RESUMO
Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1, FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and reduced levels of viral antigen in lungs during the early stages of infection. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins and provides molecular insight into the possible underlying molecular defects in fragile X syndrome.
Assuntos
COVID-19 , Síndrome do Cromossomo X Frágil , Humanos , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Peptídeos/metabolismo , Proteínas de Ligação a RNA/genética , SARS-CoV-2RESUMO
Tick-borne encephalitis virus is an enveloped, pathogenic, RNA virus in the family Flaviviridae, genus Flavivirus. Viral particles are formed when the nucleocapsid, consisting of an RNA genome and multiple copies of the capsid protein, buds through the endoplasmic reticulum membrane and acquires the viral envelope and the associated proteins. The coordination of the nucleocapsid components to the sites of assembly and budding are poorly understood. Here, we investigate the interactions of the wild-type and truncated capsid proteins with membranes with biophysical methods and model membrane systems. We show that capsid protein initially binds membranes via electrostatic interactions with negatively-charged lipids, which is followed by membrane insertion. Additionally, we show that membrane-bound capsid protein can recruit viral genomic RNA. We confirm the biological relevance of the biophysical findings by using mass spectrometry to show that purified virions contain negatively-charged lipids. Our results suggest that nucleocapsid assembly is coordinated by negatively-charged membrane patches on the endoplasmic reticulum and that the capsid protein mediates direct contacts between the nucleocapsid and the membrane.
Assuntos
Proteínas do Capsídeo , Vírus da Encefalite Transmitidos por Carrapatos , Proteínas do Capsídeo/metabolismo , Vírus da Encefalite Transmitidos por Carrapatos/genética , Montagem de Vírus , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Membrana/metabolismo , Lipídeos , Ligação ProteicaRESUMO
BackgroundThe current SARS-CoV-2 pandemic has highlighted a need for easy and safe blood sampling in combination with accurate serological methodology. Venipuncture for testing is usually performed by trained staff at healthcare centres. Long travel distances to healthcare centres in rural regions may introduce a bias of testing towards relatively large communities with closer access. Rural regions are therefore often not represented in population-based data.AimThe aim of this retrospective cohort study was to develop and implement a strategy for at-home testing in a rural region of Sweden during spring 2021, and to evaluate its role to provide equal health care for its inhabitants.MethodsWe developed a sensitive method to measure antibodies to the S-protein of SARS-CoV-2 and optimised this assay for clinical use together with a strategy of at-home capillary blood sampling.ResultsWe demonstrated that our ELISA gave comparable results after analysis of capillary blood or serum from SARS-CoV-2-experienced individuals. We demonstrated stability of the assay under conditions that reflected temperature and humidity during winter or summer. By assessment of capillary blood samples from 4,122 individuals, we could show both feasibility of the strategy and that implementation shifted the geographical spread of testing in favour of rural areas.ConclusionImplementation of at-home sampling enabled citizens living in remote rural areas access to centralised and sensitive laboratory antibody tests. The strategy for testing used here could therefore enable disease control authorities to get rapid access to information concerning immunity to infectious diseases, even across vast geographical distance.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/diagnóstico , COVID-19/epidemiologia , Estudos Retrospectivos , Suécia/epidemiologia , Teste para COVID-19 , Anticorpos AntiviraisRESUMO
Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immunoprecipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both requires nuclear steps. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Vírus Uukuniemi/fisiologia , Animais , Antivirais/farmacologia , Infecções por Bunyaviridae/virologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Glicoproteínas/metabolismo , Interações entre Hospedeiro e Microrganismos , Humanos , Espectrometria de Massas , Proteômica , Piridinas/farmacologia , Quinolinas/farmacologia , Interferência de RNA , Vírus de RNA/fisiologia , Vírus Uukuniemi/efeitos dos fármacos , Células Vero , Proteínas do Envelope Viral/metabolismo , Liberação de Vírus , Replicação ViralRESUMO
