Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 22
Filtrar
Más filtros












Base de datos
Intervalo de año de publicación
1.
PLoS Pathog ; 20(9): e1012509, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39241103

RESUMEN

The replication organelle of hepatitis C virus (HCV), called membranous web, is derived from the endoplasmic reticulum (ER) and mainly comprises double membrane vesicles (DMVs) that concentrate the viral replication complexes. It also tightly associates with lipid droplets (LDs), which are essential for virion morphogenesis. In particular acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), a rate-limiting enzyme in triglyceride synthesis, promotes early steps of virus assembly. The close proximity between ER membranes, DMVs and LDs therefore permits the efficient coordination of the HCV replication cycle. Here, we demonstrate that exaggerated LD accumulation due to the excessive expression of the DGAT1 isozyme, DGAT2, dramatically impairs the formation of the HCV membranous web. This effect depended on the enzymatic activity and ER association of DGAT2, whereas the mere LD accumulation was not sufficient to hamper HCV RNA replication. Our lipidomics data indicate that both HCV infection and DGAT2 overexpression induced membrane lipid biogenesis and markedly increased phospholipids with long chain polyunsaturated fatty acids, suggesting a dual use of these lipids and their possible competition for LD and DMV biogenesis. On the other hand, overexpression of DGAT2 depleted specific phospholipids, particularly oleyl fatty acyl chain-containing phosphatidylcholines, which, in contrast, are increased in HCV-infected cells and likely essential for viral infection. In conclusion, our results indicate that lipid exchanges occurring during LD biogenesis regulate the composition of intracellular membranes and thereby affect the formation of the HCV replication organelle. The potent antiviral effect observed in our DGAT2 overexpression system unveils lipid flux that may be relevant in the context of steatohepatitis, a hallmark of HCV infection, but also in physiological conditions, locally in specific subdomains of the ER membrane. Thus, LD formation mediated by DGAT1 and DGAT2 might participate in the spatial compartmentalization of HCV replication and assembly factories within the membranous web.


Asunto(s)
Diacilglicerol O-Acetiltransferasa , Retículo Endoplásmico , Hepacivirus , Triglicéridos , Replicación Viral , Diacilglicerol O-Acetiltransferasa/metabolismo , Diacilglicerol O-Acetiltransferasa/genética , Humanos , Hepacivirus/fisiología , Replicación Viral/fisiología , Triglicéridos/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Hepatitis C/metabolismo , Hepatitis C/virología , Gotas Lipídicas/metabolismo , Gotas Lipídicas/virología
2.
PLoS Biol ; 22(9): e3002767, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39316623

RESUMEN

Interferons (IFNs) play a crucial role in the regulation and evolution of host-virus interactions. Here, we conducted a genome-wide arrayed CRISPR knockout screen in the presence and absence of IFN to identify human genes that influence Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. We then performed an integrated analysis of genes interacting with SARS-CoV-2, drawing from a selection of 67 large-scale studies, including our own. We identified 28 genes of high relevance in both human genetic studies of Coronavirus Disease 2019 (COVID-19) patients and functional genetic screens in cell culture, with many related to the IFN pathway. Among these was the IFN-stimulated gene PLSCR1. PLSCR1 did not require IFN induction to restrict SARS-CoV-2 and did not contribute to IFN signaling. Instead, PLSCR1 specifically restricted spike-mediated SARS-CoV-2 entry. The PLSCR1-mediated restriction was alleviated by TMPRSS2 overexpression, suggesting that PLSCR1 primarily restricts the endocytic entry route. In addition, recent SARS-CoV-2 variants have adapted to circumvent the PLSCR1 barrier via currently undetermined mechanisms. Finally, we investigate the functional effects of PLSCR1 variants present in humans and discuss an association between PLSCR1 and severe COVID-19 reported recently.


