , Infecção por Zika virus/virologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
12.
PLoS Negl Trop Dis ; 16(2): e0010166, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35171909

RESUMO

The tropism of Zika virus (ZIKV) has been described in the nervous system, blood, placenta, thymus, and skeletal muscle. We investigated the mechanisms of skeletal muscle susceptibility to ZIKV using an in vitro model of human skeletal muscle myogenesis, in which myoblasts differentiate into myotubes. Myoblasts were permissive to ZIKV infection, generating productive viral particles, while myotubes controlled ZIKV replication. To investigate the underlying mechanisms, we used gene expression profiling. First, we assessed gene changes in myotubes compared with myoblasts in the model without infection. As expected, we observed an increase in genes and pathways related to the contractile muscle system in the myotubes, a reduction in processes linked to proliferation, migration and cytokine production, among others, confirming the myogenic capacity of our system in vitro. A comparison between non-infected and infected myoblasts revealed more than 500 differentially expressed genes (DEGs). In contrast, infected myotubes showed almost 2,000 DEGs, among which we detected genes and pathways highly or exclusively expressed in myotubes, including those related to antiviral and innate immune responses. Such gene modulation could explain our findings showing that ZIKV also invades myotubes but does not replicate in these differentiated cells. In conclusion, we showed that ZIKV largely (but differentially) disrupts gene expression in human myoblasts and myotubes. Identifying genes involved in myotube resistance can shed light on potential antiviral mechanisms against ZIKV infection.


Assuntos
Infecção por Zika virus , Zika virus , Antivirais/metabolismo , Feminino , Expressão Gênica , Humanos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Gravidez , Zika virus/fisiologia , Infecção por Zika virus/genética
13.
Sci Rep ; 12(1): 1054, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-35058496

RESUMO

Neonatal microcephaly and adult Guillain-Barré syndrome are severe complications of Zika virus (ZIKV) infection. The robustly induced inflammatory cytokine expressions in ZIKV-infected patients may constitute a hallmark for severe disease. In the present study, the potential role of high mobility group box 1 protein (HMGB1) in ZIKV infection was investigated. HMGB1 protein expression was determined by the enzyme-linked immunosorbent assay (ELISA) and immunoblot assay. HMGB1's role in ZIKV infection was also explored using treatment with dexamethasone, an immunomodulatory drug, and HMGB1-knockdown (shHMGB1) Huh7 cells. Results showed that the Huh7 cells were highly susceptible to ZIKV infection. The infection was found to induce HMGB1 nuclear-to-cytoplasmic translocation, resulting in a > 99% increase in the cytosolic HMGB1 expression at 72-h post-infection (h.p.i). The extracellular HMGB1 level was elevated in a time- and multiplicity of infection (MOI)-dependent manner. Treatment of the ZIKV-infected cells with dexamethasone (150 µM) reduced HMGB1 extracellular release in a dose-dependent manner, with a maximum reduction of 71 ± 5.84% (P < 0.01). The treatment also reduced virus titers by over 83 ± 0.50% (P < 0.01). The antiviral effects, however, were not observed in the dexamethasone-treated shHMGB1 cells. These results suggest that translocation of HMGB1 occurred during ZIKV infection and inhibition of the translocation by dexamethasone coincided with a reduction in ZIKV replication. These findings highlight the potential of targeting the localization of HMGB1 in affecting ZIKV infection.


Assuntos
Dexametasona/farmacocinética , Proteína HMGB1/metabolismo , Infecção por Zika virus/tratamento farmacológico , Zika virus/efeitos dos fármacos , Linhagem Celular Tumoral , Dexametasona/metabolismo , Técnicas de Silenciamento de Genes , Proteína HMGB1/genética , Humanos , Transporte Proteico/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Zika virus/fisiologia
14.
PLoS One ; 17(1): e0261821, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35041689

RESUMO

The global health emergency posed by the outbreak of Zika virus (ZIKV), an arthropod-borne flavivirus causing severe neonatal neurological conditions, has subsided, but there continues to be transmission of ZIKV in endemic regions. As such, there is still a medical need for discovering and developing therapeutical interventions against ZIKV. To identify small-molecule compounds that inhibit ZIKV disease and transmission, we screened multiple small-molecule collections, mostly derived from natural products, for their ability to inhibit wild-type ZIKV. As a primary high-throughput screen, we used a viral cytopathic effect (CPE) inhibition assay conducted in Vero cells that was optimized and miniaturized to a 1536-well format. Suitably active compounds identified from the primary screen were tested in a panel of orthogonal assays using recombinant Zika viruses, including a ZIKV Renilla luciferase reporter assay and a ZIKV mCherry reporter system. Compounds that were active in the wild-type ZIKV inhibition and ZIKV reporter assays were further evaluated for their inhibitory effects against other flaviviruses. Lastly, we demonstrated that wild-type ZIKV is able to infect a 3D-bioprinted outer-blood-retina barrier tissue model and disrupt its barrier function, as measured by electrical resistance. One of the identified compounds (3-Acetyl-13-deoxyphomenone, NCGC00380955) was able to prevent the pathological effects of the viral infection on this clinically relevant ZIKV infection model.


