RESUMEN
Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions.
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Virus del Dengue , Dengue , Vesículas Extracelulares , Infección por el Virus Zika , Virus Zika , Animales , Ratones , Infección por el Virus Zika/prevención & control , Proteínas Virales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Dengue/prevención & controlRESUMEN
Induction and suppression of antiviral RNA interference (RNAi) has been observed in mammals during infection with at least seven distinct RNA viruses, including some that are pathogenic in humans. However, while the cell-autonomous immune response mediated by antiviral RNAi is gradually being recognized, little is known about systemic antiviral RNAi in mammals. Furthermore, extracellular vesicles (EVs) also function in viral signal spreading and host immunity. Here, we show that upon antiviral RNAi activation, virus-derived small-interfering RNAs (vsiRNAs) from Nodamura virus (NoV), Sindbis virus (SINV), and Zika virus (ZIKV) enter the murine bloodstream via EVs for systemic circulation. vsiRNAs in the EVs are biologically active, since they confer RNA-RNA homology-dependent antiviral activity in both cultured cells and infant mice. Moreover, we demonstrate that vaccination with a live-attenuated virus, rendered deficient in RNAi suppression, induces production of stably maintained vsiRNAs and confers protective immunity against virus infection in mice. This suggests that vaccination with live-attenuated VSR (viral suppressor of RNAi)-deficient mutant viruses could be a new strategy to induce immunity.
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Vesículas Extracelulares , Infección por el Virus Zika , Virus Zika , Animales , Antivirales , Vesículas Extracelulares/genética , Humanos , Mamíferos/genética , Ratones , Interferencia de ARN , ARN Bicatenario , ARN Interferente Pequeño/genética , Virus Zika/genética , Infección por el Virus Zika/genética , Infección por el Virus Zika/prevención & controlRESUMEN
Hepatitis C virus (HCV) infection remains a major worldwide health problem despite development of highly effective direct-acting antivirals. HCV rapidly evolves upon acute infection and generates multiple viral variants (quasispecies), leading to immune evasion and persistent viral infection. Identification of epitopes of broadly neutralizing anti-HCV antibodies (nAbs) is critical to guide HCV vaccine development. In this study, we developed a new reverse genetics system for HCV infection based on trans-complementation of viral structural proteins. The HCV genome (JFH1 strain) lacking the structural protein-coding sequence can be efficiently rescued by ectopic expression of core-E1-E2-p7-NS2 (core-NS2) or core-E1-E2-p7 (core-p7) in trans, leading to production of single-round infectious virions designated HCVΔS. JFH1-based HCVΔS can be also rescued by expressing core-NS2 of other HCV genotypes, rendering it an efficient tool to display the structural proteins of HCV strains of interests. Furthermore, we successfully rescued HCVΔS with structural proteins from clinical isolates. Multiple viral structural proteins with different sensitivities to nAbs were identified from a same patient serum, demonstrating the genetic diversity of HCV quasispecies in vivo Interestingly, the structural protein-coding sequences of highly divergent viral quasispecies from the same patient can be clustered based on their hypervariable region 1 (HVR1) in viral envelope protein E2, which critically dictates the sensitivity to neutralizing antibodies. In summary, we developed a novel reverse genetics system that efficiently displays viral structural proteins from HCV clinical isolates, and analysis of quasispecies from the same patient using this system demonstrated that E2 HVR1 is the major determinant of viral evolution in vivoIMPORTANCE A cell culture model that can recapitulate the diversity of HCV quasispecies in patients is important for analysis of neutralizing epitopes and HCV vaccine development. In this study, we developed a new reverse genetics system for HCV infection based on trans-complementation of viral structural proteins (HCVΔS). This system can be used to display structural proteins of HCV strains of multiple genotypes as well as clinical isolates. By using this system, we showed that multiple different HCV structural proteins from a same patient were displayed on HCVΔS. Interestingly, these variant structural proteins within the same patient can be classified according to the sequence of HVR1in E2, which dictates viral sensitivity to nAbs and viral evolution in vivo Our work provided a new tool to study highly divergent HCV quasispecies and shed light on underlying mechanisms driving HCV evolution.