Asunto(s)
COVID-19 , SARS-CoV-2 , Internalización del Virus , Humanos , SARS-CoV-2/genética , COVID-19/virología , COVID-19/genética , Células HEK293 , Sistemas CRISPR-Cas/genética , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Interferones/metabolismo , Interferones/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Antígenos de Diferenciación
4.
Hepatology ; 80(5): 1239-1251, 2024 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-38728662

RESUMEN

BACKGROUND AND AIMS: HEV is estimated to be responsible for 70,000 deaths annually, yet therapy options remain limited. In the pursuit of effective antiviral therapies, targeting viral entry holds promise and has proven effective for other viruses. However, the precise mechanisms and host factors required during HEV entry remain unclear. Cellular proteases have emerged as host factors required for viral surface protein activation and productive cell entry by many viruses. Hence, we investigated the functional requirement and therapeutic potential of cellular protease during HEV infection. APPROACH AND RESULTS: Using our established HEV cell culture model and subgenomic HEV replicons, we found that blocking lysosomal cathepsins (CTS) with small molecule inhibitors impedes HEV infection without affecting replication. Most importantly, the pan-cathepsin inhibitor K11777 suppressed HEV infections with an EC 50 of ~0.02 nM. Inhibition by K11777, devoid of notable toxicity in hepatoma cells, was also observed in HepaRG and primary human hepatocytes. Furthermore, through time-of-addition and RNAscope experiments, we confirmed that HEV entry is blocked by inhibition of cathepsins. Cathepsin L (CTSL) knockout cells were less permissive to HEV, suggesting that CTSL is critical for HEV infection. Finally, we observed cleavage of the glycosylated ORF2 protein and virus particles by recombinant CTSL. CONCLUSIONS: In summary, our study highlights the pivotal role of lysosomal cathepsins, especially CTSL, in the HEV entry process. The profound anti-HEV efficacy of the pan-cathepsin inhibitor K11777, especially with its notable safety profile in primary cells, further underscores its potential as a therapeutic candidate.


Asunto(s)
Catepsinas , Virus de la Hepatitis E , Internalización del Virus , Humanos , Internalización del Virus/efectos de los fármacos , Virus de la Hepatitis E/efectos de los fármacos , Virus de la Hepatitis E/fisiología , Catepsinas/antagonistas & inhibidores , Catepsinas/metabolismo , Catepsina L/antagonistas & inhibidores , Catepsina L/metabolismo , Hepatitis E/tratamiento farmacológico , Hepatitis E/virología , Replicación Viral/efectos de los fármacos , Hepatocitos/virología , Hepatocitos/efectos de los fármacos , Antivirales/farmacología , Antivirales/uso terapéutico
6.
Antiviral Res ; 217: 105690, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37517633

RESUMEN

Hepatitis E virus (HEV) usually causes a self-limiting disease, but especially immunocompromised individuals are at risk to develop a chronic and severe course of infection. Janus kinase (JAK) inhibitors (JAKi) are a novel drug class for the treatment of autoimmune inflammatory rheumatic disease (AIRD). As JAKs play a key role in innate immunity, viral infections and reactivations are frequently reported during JAKi treatment in AIRD patients. The aim of this study was to characterize the influence of JAKis on HEV replication. To this end, we evaluated liver enzymes of an AIRD patient under JAKi therapy with hepatitis E. Further, experiments with HEV (Kernow-C1 p6) were performed by infection of primary human hepatocytes (PHHs) followed by immunofluorescence staining of viral markers and transcriptomic analysis. Infection experiments in PHHs displayed an up to 50-fold increase of progeny virus production during JAKi treatment and transcriptomic analysis revealed induction of antiviral programs during infection. Upregulation of interferon-stimulated genes (ISG) was perturbed in the presence of JAKis, concomitant with elevated HEV RNA levels. The obtained results suggest that therapeutic JAK inhibition increases HEV replication by modulating the HEV-triggered immune response. Therefore, JAKi treatment and the occurrence of elevated liver enzymes requires a monitoring of potential HEV infections.