BACKGROUND: Tick-borne encephalitis virus (TBEV) is considered to be the medically most important arthropod-borne virus in Europe. The symptoms of an infection range from subclinical to mild flu-like disease to lethal encephalitis. The exact determinants of disease severity are not known; however, the virulence of the strain as well as the immune status of the host are thought to be important factors for the outcome of the infection. Here we investigated virulence determinants in TBEV infection. METHOD: Mice were infected with different TBEV strains, and high virulent and low virulent TBEV strains were chosen. Sequence alignment identified differences that were cloned to generate chimera virus. The infection rate of the parental and chimeric virus were evaluated in primary mouse neurons, astrocytes, mouse embryonic fibroblasts, and in vivo. Neutralizing capacity of serum from individuals vaccinated with the FSME-IMMUN® and Encepur® or combined were evaluated. RESULTS: We identified a highly pathogenic and neurovirulent TBEV strain, 93/783. Using sequence analysis, we identified the envelope (E) protein of 93/783 as a potential virulence determinant and cloned it into the less pathogenic TBEV strain Torö. We found that the chimeric virus specifically infected primary neurons more efficiently compared to wild-type (WT) Torö and this correlated with enhanced pathogenicity and higher levels of viral RNA in vivo. The E protein is also the major target of neutralizing antibodies; thus, genetic variation in the E protein could influence the efficiency of the two available vaccines, FSME-IMMUN® and Encepur®. As TBEV vaccine breakthroughs have occurred in Europe, we chose to compare neutralizing capacity from individuals vaccinated with the two different vaccines or a combination of them. Our data suggest that the different vaccines do not perform equally well against the two Swedish strains. CONCLUSIONS: Our findings show that two amino acid substitutions of the E protein found in 93/783, A83T, and A463S enhanced Torö infection of neurons as well as pathogenesis and viral replication in vivo; furthermore, we found that genetic divergence from the vaccine strain resulted in lower neutralizing antibody titers in vaccinated individuals.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/genética , Encefalite Transmitida por Carrapatos/genética , Neurônios/fisiologia , Neurônios/virologia , Proteínas do Envelope Viral/genética , Vacinas Virais/administração & dosagem , Sequência de Aminoácidos , Animais , Células Cultivadas , Chlorocebus aethiops , Vírus da Encefalite Transmitidos por Carrapatos/efeitos dos fármacos , Vírus da Encefalite Transmitidos por Carrapatos/metabolismo , Encefalite Transmitida por Carrapatos/metabolismo , Encefalite Transmitida por Carrapatos/prevenção & controle , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neurônios/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Células Vero , Proteínas do Envelope Viral/metabolismo , Carga Viral/efeitos dos fármacos , Carga Viral/genética , Vacinas Virais/metabolismoRESUMO
Flavivirus is a positive-sense, single-stranded RNA viral genus, with members causing severe diseases in humans such as tick-borne encephalitis, yellow fever, and dengue fever. Flaviviruses are known to cause remodeling of intracellular membranes into small cavities, where replication of the viral RNA takes place. Nonstructural (NS) proteins are not part of the virus coat and are thought to participate in the formation of these viral replication compartments (RCs). Here, we used tick-borne encephalitis virus (TBEV) as a model for the flaviviruses and developed a stable human cell line in which the expression of NS proteins can be induced without viral RNA replication. The model system described provides a novel and benign tool for studies of the viral components under controlled expression levels. We show that the expression of six NS proteins is sufficient to induce infection-like dilation of the endoplasmic reticulum (ER) and the formation of RC-like membrane invaginations. The NS proteins form a membrane-associated complex in the ER, and electron tomography reveals that the dilated areas of the ER are closely associated with lipid droplets and mitochondria. We propose that the NS proteins drive the remodeling of ER membranes and that viral RNA, RNA replication, viral polymerase, and TBEV structural proteins are not required.IMPORTANCE TBEV infection causes a broad spectrum of symptoms, ranging from mild fever to severe encephalitis. Similar to other flaviviruses, TBEV exploits intracellular membranes to build RCs for viral replication. The viral NS proteins have been suggested to be involved in this process; however, the mechanism of RC formation and the roles of individual NS proteins remain unclear. To study how TBEV induces membrane remodeling, we developed an inducible stable cell system expressing the TBEV NS polyprotein in the absence of viral RNA replication. Using this system, we were able to reproduce RC-like vesicles that resembled the RCs formed in flavivirus-infected cells, in terms of morphology and size. This cell system is a robust tool to facilitate studies of flavivirus RC formation and is an ideal model for the screening of antiviral agents at a lower biosafety level.