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Hepacivirus/genética , Hepacivirus/metabolismo , Cuasiespecies/genética , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas Estructurales Virales/química , Proteínas Estructurales Virales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Epítopos/inmunología , Evolución Molecular , Genotipo , Células HEK293 , Hepatitis C/virología , Anticuerpos contra la Hepatitis C/inmunología , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/virología , Humanos , Evasión Inmune , Pruebas de Neutralización , Proteínas del Envoltorio Viral/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Previous observations have been reported that viruses were inactivated using strong irradiation. Here, new evidence was disclosed by studying the effects of nanosized TiO2 on viral pathogens under a low irradiation condition (0.4 mW/cm2 at UVA band) that mimics the field setting. We showed that photo-activated TiO2 efficiently inhibits hepatitis C virus infection, and weak indoor light with intensity of 0.6 mW/cm2 at broad-spectrum wavelength and around 0.15 mW/cm2 of UVA band also lead to partial inhibition. Mechanistic studies demonstrated that hydroxyl radicals produced by photo-activated TiO2 do not destroy virion structure and contents, but attack viral RNA genome, thus inactivating the virus. Furthermore, we showed that photo-activated TiO2 inactivates a broad range of human viral pathogens, including SARS-CoV-2, a novel coronavirus responsible for the ongoing COVID-19 pandemic. In conclusion, we showed that photo-catalyzed nanosized TiO2 inactivates pathogenic viruses, paving a way to its field application in control of viral infectious diseases.
RESUMEN
Despite the emergence of new direct-acting antivirals, hepatitis C virus (HCV) chronic infection and its consequent fibrosis and hepatocarcinoma remain a significant burden for public health, thus requiring an effective preventive vaccine. Our group previously showed that a subunit vaccine based on recombinant soluble E2 (sE2) can induce broadly neutralizing antibodies. To improve the immunogenicity of sE2, we designed and produced a fusion protein (sE2-ferritin) comprising sE2 and a ferritin unit in Drosophila S2 cells, which self-assembled into a nanoparticle with sE2 displayed on the surface. The sE2 moiety on the sE2-ferritin nanoparticle not only had nearly natural conformation but also had better affinities than the unfused sE2 to neutralizing antibodies, receptor, and patient serum. Mouse immunization studies showed that sE2-ferritin was more potent than sE2 in inducing anti-HCV broadly neutralizing antibodies. Our results demonstrate that sE2-ferritin is a vaccine candidate superior to previously developed sE2, providing a new possibility for controlling HCV.