Asunto(s)
Virus de la Hepatitis E , Hepatitis E , Humanos , Virus de la Hepatitis E/genética , Quinasas Janus , Interferones/farmacología , Antivirales/farmacología , Antivirales/uso terapéutico , Replicación Viral
7.
Microbiol Spectr ; 11(4): e0128423, 2023 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-37341610

RESUMEN

Mice are refractory to infection with human-tropic hepatitis C virus (HCV), although distantly related rodent hepaciviruses (RHV) circulate in wild rodents. To investigate whether liver intrinsic host factors can exhibit broad restriction against these distantly related hepaciviruses, we focused on Shiftless (Shfl), an interferon (IFN)-regulated gene (IRG) which restricts HCV in humans. Unusually, and in contrast to selected classical IRGs, human and mouse SHFL orthologues (hSHFL and mSHFL, respectively) were highly expressed in hepatocytes in the absence of viral infection, weakly induced by IFN, and highly conserved at the amino acid level (>95%). Replication of both HCV and RHV subgenomic replicons was suppressed by ectopic expression of mSHFL in human or rodent hepatoma cell lines. Gene editing of endogenous mShfl in mouse liver tumor cells increased HCV replication and virion production. Colocalization of mSHFL protein with viral double-stranded RNA (dsRNA) intermediates was confirmed and could be ablated by mutational disruption of the SHFL zinc finger domain, concomitant with a loss of antiviral activity. In summary, these data point to an evolutionarily conserved function for this gene in humans and rodents: SHFL is an ancient antiviral effector which targets distantly related hepaciviruses via restriction of viral RNA replication. IMPORTANCE Viruses have evolved ways to evade or blunt innate cellular antiviral mechanisms within their cognate host species. However, these adaptations may fail when viruses infect new species and can therefore limit cross-species transmission. This may also prevent development of animal models for human-pathogenic viruses. HCV shows a narrow species tropism likely due to distinct human host factor usage and innate antiviral defenses limiting infection of nonhuman liver cells. Interferon (IFN)-regulated genes (IRGs) partially inhibit HCV infection of human cells by diverse mechanisms. Here, we show that mouse Shiftless (mSHFL), a protein that interferes with HCV replication factories, inhibits HCV replication and infection in human and mouse liver cells. We further report that the zinc finger domain of SHFL is important for viral restriction. These findings implicate mSHFL as a host factor that impairs HCV infection of mice and provide guidance for development of HCV animal models needed for vaccine development.


Asunto(s)
Hepacivirus , Hepatitis C , Ratones , Humanos , Animales , Hepacivirus/genética , Antivirales/farmacología , Interferones , Factores de Restricción Antivirales
8.
Hepatology ; 77(6): 2104-2117, 2023 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-36745934

RESUMEN

BACKGROUND AND AIMS: Being the most common cause of acute viral hepatitis with >20 million cases per year and 70,000 deaths annually, HEV presents a long-neglected and underinvestigated health burden. Although the entry process of viral particles is an attractive target for pharmacological intervention, druggable host factors to restrict HEV entry have not been identified so far. APPROACH AND RESULTS: Here we identify the EGF receptor (EGFR) as a novel host factor for HEV and reveal the significance of EGFR for the HEV entry process. By utilizing RNAi, chemical modulation with Food and Drug Administration-approved drugs, and ectopic expression of EGFR, we revealed that EGFR is critical for HEV infection without affecting HEV RNA replication or assembly of progeny virus. We further unveiled that EGFR itself and its ligand-binding domain, rather than its signaling function, is responsible for the proviral effect. Modulation of EGF expression in HepaRG cells and primary human hepatocytes affected HEV infection. CONCLUSIONS: Taken together, our study provides novel insights into the life cycle of HEV and identified EGFR as a possible target for future antiviral strategies against HEV.