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/metabolismo , Proteínas não Estruturais Virais/metabolismo , Estruturas Virais/metabolismo , Vírus da Encefalite Transmitidos por Carrapatos/genética , Encefalite Transmitida por Carrapatos/metabolismo , Encefalite Transmitida por Carrapatos/virologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Flavivirus/genética , Flavivirus/metabolismo , Expressão Gênica/genética , Regulação Viral da Expressão Gênica/genética , Células HeLa , Humanos , Modelos Biológicos , RNA Viral/genética , Proteínas não Estruturais Virais/fisiologia , Estruturas Virais/fisiologia , Replicação Viral/fisiologiaRESUMO
Efficient antiviral immunity requires interference with virus replication at multiple layers targeting diverse steps in the viral life cycle. We describe here a novel flavivirus inhibition mechanism that results in interferon-mediated obstruction of tick-borne encephalitis virus particle assembly and involves release of malfunctioning membrane-associated capsid (C) particles. This mechanism is controlled by the activity of the interferon-induced protein viperin, a broad-spectrum antiviral interferon-stimulated gene. Through analysis of the viperin-interactome, we identified the Golgi brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) as the cellular protein targeted by viperin. Viperin-induced antiviral activity, as well as C-particle release, was stimulated by GBF1 inhibition and knockdown and reduced by elevated levels of GBF1. Our results suggest that viperin targets flavivirus virulence by inducing the secretion of unproductive noninfectious virus particles via a GBF1-dependent mechanism. This as-yet-undescribed antiviral mechanism allows potential therapeutic intervention.IMPORTANCE The interferon response can target viral infection on almost every level; however, very little is known about the interference of flavivirus assembly. We show here that interferon, through the action of viperin, can disturb the assembly of tick-borne encephalitis virus. The viperin protein is highly induced after viral infection and exhibit broad-spectrum antiviral activity. However, the mechanism of action is still elusive and appears to vary between the different viruses, indicating that cellular targets utilized by several viruses might be involved. In this study, we show that viperin induces capsid particle release by interacting and inhibiting the function of the cellular protein Golgi brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1). GBF1 is a key protein in the cellular secretory pathway and is essential in the life cycle of many viruses, also targeted by viperin, implicating GBF1 as a novel putative drug target.
Assuntos
Infecções por Flavivirus/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Interferon Tipo I/farmacologia , Proteínas/metabolismo , Células A549 , Animais , Proteínas do Capsídeo/metabolismo , Chlorocebus aethiops , Flavivirus/efeitos dos fármacos , Flavivirus/patogenicidade , Infecções por Flavivirus/tratamento farmacológico , Infecções por Flavivirus/virologia , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Células HeLa , Humanos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Células Vero , Virulência , Montagem de Vírus/efeitos dos fármacosRESUMO
Flaviviruses are arthropod-borne viruses that constitute a major global health problem, with millions of human infections annually. Their pathogenesis ranges from mild illness to severe manifestations such as hemorrhagic fever and fatal encephalitis. Type I interferons (IFNs) are induced in response to viral infection and stimulate the expression of interferon-stimulated genes (ISGs), including that encoding viperin (virus-inhibitory protein, endoplasmic reticulum associated, IFN inducible), which shows antiviral activity against a broad spectrum of viruses, including several flaviviruses. Here we describe a novel antiviral mechanism employed by viperin against two prominent flaviviruses, tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). Viperin was found to interact and colocalize with the structural proteins premembrane (prM) and envelope (E) of TBEV, as well as with nonstructural (NS) proteins NS2A, NS2B, and NS3. Interestingly, viperin expression reduced the NS3 protein level, and the stability of the other interacting viral proteins, but only in the presence of NS3. We also found that although viperin