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Hepacivirus/inmunología , Hepatitis C Crónica/microbiología , Nanopartículas/química , Vacunas contra Hepatitis Viral/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Drosophila/inmunología , Genotipo , Anticuerpos contra la Hepatitis C/inmunología , Hepatitis C Crónica/virología , Inmunización/métodos , Ratones , Proteínas Recombinantes/inmunología , Vacunas de Subunidad/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas contra Hepatitis Viral/químicaRESUMEN
Defective viral genomes (DVGs) of hepatitis C virus (HCV) exist, but their biological significances have not been thoroughly investigated. Here, we analyzed HCV DVGs circulating in patient sera that possess deletions in the structural protein-encoding region. About 30% of 41 HCV clinical isolates possess DVGs that originated from the full-length genome in the same patients. No correlation between DVGs, viremia, and alanine aminotransferase (ALT) levels was found. Sequencing analysis of DVGs revealed the existence of deletion hot spots, with upstream sites in E1 and downstream sites in E2 and NS2. Interestingly, the coding sequences for the core protein and the C-terminal protease domain of NS2 were always intact in DVGs despite the fact that both proteins are dispensable for HCV genome replication. Mechanistic studies showed that transmembrane segment 3 (TMS3) of NS2, located immediately upstream of its protease domain, was required for the cleavage of NS2-NS3 and the replication of DVGs. Moreover, we identified a highly conserved secondary structure (SL750) within the core domain 2-coding region that is critical for HCV genome packaging. In summary, our analysis of serum-derived HCV DVGs revealed novel viral cis elements that play important roles in virus replication and assembly.IMPORTANCE HCV DVGs have been identified in vivo and in vitro, but their biogenesis and physiological significances remain elusive. In addition, a conventional packaging signal has not yet been identified on the HCV RNA genome, and mechanisms underlying the specificity in the encapsidation of the HCV genome into infectious particles remain to be uncovered. Here, we identified new viral cis elements critical for the HCV life cycle by determining genetic constraints that define the boundary of serum-derived HCV DVGs. We found that transmembrane segment 3 of NS2, located immediately upstream of its protease domain, was required for the cleavage of NS2-NS3 and the replication of DVGs. We identified a highly conserved secondary structure (SL750) within the core-coding region that is critical for HCV genome packaging. In summary, our analysis of serum-derived HCV DVGs revealed previously unexpected novel cis elements critical for HCV replication and morphogenesis.
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Secuencia de Bases , Genoma Viral , Hepacivirus/fisiología , ARN Viral/genética , Eliminación de Secuencia , Proteínas del Envoltorio Viral/genética , Ensamble de Virus/genética , Humanos , Dominios Proteicos , Estructura Secundaria de Proteína , ARN Viral/sangre , Proteínas del Envoltorio Viral/sangreRESUMEN
Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV can be sensed by host innate immunity to induce expression of interferons (IFNs) and a number of antiviral effectors. In this study, we found HCV infection induced the expression of neuralized E3 ubiquitin protein ligase 3 (NEURL3), a putative E3 ligase, in a manner that requires the involvement of innate immune sensing but is independent of the IFN action. Furthermore, we showed that NEURL3 inhibited HCV infection while it had little effect on other RNA viruses, including Zika virus (ZIKV), dengue virus (DENV), and vesicular stomatitis virus (VSV). Mechanistic studies demonstrated that NEURL3 inhibited HCV assembly by directly binding HCV envelope glycoprotein E1 to interfere with the E1/E2 heterodimerization, an important prerequisite for virion morphogenesis. Finally, we showed that knockout of NEURL3 significantly enhanced HCV infection. In summary, we identified NEURL3 as a novel inducible antiviral host factor that suppresses HCV assembly. Our results not only shed new insight into how host innate immunity acts against HCV but also revealed a new important biological function for NEURL3.IMPORTANCE The exact biological function of NEURL3, a putative E3 ligase, remains largely unknown. In this study, we found that NEURL3 could be upregulated upon HCV infection in a manner dependent on pattern recognition receptor-mediated innate immune response. NEURL3 inhibits HCV assembly by directly binding viral E1 envelope glycoprotein to disrupt its interaction with E2, an action that requires its Neuralized homology repeat (NHR) domain but not the RING domain. Furthermore, we found that NEURL3 has a pangenotypic anti-HCV activity and interacts with E1 of genotypes 2a, 1b, 3a, and 6a but does not inhibit other closely related RNA viruses, such as ZIKV, DENV, and VSV. To our knowledge, our study is the first report to demonstrate that NEURL3 functions as an antiviral host factor. Our results not only shed new insight into how host innate immunity acts against HCV, but also revealed a new important biological function for NEURL3.