Asunto(s)
Virus de la Hepatitis E , Hepatocitos , Humanos , Hepatocitos/metabolismo , Antivirales/farmacología , Receptores ErbB/metabolismo , Interferencia de ARN , Transducción de Señal , Virus de la Hepatitis E/genética , Replicación Viral
9.
Curr Opin Virol ; 56: 101274, 2022 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-36283248

RESUMEN

The intimate relationship between virus and host cell can result in highly adapted viruses that are restricted to a single host. However, some viruses have the ability to infect multiple host species. Remarkably, hepatitis E viruses (HEV) comprise genotypes that are either 'single-host' or 'multi-host' genotypes, a trait that raises fundamental questions: Why do different genotypes differ in their host range, despite a high degree of genomic similarity? What are the underlying molecular determinants that shape species barriers? Here, we review the current knowledge of viral and host determinants that may affect the evolutionary trajectories of HEV. We also provide a perspective on techniques and methods that address open questions of HEV host range and adaptation.

10.
Transfusion ; 62(11): 2200-2204, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36125237

RESUMEN

BACKGROUND AND OBJECTIVES: Photodynamic treatment with methylene blue (MB) and visible light is a well-established pathogen inactivation system for human plasma. This technique is routinely used in different countries. MB/light treatment was shown to inactivate several transfusion-transmittable viruses, but its efficiency for the inactivation of the quasi-enveloped hepatitis E virus (HEV) has not yet been investigated. MATERIALS AND METHODS: Plasma units were spiked with cell culture-derived HEV and treated with the THERAFLEX MB-Plasma system using various light doses (30, 60, 90, and 120 J/cm2 ). HEV titers in pre- and post-treatment samples were determined by virus titration and a large-volume plating assay to improve the detection limit of the virus assay. RESULTS: THERAFLEX MB-Plasma efficiently inactivated HEV in human plasma. Even the lowest light dose of 30 J/cm2 inactivated HEV down to the limit of detection, with a mean log reduction factor of greater than 2.4 for the total process. CONCLUSION: Our study demonstrates that the THERAFLEX MB-Plasma system effectively inactivates HEV in human plasma.


Asunto(s)
Virus de la Hepatitis E , Azul de Metileno , Humanos , Azul de Metileno/farmacología , Inactivación de Virus , Rayos Ultravioleta , Luz
11.
Antiviral Res ; 204: 105359, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35728703

RESUMEN

Hepatitis E virus (HEV) infections are a leading cause of acute viral hepatitis in humans and pose a considerable threat to public health. Current standard of care treatment is limited to the off-label use of nucleoside-analog ribavirin (RBV) and PEGylated interferon-α, both of which are associated with significant side effects and provide limited efficacy. In the past few years, a promising natural product compound class of eukaryotic initiation factor 4A (eIF4A) inhibitors (translation initiation inhibitors), called rocaglates, were identified as antiviral agents against RNA virus infections. In the present study, we evaluated a total of 205 synthetic rocaglate derivatives from the BU-CMD compound library for their antiviral properties against HEV. At least eleven compounds showed inhibitory activities against the HEV genotype 3 (HEV-3) subgenomic replicon below 30 nM (EC50 value) as determined by Gaussia luciferase assay. Three amidino-rocaglates (ADRs) (CMLD012073, CMLD012118, and CMLD012612) possessed antiviral activity against HEV with EC50 values between 1 and 9 nM. In addition, these three selected compounds inhibited subgenomic replicons of different genotypes (HEV-1 [Sar55], wild boar HEV-3 [83-2] and human HEV-3 [p6]) in a dose-dependent manner and at low nanomolar concentrations. Furthermore, tested ADRs tend to be better tolerated in primary hepatocytes than hepatoma cancer cell lines and combination treatment of CMLD012118 with RBV and interferon-α (IFN-α) showed that CMLD012118 acts additive to RBV and IFN-α treatment. In conclusion, our results indicate that ADRs, especially CMLD012073, CMLD012118, and CMLD012612 may prove to be potential therapeutic candidates for the treatment of HEV infections and may contribute to the discovery of pan-genotypic inhibitors in the future.