interacted with NS3 of mosquito-borne flaviviruses (ZIKV, Japanese encephalitis virus, and yellow fever virus), only ZIKV was sensitive to the antiviral effect of viperin. This sensitivity correlated with viperin's ability to induce proteasome-dependent degradation of NS3. ZIKV and TBEV replication was rescued completely when NS3 was overexpressed, suggesting that the viral NS3 is the specific target of viperin. In summary, we present here a novel antiviral mechanism of viperin that is selective for specific viruses in the genus Flavivirus, affording the possible availability of new drug targets that can be used for therapeutic intervention.IMPORTANCE Flaviviruses are a group of enveloped RNA viruses that cause severe diseases in humans and animals worldwide, but no antiviral treatment is yet available. Viperin, a host protein produced in response to infection, effectively restricts the replication of several flaviviruses, but the exact molecular mechanisms have not been elucidated. Here we have identified a novel mechanism employed by viperin to inhibit the replication of two flaviviruses: tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). Viperin induced selective degradation via the proteasome of TBEV and ZIKV nonstructural 3 (NS3) protein, which is involved in several steps of the viral life cycle. Furthermore, viperin also reduced the stability of several other viral proteins in a NS3-dependent manner, suggesting a central role of NS3 in viperin's antiflavivirus activity. Taking the results together, our work shows important similarities and differences among the members of the genus Flavivirus and could lead to the possibility of therapeutic intervention.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Regulação da Expressão Gênica , Imunidade Inata , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/metabolismo , Proteólise , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/fisiologia , Zika virus/fisiologia , Células HEK293 , Células HeLa , Humanos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Complexo de Endopeptidases do Proteassoma/genética , Proteínas/genética , Proteínas/imunologia , RNA Helicases/genética , RNA Helicases/imunologia , RNA Helicases/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologiaRESUMO
Viperin (RSAD2) is an interferon-stimulated antiviral protein that belongs to the radical S-adenosylmethionine (SAM) enzyme family. Viperin's iron-sulfur (Fe/S) cluster is critical for its antiviral activity against many different viruses. CIA1 (CIAO1), an essential component of the cytosolic iron-sulfur protein assembly (CIA) machinery, is crucial for Fe/S cluster insertion into viperin and hence for viperin's antiviral activity. In the CIA pathway, CIA1 cooperates with CIA2A, CIA2B, and MMS19 targeting factors to form various complexes that mediate the dedicated maturation of specific Fe/S recipient proteins. To date, however, the mechanisms of how viperin acquires its radical SAM Fe/S cluster to gain antiviral activity are poorly understood. Using co-immunoprecipitation and 55Fe-radiolabeling experiments, we therefore studied the roles of CIA2A, CIA2B, and MMS19 for Fe/S cluster insertion. CIA2B and MMS19 physically interacted with the C terminus of viperin and used CIA1 as the primary viperin-interacting protein. In contrast, CIA2A bound to viperin's N terminus in a CIA1-, CIA2B-, and MMS19-independent fashion. Of note, the observed interaction of both CIA2 isoforms with a single Fe/S target protein is unprecedented in the CIA pathway. 55Fe-radiolabeling experiments with human cells depleted of CIA1, CIA2A, CIA2B, or MMS19 revealed that CIA1, but none of the other CIA factors, is predominantly required for 55Fe/S cluster incorporation into viperin. Collectively, viperin maturation represents a novel CIA pathway with a minimal requirement of the CIA-targeting factors and represents a new paradigm for the insertion of the Fe/S cofactor into a radical SAM protein.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Metalochaperonas/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Substituição de Aminoácidos , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/química , Proteínas de Transporte/genética , Células HEK293 , Humanos , Imunoprecipitação , Ferro/química , Ferro/metabolismo , Radioisótopos de Ferro , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Metalochaperonas/antagonistas & inibidores , Metalochaperonas/química , Metalochaperonas/genética , Metaloproteínas , Mutação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Proteínas Nucleares/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Proteínas/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