Assuntos
Antivirais/farmacologia , Modelos Biológicos , Impressão Tridimensional , Retina , Replicação Viral/efeitos dos fármacos , Infecção por Zika virus , Zika virus/fisiologia , Animais , Antivirais/química , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos , Células Hep G2 , Humanos , Retina/metabolismo , Retina/virologia , Células Vero , Replicação Viral/genética , Infecção por Zika virus/tratamento farmacológico , Infecção por Zika virus/genética , Infecção por Zika virus/metabolismo
15.
J Virol ; 96(1): e0130121, 2022 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-34643427

RESUMO

The ability of viruses to evade the host antiviral immune system determines their level of replication fitness, species specificity, and pathogenic potential. Flaviviruses rely on the subversion of innate immune barriers, including the type I and type III interferon (IFN) antiviral systems. Zika virus infection induces the degradation of STAT2, an essential component of the IFN-stimulated gene transcription factor ISGF3. The mechanisms that lead to STAT2 degradation by Zika virus are poorly understood, but it is known to be mediated by the viral NS5 protein that binds to STAT2 and targets it for proteasome-mediated destruction. To better understand how NS5 engages and degrades STAT2, functional analysis of the protein interactions that lead to Zika virus and NS5-dependent STAT2 proteolysis were investigated. Data implicate the STAT2 coiled-coil domain as necessary and sufficient for NS5 interaction and proteasome degradation after Zika virus infection. Molecular dissection reveals that the first two α-helices of the STAT2 coiled-coil domain contain a specific targeting region for IFN antagonism. These functional interactions provide a more complete understanding of the essential protein-protein interactions needed for Zika virus evasion of the host antiviral response and identify new targets for antiviral therapeutic approaches. IMPORTANCE Zika virus infection can cause mild fever, rash, and muscle pain and in rare cases can lead to brain or nervous system diseases, including Guillain-Barré syndrome. Infections in pregnant women can increase the risk of miscarriage or serious birth defects, including brain anomalies and microcephaly. There are no drugs or vaccines for Zika disease. Zika virus is known to break down the host antiviral immune response, and this research project reveals how the virus suppresses interferon signaling, and may reveal therapeutic vulnerabilities.


Assuntos
Interações Hospedeiro-Patógeno , Interferons/metabolismo , Fator de Transcrição STAT2/metabolismo , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologia , Zika virus/fisiologia , Suscetibilidade a Doenças , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise , Fator de Transcrição STAT2/química , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
16.
Sci China Life Sci ; 65(3): 540-549, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34536207

RESUMO

Apoptosis is a form of programmed cell death that is essential for maintaining internal environmental stability. Disordered apoptosis can cause a variety of diseases; therefore, sensing apoptosis can provide help in study of mechanism of the relevant diseases and drug development. It is known that caspase-3 is a key enzyme involved in apoptosis and the expression of its activity is an indication of apoptosis. Here, we present a genetically encoded switch-on mNeonGreen2-based molecular biosensor. mNeonGreen2 is the brightest monomeric green fluorescent protein. The substrate of caspase-3, DEVD amino acid residues, is inserted in it, while cyclized by insertion of Nostoc punctiforme DnaE intein to abolish the fluorescence (inactive state). Caspase-3-catalyzed cleavage of DEVD linearizes mNeonGreen2 and rebuilds the natural barrel structure to restore the fluorescence (activated state). The characterization exhibited that the Caspase-3 biosensor has shortened response time, higher sensitivity, and prolonged functional shelf life in detection of caspase-3 amongst the existing counterparts. We also used the Caspase-3 biosensor to evaluate the effect of several drugs on the induction of apoptosis of HeLa and MCF-7 tumor cells and inhibition of Zika virus invasion.