Asunto(s)
Virus de la Hepatitis E , Hepatitis E , Antivirales/farmacología , Antivirales/uso terapéutico , Hepatitis E/tratamiento farmacológico , Humanos , Interferón-alfa/farmacología , Interferón-alfa/uso terapéutico , Ribavirina/farmacología , Ribavirina/uso terapéutico , Replicación Viral
12.
J Hepatol ; 76(5): 1062-1069, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35085595

RESUMEN

BACKGROUND & AIMS: Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide and is mainly transmitted via the fecal-oral route or through consumption of contaminated food products. Due to the lack of efficient cell culture systems for the propagation of HEV, limited data regarding its sensitivity to chemical disinfectants are available. Consequently, preventive and evidence-based hygienic guidelines on HEV disinfection are lacking. METHODS: We used a robust HEV genotype 3 cell culture model which enables quantification of viral infection of quasi-enveloped and naked HEV particles. For HEV genotype 1 infections, we used the primary isolate Sar55 in a fecal suspension. Standardized quantitative suspension tests using end point dilution and large-volume plating were performed for the determination of virucidal activity of alcohols (1-propanol, 2-propanol, ethanol), WHO disinfectant formulations and 5 different commercial hand disinfectants against HEV. Iodixanol gradients were conducted to elucidate the influence of ethanol on quasi-enveloped viral particles. RESULTS: Naked and quasi-enveloped HEV was resistant to alcohols as well as alcohol-based formulations recommended by the WHO. Of the tested commercial hand disinfectants only 1 product displayed virucidal activity against HEV. This activity could be linked to phosphoric acid as an essential ingredient. Finally, we observed that ethanol and possibly non-active alcohol-based disinfectants disrupt the quasi-envelope structure of HEV particles, while leaving the highly transmissible and infectious naked virions intact. CONCLUSIONS: Different alcohols and alcohol-based hand disinfectants were insufficient to eliminate HEV infectivity with the exception of 1 commercial ethanol-based product that included phosphoric acid. These findings have major implications for the development of measures to reduce viral transmission in clinical practice. LAY SUMMARY: Hepatitis E virus (HEV) showed a high level of resistance to alcohols and alcohol-based hand disinfectants. The addition of phosphoric acid to alcohol was essential for virucidal activity against HEV. This information should be used to guide improved hygiene measures for the prevention of HEV transmission.


Asunto(s)
Desinfectantes , Desinfectantes para las Manos , Virus de la Hepatitis E , Hepatitis E , Desinfectantes/farmacología , Etanol/farmacología , Virus de la Hepatitis E/genética , Humanos
13.
iScience ; 24(8): 102908, 2021 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-34337354

RESUMEN

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created a significant threat to global health. While respiratory aerosols or droplets are considered as the main route of human-to-human transmission, secretions expelled by infected individuals can also contaminate surfaces and objects, potentially creating the risk of fomite-based transmission. Consequently, frequently touched objects such as paper currency and coins have been suspected as potential transmission vehicle. To assess the risk of SARS-CoV-2 transmission by banknotes and coins, we examined the stability of SARS-CoV-2 and bovine coronavirus, as surrogate with lower biosafety restrictions, on these different means of payment and developed a touch transfer method to examine transfer efficiency from contaminated surfaces to fingertips. Although we observed prolonged virus stability, our results indicate that transmission of SARS-CoV-2 via contaminated coins and banknotes is unlikely and requires high viral loads and a timely order of specific events.