We compared 2 tick-borne encephalitis virus strains isolated from 2 different foci that cause different symptoms in tick-borne encephalitis patients, from neurologic to mild gastrointestinal symptoms. We compared neuroinvasiveness, neurovirulence, and proinflammatory cytokine response in mice and found unique differences that contribute to our understanding of pathogenesis.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Encefalite Transmitida por Carrapatos/virologia , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Índice de Gravidade de Doença , Especificidade da Espécie , VirulênciaRESUMO
BACKGROUND: Flaviviruses are a group of diverse and emerging arboviruses and an immense global health problem. A number of flaviviruses are neurotropic, causing severe encephalitis and even death. Type I interferons (IFNs) are the first line of defense of the innate immune system against flavivirus infection. IFNs elicit the concerted action of numerous interferon-stimulated genes (ISGs) to restrict both virus infection and replication. Viperin (virus-inhibitory protein, endoplasmic reticulum-associated, IFN-inducible) is an ISG with broad-spectrum antiviral activity against multiple flaviviruses in vitro. Its activity in vivo restricts neurotropic infections to specific regions of the central nervous system (CNS). However, the cell types in which viperin activity is required are unknown. Here we have examined both the regional and cell-type specificity of viperin in the defense against infection by several model neurotropic flaviviruses. METHODS: Viral burden and IFN induction were analyzed in vivo in wild-type and viperin-/- mice infected with Langat virus (LGTV). The effects of IFN pretreatment were tested in vitro in primary neural cultures from different brain regions in response to infection with tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Zika virus (ZIKV). RESULTS: Viperin activity restricted nonlethal LGTV infection in the spleen and the olfactory bulb following infection via a peripheral route. Viperin activity was also necessary to restrict LGTV replication in the olfactory bulb and the cerebrum following CNS infection, but not in the cerebellum. In vitro, viperin could restrict TBEV replication in primary cortical neurons, but not in the cerebellar granule cell neurons. Interferon-induced viperin was also very important in primary cortical neurons to control TBEV, WNV, and ZIKV. CONCLUSIONS: Our findings show that viperin restricts replication of neurotropic flaviviruses in the CNS in a region- and cell-type-specific manner. The most important sites of activity are the olfactory bulb and cerebrum. Activity within the cerebrum is required in the cortical neurons in order to restrict spread. This study exemplifies cell type and regional diversity of the IFN response within the CNS and shows the importance of a potent broad-spectrum antiviral ISG.
Assuntos
Antivirais/metabolismo , Infecções por Flavivirus/patologia , Flavivirus/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Proteínas/metabolismo , Animais , Antivirais/uso terapêutico , Astrócitos/patologia , Astrócitos/virologia , Células Cultivadas , Cérebro/patologia , Cérebro/virologia , Modelos Animais de Doenças , Interferon-alfa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/patologia , Neurônios/virologia , Bulbo Olfatório/efeitos dos fármacos , Bulbo Olfatório/patologia , Bulbo Olfatório/virologia , Proteínas/genética , Proteínas/uso terapêutico , Replicação Viral/efeitos dos fármacosRESUMO
We report a case of human tick-borne encephalitis (TBE) in which the TBE virus was isolated from the biting tick. Viral growth and sequence were characterized and compared with those of a reference strain. Virus isolation from ticks from patients with TBE may offer a new approach for studies of epidemiology and pathogenicity.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/genética , Encefalite Transmitida por Carrapatos/virologia , Ixodes/virologia , Células A549 , Idoso , Animais , Encefalite Transmitida por Carrapatos/diagnóstico , Encefalite Transmitida por Carrapatos/patologia , Humanos , Masculino , Ninfa/virologia , RNA Viral/isolamento & purificação , CarrapatosRESUMO