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Antivirales/farmacología , Hepatitis C/prevención & control , Inmunidad Innata/inmunología , Infecciones por Virus ARN/virología , Ubiquitina-Proteína Ligasas/farmacología , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Virus del Dengue/efectos de los fármacos , Células HEK293 , Hepacivirus/clasificación , Hepacivirus/genética , Hepacivirus/inmunología , Hepatitis C/inmunología , Hepatitis C/virología , Humanos , Infecciones por Virus ARN/tratamiento farmacológico , Infecciones por Virus ARN/inmunología , Virus ARN/inmunología , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacos , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Ensamble de Virus , Virus Zika/efectos de los fármacosRESUMEN
Ebola virus (EBOV) causes severe hemorrhagic fever in humans and other primates with a high case fatality rate. No approved drug or vaccine of EBOV is available, which necessitates better understanding of the virus life cycle. Studies on EBOV have been hampered because experimentations involving live virus are restricted to biosafety level 4 (BSL4) laboratories. The EBOV minigenome system has provided researchers with the opportunity to study EBOV under BSL2 conditions. Here, we developed a novel EBOV minigenome replicon which, to our knowledge, is the first EBOV cell culture system that can stably replicate and transcribe the EBOV minigenome. The minigenomic RNA harboring a Gaussia luciferase and hygromycin-resistant marker can replicate for months in a helper cell stably expressing viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and L proteins. Quantification of viral RNA (vRNA), cRNA, and mRNA levels of the EBOV minigenome demonstrated that the stable EBOV replicon had much-more-active minigenome replication than previously developed transient-transfection-based EBOV minigenome systems, which recapitulate viral primary transcription more than genome replication. Interestingly, minigenome replication in the stable EBOV replicon cells was insensitive to interferon treatment or RNA interference. Moreover, RNase digestion of the replicon cell lysates revealed the remarkably stable nature of the EBOV minigenomic vRNA ribonucleoprotein complex, which may help improve understanding of EBOV persistence in convalescent patients.IMPORTANCE The scope and severity of the recent Ebola outbreak in Western Africa justified a more comprehensive investigation of the causative risk group 4 agent Ebola virus (EBOV). Study of EBOV replication and antiviral development can be facilitated by developing a cell culture system that allows experimentation under biosafety level 2 conditions. Here, we developed a novel stable EBOV minigenome replicon which, to our knowledge, is the first EBOV cell culture system that can stably replicate and transcribe the EBOV minigenome. The replicon system had more-active genome replication than previously developed transient-transfection-based EBOV minigenome systems, providing a convenient surrogate system to study EBOV replication. Furthermore, self-replicating minigenomic vRNA in the replicon cells displayed strong stability in response to interferon treatment, RNA silencing, and RNase digestion, which may provide an explanation for the persistence of EBOV in survivors.
RESUMEN
BACKGROUND/AIMS: Sphingosine kinase 1 (SphK1), which phosphorylates sphingosine to sphingosine-1-phosphate (S1P), is overexpressed in various types of cancers, and may act as an oncogene in tumorigenesis. However, little is known about the precise role of the SphK1/S1P pathway in human liver cancer, especially regarding the metastasis of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: The expression of SphK1 was detected by quantitative reverse-transcription PCR. In addition, transwell cell migration and invasion assay were carried out for functional analysis. Furthermore, the level of S1P was quantified by ELISA and Rac1/Cdc42 GTPase activation was assessed by western blot analysis. RESULTS: The levels of SphK1 mRNA are commonly up-regulated in HCC patients and human liver cancer cell migration and invasion can be promoted by the overexpression of SphK1. In addition, inhibition of SphK1 with either a SphK1 inhibitor or siRNA reduced human liver cancer cell migration and invasion. Furthermore, overexpression of SphK1 increased S1P levels, and the exogenous addition of S1P increased liver cell migration and invasion through the EDG1 receptor. DISCUSSION AND CONCLUSION: The results from this study provide strong evidence of a role for the SphK1/S1P/EDG1 pathway in liver metastasis, thus making it an attractive therapeutic target for the development of new anti-HCC drugs.