14.
Viruses ; 13(8)2021 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-34452320

RESUMEN

Since its first discovery by Arnold Theiler in 1918, serum hepatitis also known as Theiler's disease has been reported worldwide, causing idiopathic acute hepatitis and liver failure in horses. Recent studies have suggested a novel parvovirus, named equine parvovirus hepatitis (EqPV-H), to be associated with Theiler's disease. Despite the severity and potential fatality of EqPV-H infection, little is known about the possibility of developing chronic infections and putative cross-species infection of equine sister species. In the present longitudinal study, we employed qPCR analysis, serology, and biochemical testing as well as pathology examination of liver biopsies and sequence analysis to investigate potential chronic EqPV-H infection in an isolated study cohort of in total 124 horses from Germany over five years (2013-2018). Importantly, our data suggest that EqPV-H viremia can become chronic in infected horses that do not show biochemical and pathological signs of liver disease. Phylogenetic analysis by maximum likelihood model also confirms high sequence similarity and nucleotide conservation of the multidomain nuclear phosphoprotein NS1 sequences from equine serum samples collected between 2013-2018. Moreover, by examining human, zebra, and donkey sera for the presence of EqPV-H DNA and VP1 capsid protein antibodies, we found evidence for cross-species infection in donkey, but not to human and zebra. In conclusion, this study provides proof for the occurrence of persistent EqPV-H infection in asymptomatic horses and cross-species EqPV-H detection in donkeys.


Asunto(s)
Hepatitis Viral Animal/sangre , Hepatitis Viral Animal/fisiopatología , Infecciones por Parvoviridae/fisiopatología , Infecciones por Parvoviridae/veterinaria , Parvovirus/genética , Viremia/veterinaria , Animales , Biopsia , Estudios de Cohortes , ADN Viral/genética , Enfermedades de los Caballos/virología , Caballos , Hígado/patología , Hígado/virología , Estudios Longitudinales , Infecciones por Parvoviridae/sangre , Parvovirus/clasificación , Infección Persistente , Filogenia
15.
J Hepatol ; 73(3): 549-558, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32294532

RESUMEN

BACKGROUND & AIMS: HCV is a positive-strand RNA virus that primarily infects human hepatocytes. Recent studies have reported that C19orf66 is expressed as an interferon (IFN)-stimulated gene; however, the intrinsic regulation of this gene within the liver as well as its antiviral effects against HCV remain elusive. METHODS: Expression of C19orf66 was quantified in both liver biopsies and primary human hepatocytes, with or without HCV infection. Mechanistic studies of the potent anti-HCV phenotype mediated by C19orf66 were conducted using state-of-the-art virological, biochemical and genetic approaches, as well as correlative light and electron microscopy and transcriptome and proteome analysis. RESULTS: Upregulation of C19orf66 mRNA was observed in both primary human hepatocytes upon HCV infection and in the livers of patients with chronic hepatitis C (CHC). In addition, pegIFNα/ribavirin therapy induced C19orf66 expression in patients with CHC. Transcriptomic profiling and whole cell proteomics of hepatoma cells ectopically expressing C19orf66 revealed no induction of other antiviral genes. Expression of C19orf66 restricted HCV infection, whereas CRIPSPR/Cas9 mediated knockout of C19orf66 attenuated IFN-mediated suppression of HCV replication. Co-immunoprecipitation followed by mass spectrometry identified a stress granule protein-dominated interactome of C19orf66. Studies with subgenomic HCV replicons and an expression system revealed that C19orf66 expression impairs HCV-induced elevation of phosphatidylinositol-4-phosphate, alters the morphology of the viral replication organelle (termed the membranous web) and thereby targets viral RNA replication. CONCLUSION: C19orf66 is an IFN-stimulated gene, which is upregulated in hepatocytes within the first hours post IFN treatment or HCV infection in vivo. The encoded protein possesses specific antiviral activity against HCV and targets the formation of the membranous web. Our study identifies C19orf66 as an IFN-inducible restriction factor with a novel antiviral mechanism that specifically targets HCV replication. LAY SUMMARY: Interferon-stimulated genes are thought to be important to for antiviral immune responses to HCV. Herein, we analysed C19orf66, an interferon-stimulated gene, which appears to inhibit HCV replication. It prevents the HCV-induced elevation of phosphatidylinositol-4-phosphate and alters the morphology of HCV's replication organelle.