BACKGROUND: Neurotropic flaviviruses such as tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) are causative agents of severe brain-related diseases including meningitis, encephalitis, and microcephaly. We have previously shown that local type I interferon response within the central nervous system (CNS) is involved in the protection of mice against tick-borne flavivirus infection. However, the cells responsible for mounting this protective response are not defined. METHODS: Primary astrocytes were isolated from wild-type (WT) and interferon alpha receptor knock out (IFNAR-/-) mice and infected with neurotropic flaviviruses. Viral replication and spread, IFN induction and response, and cellular viability were analyzed. Transcriptional levels in primary astrocytes treated with interferon or supernatant from virus-infected cells were analyzed by RNA sequencing and evaluated by different bioinformatics tools. RESULTS: Here, we show that astrocytes control viral replication of different TBEV strains, JEV, WNV, and ZIKV. In contrast to fibroblast, astrocytes mount a rapid interferon response and restrict viral spread. Furthermore, basal expression levels of key interferon-stimulated genes are high in astrocytes compared to mouse embryonic fibroblasts. Bioinformatic analysis of RNA-sequencing data reveals that astrocytes have established a basal antiviral state which contributes to the rapid viral recognition and upregulation of interferons. The most highly upregulated pathways in neighboring cells were linked to type I interferon response and innate immunity. The restriction in viral growth was dependent on interferon signaling, since loss of the interferon receptor, or its blockade in wild-type cells, resulted in high viral replication and virus-induced cytopathic effects. Astrocyte supernatant from TBEV-infected cells can restrict TBEV growth in astrocytes already 6 h post infection, the effect on neurons is highly reinforced, and astrocyte supernatant from 3 h post infection is already protective. CONCLUSIONS: These findings suggest that the combination of an intrinsic constitutive antiviral response and the fast induction of type I IFN production by astrocytes play an important role in self-protection of astrocytes and suppression of flavivirus replication in the CNS.
Assuntos
Astrócitos , Flavivirus/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Receptor de Interferon alfa e beta/metabolismo , Animais , Animais Recém-Nascidos , Antivirais/farmacologia , Astrócitos/metabolismo , Astrócitos/patologia , Astrócitos/virologia , Biologia Computacional , Imunoglobulina G/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/virologia , Oxazinas/farmacologia , RNA Mensageiro/metabolismo , Receptor de Interferon alfa e beta/genética , Xantenos/farmacologiaRESUMO
BACKGROUND: Although type I interferons (IFNs)-key effectors of antiviral innate immunity are known to be induced via different pattern recognition receptors (PRRs), the cellular source and the relative contribution of different PRRs in host protection against viral infection is often unclear. IPS-1 is a downstream adaptor for retinoid-inducible gene I (RIG-I)-like receptor signaling. In this study, we investigate the relative contribution of IPS-1 in the innate immune response in the different brain regions during infection with tick-borne encephalitis virus (TBEV), a flavivirus that causes a variety of severe symptoms like hemorrhagic fevers, encephalitis, and meningitis in the human host. METHODS: IPS-1 knockout mice were infected with TBEV/Langat virus (LGTV), and viral burden in the peripheral and the central nervous systems, type I IFN induction, brain infiltrating cells, and inflammatory response was analyzed. RESULTS: We show that IPS-1 is indispensable for controlling TBEV and LGTV infections in the peripheral and central nervous system. Our data indicate that IPS-1 regulates neuropathogenicity in mice. IFN response is differentially regulated in distinct regions of the central nervous system (CNS) influencing viral tropism, as LGTV replication was mainly restricted to olfactory bulb in wild-type (WT) mice. In contrast to the other brain regions, IFN upregulation in the olfactory bulb was dependent on IPS-1 signaling. IPS-1 regulates basal levels of antiviral interferon-stimulated genes (ISGs) like viperin and IRF-1 which contributes to the establishment of early viral replication which inhibits STAT1 activation. This diminishes the antiviral response even in the presence of high IFN-ß levels. Consequently, the absence of IPS-1 causes uncontrolled virus replication, in turn resulting in apoptosis, activation of microglia and astrocytes, elevated proinflammatory response, and recruitment of inflammatory cells into the CNS. CONCLUSIONS: We show that LGTV replication is restricted to the olfactory bulb and that IPS-1 is a very important player in the olfactory bulb in shaping the innate immune response by inhibiting early viral replication and viral spread throughout the central nervous system. In the absence of IPS-1, higher viral replication leads to the evasion of antiviral response by inhibiting interferon signaling. Our data suggest that the local microenvironment of distinct brain regions is critical to determine virus permissiveness.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Encefalite Transmitida por Carrapatos/patologia , Interferon Tipo I/metabolismo , Bulbo Olfatório/metabolismo , Transdução de Sinais/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígenos CD , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Regulação Viral da Expressão Gênica/genética , Hipocampo/citologia , Interferon Tipo I/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Bulbo Olfatório/patologia , Bulbo Olfatório/virologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Fatores de Tempo , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