Assuntos
Apoptose , Técnicas Biossensoriais/métodos , Caspase 3/análise , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cicloeximida/farmacologia , Células HEK293 , Células HeLa , Humanos , Fator de Necrose Tumoral alfa/farmacologia , Zika virus/fisiologia
17.
J Virol ; 96(2): e0118921, 2022 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-34730391

RESUMO

Zika virus (ZIKV) belongs to mosquito-borne flaviviruses. Unlike other members in the family, ZIKV can be sexually transmitted, and the female genital tracts are susceptible to ZIKV. However, the impact of ZIKV infection on nonpregnant female reproductive health is not understood. In this study, we investigated the effects of ZIKV infection on the ovary by using nonpregnant female interferon α/ß receptor-deficient (Ifnar1-/-) mice. The results showed that the ovary supported ZIKV replication, and the granulosa and theca cells of antral follicles were susceptible. ZIKV replication in situ significantly reduced the numbers of antral follicles, aggravated follicular atresia, and disrupted folliculogenesis. Notably, ZIKV replication in the ovary caused disordered ovarian steroidogenesis manifested by decreased expression of key enzymes linked to sex hormone synthesis, including the cytochrome P450 17A1 (CYP17A1) and aromatase (CYP19A1). Further, we observed that ZIKV infection disrupted the estrous cycle and thus prolonged the time to conceive. More importantly, although ZIKV RNA could not be detected at 3 months postinfection, damaged ovarian structure and dysfunction were also observed. Taken together, our study demonstrates that ZIKV infection in nonpregnant female mice cause ovarian damage and dysfunction, even long after ZIKV clearance. These data provide important information to understand the effects of ZIKV infection in female reproductive tissues and basic evidence for further studies. IMPORTANCE Zika virus (ZIKV), a flavivirus, is primarily transmitted by mosquito bites. But it can also be transmitted vertically and sexually. Although ZIKV-associated Guillain-Barré syndrome and microcephaly have drawn great attention, there have been few studies on the potential effects of ZIKV on the genital tract of nonpregnant females. This study investigated the effects of ZIKV on the ovaries in mice. We found that ZIKV replicated in the ovary and the granulosa and theca cells of antral follicles were susceptible. ZIKV replication in situ significantly damaged ovarian structure and function and disrupted folliculogenesis. Notably, ZIKV infection further disrupted the estrous cycle and prolonged the time to conceive in mice by causing disordered ovarian steroidogenesis. These effects were observed in both the acute phase and the recovery phase after viral elimination. Overall, the new findings provide important additions to make out the potential adverse impacts of ZIKV on reproductive health in females.


Assuntos
Fertilização , Ovário/virologia , Progesterona/sangue , Zika virus/patogenicidade , Animais , Modelos Animais de Doenças , Ciclo Estral , Feminino , Atresia Folicular , Camundongos , Ovário/patologia , Ovário/fisiopatologia , Receptor de Interferon alfa e beta/deficiência , Especificidade da Espécie , Replicação Viral , Zika virus/fisiologia , Infecção por Zika virus/sangue , Infecção por Zika virus/virologia
18.
J Virol ; 96(2): e0177421, 2022 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-34757841

RESUMO

Alphaviruses and flaviviruses have class II fusion glycoproteins that are essential for virion assembly and infectivity. Importantly, the tip of domain II is structurally conserved between the alphavirus and flavivirus fusion proteins, yet whether these structural similarities between virus families translate to functional similarities is unclear. Using in vivo evolution of Zika virus (ZIKV), we identified several novel emerging variants, including an envelope glycoprotein variant in ß-strand c (V114M) of domain II. We have previously shown that the analogous ß-strand c and the ij loop, located in the tip of domain II of the alphavirus E1 glycoprotein, are important for infectivity. This led us to hypothesize that flavivirus E ß-strand c also contributes to flavivirus infection. We generated this ZIKV glycoprotein variant and found that while it had little impact on infection in mosquitoes, it reduced replication in human cells and mice and increased virus sensitivity to ammonium chloride, as seen for alphaviruses. In light of these results and given our alphavirus ij loop studies, we mutated a conserved alanine at the tip of the flavivirus ij loop to valine to test its effect on ZIKV infectivity. Interestingly, this mutation inhibited infectious virion production of ZIKV and yellow fever virus, but not West Nile virus. Together, these studies show that shared domains of the alphavirus and flavivirus class II fusion glycoproteins harbor structurally analogous residues that are functionally important and contribute to virus infection in vivo. IMPORTANCE Arboviruses are a significant global public health threat, yet there are no antivirals targeting these viruses. This problem is in part due to our lack of knowledge of the molecular mechanisms involved in the arbovirus life cycle. In particular, virus entry and assembly are essential processes in the virus life cycle and steps that can be targeted for the development of antiviral therapies. Therefore, understanding common, fundamental mechanisms used by different arboviruses for entry and assembly is essential. In this study, we show that flavivirus and alphavirus residues located in structurally conserved and analogous regions of the class II fusion proteins contribute to common mechanisms of entry, dissemination, and infectious-virion production. These studies highlight how class II fusion proteins function and provide novel targets for development of antivirals.