Asunto(s)
Antivirales/uso terapéutico , Hepacivirus/genética , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/metabolismo , Interferones/uso terapéutico , Orgánulos/virología , Proteínas de Unión al ARN/metabolismo , Compartimentos de Replicación Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Adulto , Línea Celular Tumoral , Femenino , Técnicas de Inactivación de Genes , Genotipo , Células HEK293 , Hepatitis C Crónica/patología , Hepatitis C Crónica/virología , Hepatocitos/metabolismo , Humanos , Hígado/patología , Masculino , Persona de Mediana Edad , Orgánulos/efectos de los fármacos , Orgánulos/metabolismo , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , Replicón/efectos de los fármacos , Replicón/genética , Ribavirina/uso terapéutico , Resultado del Tratamiento , Replicación Viral/genética
16.
Proc Natl Acad Sci U S A ; 117(3): 1731-1741, 2020 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-31896581

RESUMEN

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and the leading cause for acute viral hepatitis worldwide. The virus is classified as a member of the genus Orthohepevirus A within the Hepeviridae family. Due to the absence of a robust cell culture model for HEV infection, the analysis of the viral life cycle, the development of effective antivirals and a vaccine is severely limited. In this study, we established a protocol based on the HEV genotype 3 p6 (Kernow C-1) and the human hepatoma cell lines HepG2 and HepG2/C3A with different media conditions to produce intracellular HEV cell culture-derived particles (HEVcc) with viral titers between 105 and 106 FFU/mL. Viral titers could be further enhanced by an HEV variant harboring a mutation in the RNA-dependent RNA polymerase. These HEVcc particles were characterized in density gradients and allowed the trans-complementation of subgenomic reporter HEV replicons. In addition, in vitro produced intracellular-derived particles were infectious in liver-humanized mice with high RNA copy numbers detectable in serum and feces. Efficient infection of primary human and swine hepatocytes using the developed protocol could be observed and was inhibited by ribavirin. Finally, RNA sequencing studies of HEV-infected primary human hepatocytes demonstrated a temporally structured transcriptional defense response. In conclusion, this robust cell culture model of HEV infection provides a powerful tool for studying viral-host interactions that should facilitate the discovery of antiviral drugs for this important zoonotic pathogen.


Asunto(s)
Virus de la Hepatitis E/genética , Virus de la Hepatitis E/fisiología , Hepatitis E/metabolismo , Hepatocitos/virología , Animales , Antivirales/farmacología , Carcinoma Hepatocelular , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Genotipo , Células Hep G2 , Hepatitis E/virología , Virus de la Hepatitis E/efectos de los fármacos , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Ratones , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Replicón , Ribavirina/metabolismo , Porcinos , Carga Viral , Replicación Viral
17.
Pathogens ; 8(3)2019 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-31443360