UNLABELLED: Uukuniemi virus (UUKV) is a tick-borne member of the Phlebovirus genus (family Bunyaviridae) and has been widely used as a safe laboratory model to study aspects of bunyavirus replication. Recently, a number of new tick-borne phleboviruses have been discovered, some of which, like severe fever with thrombocytopenia syndrome virus and Heartland virus, are highly pathogenic in humans. UUKV could now serve as a useful comparator to understand the molecular basis for the different pathogenicities of these related viruses. We established a reverse-genetics system to recover UUKV entirely from cDNA clones. We generated two recombinant viruses, one in which the nonstructural protein NSs open reading frame was deleted from the S segment and one in which the NSs gene was replaced with green fluorescent protein (GFP), allowing convenient visualization of viral infection. We show that the UUKV NSs protein acts as a weak interferon antagonist in human cells but that it is unable to completely counteract the interferon response, which could serve as an explanation for its inability to cause disease in humans. IMPORTANCE: Uukuniemi virus (UUKV) is a tick-borne phlebovirus that is apathogenic for humans and has been used as a convenient model to investigate aspects of phlebovirus replication. Recently, new tick-borne phleboviruses have emerged, such as severe fever with thrombocytopenia syndrome virus in China and Heartland virus in the United States, that are highly pathogenic, and UUKV will now serve as a comparator to aid in the understanding of the molecular basis for the virulence of these new viruses. To help such investigations, we have developed a reverse-genetics system for UUKV that permits manipulation of the viral genome. We generated viruses lacking the nonstructural protein NSs and show that UUKV NSs is a weak interferon antagonist. In addition, we created a virus that expresses GFP and thus allows convenient monitoring of virus replication. These new tools represent a significant advance in the study of tick-borne phleboviruses.
Assuntos
Interferons/antagonistas & inibidores , Vírus Uukuniemi/imunologia , Vírus Uukuniemi/fisiologia , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular , DNA Complementar/genética , DNA Viral/genética , Deleção de Genes , Humanos , Recombinação Genética , Genética Reversa , Vírus Uukuniemi/genética , Proteínas não Estruturais Virais/genéticaRESUMO
UNLABELLED: Vector-borne flaviviruses, such as tick-borne encephalitis virus (TBEV), West Nile virus, and dengue virus, cause millions of infections in humans. TBEV causes a broad range of pathological symptoms, ranging from meningitis to severe encephalitis or even hemorrhagic fever, with high mortality. Despite the availability of an effective vaccine, the incidence of TBEV infections is increasing. Not much is known about the role of the innate immune system in the control of TBEV infections. Here, we show that the type I interferon (IFN) system is essential for protection against TBEV and Langat virus (LGTV) in mice. In the absence of a functional IFN system, mice rapidly develop neurological symptoms and succumb to LGTV and TBEV infections. Type I IFN system deficiency results in severe neuroinflammation in LGTV-infected mice, characterized by breakdown of the blood-brain barrier and infiltration of macrophages into the central nervous system (CNS). Using mice with tissue-specific IFN receptor deletions, we show that coordinated activation of the type I IFN system in peripheral tissues as well as in the CNS is indispensable for viral control and protection against virus induced inflammation and fatal encephalitis. IMPORTANCE: The type I interferon (IFN) system is important to control viral infections; however, the interactions between tick-borne encephalitis virus (TBEV) and the type I IFN system are poorly characterized. TBEV causes severe infections in humans that are characterized by fever and debilitating encephalitis, which can progress to chronic illness or death. No treatment options are available. An improved understanding of antiviral innate immune responses is pivotal for the development of effective therapeutics. We show that type I IFN, an effector molecule of the innate immune system, is responsible for the extended survival of TBEV and Langat virus (LGTV), an attenuated member of the TBE serogroup. IFN production and signaling appeared to be essential in two different phases during infection. The first phase is in the periphery, by reducing systemic LGTV replication and spreading into the central nervous system (CNS). In the second phase, the local IFN response in the CNS prevents virus-induced inflammation and the development of encephalitis.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/mortalidade , Interferon Tipo I/imunologia , Animais , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interferon/deficiência , Análise de SobrevidaRESUMO