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Carcinoma Hepatocelular/enzimología , Neoplasias Hepáticas/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Invasividad Neoplásica/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , ARN Interferente Pequeño/farmacología , Receptores de Lisoesfingolípidos/genética , Receptores de Esfingosina-1-Fosfato , Transfección , Células Tumorales CultivadasRESUMEN
Hepatitis C virus (HCV) remains a major human pathogen that requires better understanding of virus-host interactions. In this study, we performed a genome-wide CRISPR-Cas9 screening and identified TRIM26, an E3 ligase, as a critical HCV host factor. Deficiency of TRIM26 specifically impairs HCV genome replication. Mechanistic studies showed that TRIM26 interacts with HCV-encoded NS5B protein and mediates its K27-linked ubiquitination at residue K51, and thus promotes the NS5B-NS5A interaction. Moreover, mouse TRIM26 does not support HCV replication because of its unique six-amino acid insert that prevents its interaction with NS5B. Ectopic expression of human TRIM26 in a mouse hepatoma cell line that has been reconstituted with other essential HCV host factors promotes HCV infection. In conclusion, we identified TRIM26 as a host factor for HCV replication and a new determinant of host tropism. These results shed light on HCV-host interactions and may facilitate the development of an HCV animal model.
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Induction of broadly neutralizing monoclonal antibodies (bNAbs) that bind to the viral envelope glycoproteins is a major goal of hepatitis C virus (HCV) vaccine research. The study of bNAbs arising in natural infection is essential in this endeavor. We generated a human antibody, 8D6, recognizing the E2 protein of HCV isolated from a chronic hepatitis C patient. This antibody shows broadly neutralizing activity, which covers a pan-genotypic panel of cell culture-derived HCV virions (HCVcc). Functional and epitope analyses demonstrated that 8D6 can block the interaction between E2 and CD81 by targeting a highly conserved epitope on E2. We describe how the 8D6 lineage evolved via somatic hypermutation to achieve broad neutralization. We found that the V(D)J recombination-generated junctional and somatic hypermutation-induced disulfide bridge (C-C) motif in the CDRH3 is critical for the broad neutralization and binding activity of 8D6. This motif is conserved among a series of broadly neutralizing HCV antibodies, indicating a common binding model. Next, the 8D6 inferred germline (iGL) was reconstructed and tested for its binding affinity and neutralization activity. Interestingly, 8D6 iGL-mediated relatively strong inhibition of the 1b genotype PR79L9 strain, suggesting that PR79L9 may serve as a potential natural viral strain that provides E2 sequences that induce bNAbs. Overall, our detailed epitope mapping and genetic studies of the HCV E2-specific mAb 8D6 have allowed for further refinement of antigenic sites on E2 and reveal a new mechanism to generate a functional CDRH3, while its iGL can serve as a probe to identify potential HCV vaccine strains.
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Anticuerpos ampliamente neutralizantes/farmacología , Regiones Determinantes de Complementariedad/genética , Epítopos/inmunología , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/inmunología , Hepatitis C/prevención & control , Proteínas del Envoltorio Viral/inmunología , Anticuerpos Monoclonales/farmacología , Regiones Determinantes de Complementariedad/inmunología , Mapeo Epitopo , Hepatitis C/genética , Hepatitis C/inmunología , Hepatitis C/virología , Humanos , Cadenas Pesadas de Inmunoglobulina , Mutación , Dominios y Motivos de Interacción de ProteínasRESUMEN
Viruses in the Flaviviridae family such as Zika virus (ZIKV), dengue virus (DENV), and Japanese encephalitis virus (JEV) are major public health concerns. The development of antiviral agents against these viruses is urgently needed. We have previously discovered that the Keggin structured polyoxometalate POM-12 has potent inhibitory activity against hepatitis C virus, another member of the Flaviviridae family. In this study, we tested its antiviral activity of DENV, JEV and ZIKV, and found that POM-12 dramatically inhibited their infection with IC50 value of 1.16 µM, 1.9 µM and 0.64 µM, respectively. Mechanistic studies indicated that POM-12 directly disrupted the integrity of these virions. Moreover, POM-12 also targeted the post-entry steps of viral replication of JEV, but having no similar activities on ZIKV and DENV. The differential actions of POM-12 on these viruses suggest that surface topology and charge of virion may have influence on its drug effect, and thus POM-12 may be modified to more efficiently inhibit these and other similar viruses.