Assuntos
Alphavirus/fisiologia , Flavivirus/fisiologia , Proteínas Virais de Fusão/metabolismo , Vírion/metabolismo , Replicação Viral , Células A549 , Alphavirus/efeitos dos fármacos , Cloreto de Amônio/farmacologia , Animais , Culicidae/virologia , Flavivirus/efeitos dos fármacos , Humanos , Interferon Tipo I/deficiência , Camundongos , Camundongos Mutantes , Mutação , Domínios Proteicos , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Vírion/genética , Montagem de Vírus/genética , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/genética , Zika virus/efeitos dos fármacos , Zika virus/fisiologia , Infecção por Zika virus/virologia
19.
J Virol ; 96(4): e0211721, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-34935441

RESUMO

Zika virus (ZIKV) is a flavivirus that is mainly transmitted by Aedes mosquitos and normally causes mild symptoms. During the outbreak in the Americas in 2015, it was associated with more severe implications, like microcephaly in newborns and the Guillain-Barré syndrome. The lack of specific vaccines and cures strengthens the need for a deeper understanding of the virus life cycle and virus-host interactions. The restriction factor tetherin (THN) is an interferon-inducible cellular protein with broad antiviral properties. It is known to inhibit the release of various enveloped viruses by tethering them to each other and the cell membrane, thereby preventing their further spread. On the other hand, different viruses have developed various escape strategies against THN. Analysis of the cross-talk between ZIKV and THN revealed that, despite a strong induction of THN mRNA expression in ZIKV-infected cells, this is not reflected by an elevated protein level of THN. Contrariwise, the THN protein level is decreased due to a reduced half-life. The increased degradation of THN in ZIKV infected cells involves the endo-lysosomal system but does not depend on the early steps of autophagy. Enrichment of THN by depletion of the ESCRT-0 protein HRS diminishes ZIKV release and spread, which points out the capacity of THN to restrict ZIKV and explains the enhanced THN degradation in infected cells as an effective viral escape strategy. IMPORTANCE Although tetherin expression is strongly induced by ZIKV infection there is a reduction in the amount of tetherin protein. This is due to enhanced lysosomal degradation. However, if the tetherin level is rescued then the release of ZIKV is impaired. This shows that tetherin is a restriction factor for ZIKV, and the induction of an efficient degradation represents a viral escape strategy. To our knowledge, this is the first study that describes and characterizes tetherin as a restriction factor for the ZIKV life cycle.


Assuntos
Antígenos CD/metabolismo , Zika virus/fisiologia , Animais , Antígenos CD/genética , /metabolismo , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Meia-Vida , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Inibidores de Proteassoma/farmacologia , RNA Mensageiro/genética , Liberação de Vírus
20.
Cell ; 184(25): 6067-6080.e13, 2021 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-34852238

RESUMO

The human monoclonal antibody (HmAb) C10 potently cross-neutralizes Zika virus (ZIKV) and dengue virus. Analysis of antibody fragment (Fab) C10 interactions with ZIKV and dengue virus serotype 2 (DENV2) particles by cryoelectron microscopy (cryo-EM) and amide hydrogen/deuterium exchange mass spectrometry (HDXMS) shows that Fab C10 binding decreases overall ZIKV particle dynamics, whereas with DENV2, the same Fab causes increased dynamics. Testing of different Fab C10:DENV2 E protein molar ratios revealed that, at higher Fab ratios, especially at saturated concentrations, the Fab enhanced viral dynamics (detected by HDXMS), and observation under cryo-EM showed increased numbers of distorted particles. Our results suggest that Fab C10 stabilizes ZIKV but that with DENV2 particles, high Fab C10 occupancy promotes E protein dimer conformational changes leading to overall increased particle dynamics and distortion of the viral surface. This is the first instance of a broadly neutralizing antibody eliciting virus-specific increases in whole virus particle dynamics.


Assuntos
Anticorpos Neutralizantes , Vírus da Dengue , Dengue , Proteínas do Envelope Viral , Infecção por Zika virus , Zika virus , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/imunologia , Reações Cruzadas , Dengue/imunologia , Dengue/virologia , Vírus da Dengue/imunologia , Vírus da Dengue/fisiologia , Humanos , Ligação Proteica , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Zika virus/imunologia , Zika virus/fisiologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia
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