Viperin is an interferon-induced protein with a broad antiviral activity. This evolutionary conserved protein contains a radical S-adenosyl-l-methionine (SAM) domain which has been shown in vitro to hold a [4Fe-4S] cluster. We identified tick-borne encephalitis virus (TBEV) as a novel target for which human viperin inhibits productionof the viral genome RNA. Wt viperin was found to require ER localization for full antiviral activity and to interact with the cytosolic Fe/S protein assembly factor CIAO1. Radiolabelling in vivo revealed incorporation of (55) Fe, indicative for the presence of an Fe-S cluster. Mutation of the cysteine residues ligating the Fe-S cluster in the central radical SAM domain entirely abolished both antiviral activity and incorporation of (55) Fe. Mutants lacking the extreme C-terminal W361 did not interact with CIAO1, were not matured, and were antivirally inactive. Moreover, intracellular removal of SAM by ectopic expression of the bacteriophage T3 SAMase abolished antiviral activity. Collectively, our data suggest that viperin requires CIAO1 for [4Fe-4S] cluster assembly, and acts through an enzymatic, Fe-S cluster- and SAM-dependent mechanism to inhibit viral RNA synthesis.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Proteínas Ferro-Enxofre/metabolismo , Proteínas/metabolismo , Bacteriófago T3/enzimologia , Linhagem Celular , Retículo Endoplasmático/química , Humanos , Ferro/metabolismo , Metalochaperonas/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Mapeamento de Interação de Proteínas , Proteínas/genética , RNA Viral/biossínteseRESUMO
Introduction: Optical Projection Tomography (OPT) and light sheet fluorescence microscopy (LSFM) are high resolution optical imaging techniques, ideally suited for ex vivo 3D whole mouse brain imaging. Although they exhibit high specificity for their targets, the anatomical detail provided by tissue autofluorescence remains limited. Methods: T1-weighted images were acquired from 19 BABB or DBE cleared brains to create an MR template using serial longitudinal registration. Afterwards, fluorescent OPT and LSFM images were coregistered/normalized to the MR template to create fusion images. Results: Volumetric calculations revealed a significant difference between BABB and DBE cleared brains, leading to develop two optimized templates, with associated tissue priors and brain atlas, for BABB (OCUM) and DBE (iOCUM). By creating fusion images, we identified virus infected brain regions, mapped dopamine transporter and translocator protein expression, and traced innervation from the eye along the optic tract to the thalamus and superior colliculus using cholera toxin B. Fusion images allowed for precise anatomical identification of fluorescent signal in the detailed anatomical context provided by MR. Discussion: The possibility to anatomically map fluorescent signals on magnetic resonance (MR) images, widely used in clinical and preclinical neuroscience, would greatly benefit applications of optical imaging of mouse brain. These specific MR templates for cleared brains enable a broad range of neuroscientific applications integrating 3D optical brain imaging.
RESUMO
The virus life cycle depends on host-virus protein-protein interactions, which often involve a disordered protein region binding to a folded protein domain. Here, we used proteomic peptide phage display (ProP-PD) to identify peptides from the intrinsically disordered regions of the human proteome that bind to folded protein domains encoded by the SARS-CoV-2 genome. Eleven folded domains of SARS-CoV-2 proteins were found to bind 281 peptides from human proteins, and affinities of 31 interactions involving eight SARS-CoV-2 protein domains were determined (KD â¼ 7-300 µM). Key specificity residues of the peptides were established for six of the interactions. Two of the peptides, binding Nsp9 and Nsp16, respectively, inhibited viral replication. Our findings demonstrate how high-throughput peptide binding screens simultaneously identify potential host-virus interactions and peptides with antiviral properties. Furthermore, the high number of low-affinity interactions suggest that overexpression of viral proteins during infection may perturb multiple cellular pathways.