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Aniones/farmacología , Antivirales/farmacología , Flavivirus/efectos de los fármacos , Polielectrolitos/farmacología , Replicación Viral/efectos de los fármacos , Animales , Chlorocebus aethiops , Virus del Dengue/efectos de los fármacos , Descubrimiento de Drogas , Virus de la Encefalitis Japonesa (Especie)/efectos de los fármacos , Flavivirus/clasificación , Flavivirus/fisiología , Concentración 50 Inhibidora , Células Vero , Virus Zika/efectos de los fármacosRESUMEN
Japanese encephalitis virus (JEV) exposure or vaccination could elicit cross-reactive CD8 T cell immunity against heterologous flaviviruses in humans. In addition, cross-reactive CD8 T cells induced by dengue virus (DENV) have been shown to play a protective role against Zika virus (ZIKV). However, how JEV exposure or vaccination affects ZIKV infection in humans remains unclear. In this report, epitope prediction algorithms were used to predict the cross-reactive CD8 T cell epitope restricted to human HLA between JEV and ZIKV. We found that these predicted CD8 T cell epitopes are immunogenic and cross-reactive in humanized HLA transgenic mice. Moreover, JEV vaccine immunization provided cross-protection against ZIKV infection. Furthermore, CD8 T cells were involved in the protection against ZKIV infection in vivo. Our results have an important clinical implication that vaccination with JEV SA14-14-2 may provide protection against ZIKV infection in humans.
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Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Antígeno HLA-A2/inmunología , Inmunidad Celular , Inmunogenicidad Vacunal , Vacunas contra la Encefalitis Japonesa/farmacología , Infección por el Virus Zika/prevención & control , Virus Zika/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Chlorocebus aethiops , Cricetinae , Reacciones Cruzadas , Modelos Animales de Enfermedad , Epítopos de Linfocito T/metabolismo , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Interacciones Huésped-Patógeno , Humanos , Epítopos Inmunodominantes , Vacunas contra la Encefalitis Japonesa/administración & dosificación , Células K562 , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Receptores de Interferón/genética , Receptores de Interferón/metabolismo , Vacunación , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/farmacología , Células Vero , Virus Zika/patogenicidad , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/metabolismo , Infección por el Virus Zika/virología , Receptor de Interferón gammaRESUMEN
Chronic hepatitis C infection is a leading cause of liver cirrhosis, which is linked to chronic hepatic inflammation. While there are multiple studies detailing the proinflammatory role of interleukin-1ß (IL-1ß) in HCV-induced inflammasome signaling, the antiviral capacity of this cytokine has not been adequately investigated in the context of HCV infection or other members of Flaviridae. Our data indicated that IL-1ß alone does not inhibit HCV replication, yet when in combination with IFN-α, it can boost the anti-HCV activity of IFN-α, which is mediated by augmented STAT1 tyrosine 701 phosphorylation. Through signaling inhibitor screening, we found that ERK2 kinase is directly linked to the enhanced activation of the STAT1 complex. Our study found that IL-1ß negatively affects ERK2 phosphorylation, which suggests that IL-1ß-mediated STAT1 tyrosine 701 phosphorylation employed kinase machinery of ERK2 other than JNK or P38 kinase. Our results identify IL-1ß as a proinflammatory cytokine possessing wide spectrum synergistic antiviral capability via enhancing IFN-α-induced interferon-stimulated genes (ISGs) expression. A more nuanced understanding of the antiviral mechanisms of this important cytokine could facilitate the development of new therapeutic options.
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Antivirales , Hepatitis C , Antivirales/farmacología , Antivirales/uso terapéutico , Hepacivirus , Humanos , Interferón-alfa/farmacología , Interferón-alfa/uso terapéutico , Interleucina-1beta/uso terapéuticoRESUMEN
Flaviviruses including Zika virus, Dengue virus, Japanese Encephalitis virus, and Yellow Fever virus cause heavy burdens to public health around the world. No specific antiviral drug is available in the clinic against these flavivirus infections. Heat-shock protein 70 (HSP70) has recently been proven to be a promising antiviral target against Zika virus and Dengue virus. Here, we report that Apoptozole, a small molecule inhibitor of ATPase activity of HSP70, has broad-spectrum anti-flavivirus potential. The mode of action analysis revealed that Apoptozole acted at the post-entry step. Transcriptome analysis revealed that genes related to cholesterol metabolism, fatty acid synthesis, and innate immunity were differentially expressed after treatment with Apoptozole. In vivo data suggested Apoptozole exerted protection effects against Zika virus (ZIKV) infection in a mouse model by enhancing the innate immune response, which suggested a novel anti-ZIKV mechanism of HSP70 inhibitors.
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Adenosina Trifosfatasas/antagonistas & inhibidores , Benzamidas/uso terapéutico , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Imidazoles/uso terapéutico , Infección por el Virus Zika/tratamiento farmacológico , Animales , Inmunidad Innata , RatonesRESUMEN
Zika virus (ZIKV) and Japanese encephalitis virus (JEV) are closely related flaviviruses, ZIKV circulates in the population that has been JEV vaccinated in Southeast Asian countries. This alerts that a pre-existing immunity to JEV would impact ZIKV infection and/or pathogenesis. Herein we showed that the pre-existing immunity to JEV SA14-14-2 vaccination provided an ample protection against non-lethal or lethal dose of ZIKV infection in mice. This was in sharp contrast to the passive immunization of JEV antibodies, which failed to affect ZIKV infection or pathogenesis in mice, albeit these antibodies exhibited cross-reactivity and antibody dependent enhancement (ADE) of ZIKV infection in vitro. Furthermore, we determined that JEV vaccine-elicited CD8+ T cells were required to mediate the heterotypic protection of ZIKV infection, which cross-reacted to ZIKV E and NS5 antigens (E294-302 and NS52839-2848). Adoptive transfer of these CD8+ T cells could partially protect the mice from ZIKV challenge. Therefore, although short of epidemiological evidence, these results suggested that cross-reactive CD8+ T cells activated by JEV vaccination could protect potential ZIKV infection in human populations.
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Hepatitis C virus (HCV), a major causative agent of chronic hepatitis, is a positive-stranded RNA virus and has a high degree of genetic diversity due to its error-prone RNA-dependent RNA polymerase. Development of direct-acting antiviral agents (DAAs) has greatly improved the therapeutic outcome of chronic hepatitis C patients. However, naturally existing resistance-associated variants (RAVs) or occurrence of resistance-associated substitutions (RASs) in the HCV genome may impose a challenge to the long-term success of the DAA-based therapies. Genotype-3 HCV is the most difficult genotype to treat by DAAs, but the underlying molecular mechanisms remain to be explored. Here we developed a novel genotype-3a subgenomic replicon PR87A7 by screening a HCV cDNA pool amplified from a patient serum RNA. PR87A7 replicon displayed strong resistance to anti-NS3 DAAs, mainly owing to a genotype-3-specific polymorphism 168Q in NS3. Introduction of NS3 168Q into a genotype-2a JFH1 strain rendered resistance to anti-NS3 DAAs while greatly diminished the viral replication, and yet this fitness defect can be rescued by additional genotype-3-specific polymorphism. In conclusion, we developed a novel genotype-3a subgenomic replicon by a functional screening approach, and revealed genotype-3-specfic amino acid residues that confer resistance to anti-NS3 DAAs while retaining viral fitness.
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Farmacorresistencia Viral , Genotipo , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Hepatitis C/virología , Polimorfismo Genético , Replicación Viral/efectos de los fármacos , Sustitución de Aminoácidos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Aptitud Genética , Genoma Viral , Hepacivirus/clasificación , Hepatitis C/tratamiento farmacológico , Humanos , Filogenia , Proteínas no Estructurales Virales/genéticaRESUMEN
Induction of long-lived antibody responses during infection or vaccination is often essential for subsequent protection, but the relative contributions of T follicular helper (Tfh) cells and T helper 1 (Th1) cells for induction of antigen specific antibody responses to viruses are unclear. Here, we establish an acute Zika virus (ZIKV) infection model in immunocompetent mice, and show that ZIKV infection elicits robust Th1-like Tfh cell and protective antibody responses. While these Th1-like Tfh cells share phenotypic and transcriptomic profiles with both Tfh and Th1 cells, they also have unique surface markers and gene expression characteristics, and are dependent on T-bet for their development. Th1-like Tfh cells, but not Th1 cells, are essential for class switching of ZIKV-specific IgG2c antibodies and maintenance of long-term neutralizing antibody responses. Our study suggests that specific modulation of the Th1-like Tfh cell response during infection or vaccination may augment the induction of antiviral antibody response to ZIKV and other viruses.
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Cambio de Clase de Inmunoglobulina/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Infección por el Virus Zika/inmunología , Virus Zika/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Chlorocebus aethiops , Interacciones Microbiota-Huesped/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Interferón gamma/genética , Interferón gamma/inmunología , Interferón gamma/metabolismo , Ratones , Ratones Noqueados , Transducción de Señal/inmunología , Proteínas de Dominio T Box/metabolismo , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Células Vero , Infección por el Virus Zika/virologíaRESUMEN
Simeprevir was developed as a small molecular drug targeting the NS3/4A protease of hepatitis C virus (HCV). Unexpectedly, our current work discovered that Simeprevir effectively promoted the transcription of IFN-ß and ISG15, inhibited the infection of host cells by multiple viruses including Zika virus (ZIKV), Enterovirus A71 (EV-A71), as well as herpes simplex virus type 1 (HSV-1). However, the inhibitory effects of Simeprevir on ZIKV, EV-A71 and HSV-1 were independent from IFN-ß and ISG15. This study thus demonstrates that the application of Simeprevir can be extended to other viruses besides HCV.
Asunto(s)
Antivirales/farmacología , Enterovirus Humano A/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Interferón beta/metabolismo , Simeprevir/farmacología , Virus Zika/efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , Citocinas/metabolismo , Infecciones por Enterovirus/tratamiento farmacológico , Hepatitis C/tratamiento farmacológico , Humanos , Inmunidad Innata , Transducción de Señal , Ubiquitinas/metabolismo , Células Vero , Replicación Viral/efectos de los fármacos , Infección por el Virus Zika/tratamiento farmacológicoRESUMEN
Toll-like receptors (TLRs) play a central role in sensing and initiating innate antiviral response. In this study, we first investigated the expression of TLR1-10 mRNA transcripts in peripheral blood mononuclear cells (PBMCs) from chronic HBV-infected (CHB) patients and healthy donors by quantitative real-time PCR. The expression of TLR1, TLR2, TLR4 and TLR6 transcripts was significantly lower in PBMCs from CHB patients, and the down-regulation of TLR2 was related to HBV genotype C. Flow cytometric analysis showed that the expression of TLR2 on PBMCs was significantly decreased in CHB patients. Furthermore, impaired cytokine production was observed in PBMCs from CHB patients after challenged with TLR2 and TLR4 ligands and was correlated with the levels of plasma hepatitis B virus surface antigen (HBsAg). In conclusion, our study reveals a possible interaction between HBsAg, TLR signaling and the innate immune response, which may partially explain the mechanism of HBV infection induced immuno-tolerance.