RESUMEN

Hepatitis E virus (HEV) is an increasingly recognised pathogen, affecting several hundred thousand individuals in western countries each year. Importantly, the majority of immunocompromised individuals are not able to clear HEV but develop a chronic course of infection. In the case of lymphoma, which is an inherent immunosuppressive disease per se, chemotherapy can even further exacerbate the immunosuppressive status. As the mechanism of HEV chronification is barely understood, it is important to gain knowledge about the influence of chemotherapeutic drugs on the HEV replication cycle to guide rational clinical management of HEV infection in such patients. In this case report, a 70 year old man was diagnosed with lymphoplasmacytic lymphoma. As we observed the occurrence of chronic HEV after treatment with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib in vivo, we investigated the influence of BTK signaling and ibrutinib treatment in the HEV replication cycle in vitro. First, we detected an HEV-induced mobilisation of BTK in human liver cells during HEV replication. A moderate antiviral effect against HEV replicating isolates including genotypes 1 and 3 was observed, suggesting that ibrutinib did not support HEV replication in a direct manner. Combinatory treatments of ibrutinib with ribavirin indicated that ibrutinib did not influence the antiviral effect of ribavirin. Taken together, chemotherapy targeting cellular factors for the treatment of lymphomas may be a neglected risk factor for the chronification of HEV. For ibrutinib, despite the upregulation of its target BTK during HEV replication, we observed neither a proviral effect on HEV replication nor an influence on the antiviral effect of ribavirin, suggesting that the chronification of HEV may be favoured by its immunosuppressive effect.

18.
Cells ; 8(6)2019 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-31216713

RESUMEN

Keratin proteins form intermediate filaments, which provide structural support for many tissues. Multiple keratin family members are reported to be associated with the progression of liver disease of multiple etiologies. For example, keratin 23 (KRT23) was reported as a stress-inducible protein, whose expression levels correlate with the severity of liver disease. Hepatitis C virus (HCV) is a human pathogen that causes chronic liver diseases including fibrosis, cirrhosis, and hepatocellular carcinoma. However, a link between KRT23 and hepatitis C virus (HCV) infection has not been reported previously. In this study, we investigated KRT23 mRNA levels in datasets from liver biopsies of chronic hepatitis C (CHC) patients and in primary human hepatocytes experimentally infected with HCV, in addition to hepatoma cells. Interestingly, in each of these specimens, we observed an HCV-dependent increase of mRNA levels. Importantly, the KRT23 protein levels in patient plasma decreased upon viral clearance. Ectopic expression of KRT23 enhanced HCV infection; however, CRIPSPR/Cas9-mediated knockout did not show altered replication efficiency. Taken together, our study identifies KRT23 as a novel, virus-induced host-factor for hepatitis C virus.


Asunto(s)
Hepatitis C/metabolismo , Factores Celulares Derivados del Huésped/metabolismo , Queratinas Tipo I/metabolismo , Carcinoma Hepatocelular/genética , Línea Celular , Células HEK293 , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatitis C/genética , Hepatitis C/fisiopatología , Hepatitis C Crónica/metabolismo , Hepatocitos/metabolismo , Interacciones Huésped-Patógeno , Humanos , Queratinas/metabolismo , Queratinas Tipo I/genética , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/genética , ARN Mensajero/metabolismo , Transcriptoma/genética , Replicación Viral
19.
Viruses ; 11(6)2019 05 28.
Artículo en Inglés | MEDLINE | ID: mdl-31141919

RESUMEN

Hepatitis E virus (HEV) is an underestimated disease, leading to estimated 20 million infections and up to 70,000 deaths annually. Infections are mostly asymptomatic but can reach mortality rates up to 25% in pregnant women or become chronic in immunocompromised patients. The current therapy options are limited to the unspecific antivirals Ribavirin (RBV) and pegylated Interferon-α (pegIFN-α). RBV leads to viral clearance in only 80% of patients treated, and is, similar to pegIFN-α, contraindicated in the major risk group of pregnant women, emphasizing the importance of new therapy options. In this review, we focus on the urgent need and current efforts in HEV drug development. We provide an overview of the current status of HEV antiviral research. Furthermore, we discuss strategies for drug development and the limitations of the approaches with respect to HEV.


Asunto(s)
Antivirales/uso terapéutico , Desarrollo de Medicamentos/tendencias , Hepatitis E/tratamiento farmacológico , Quimioterapia Combinada , Femenino , Humanos , Huésped Inmunocomprometido , Interferón-alfa/uso terapéutico , Embarazo , Ribavirina/uso terapéutico
20.
Virulence ; 9(1): 1669-1684, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30403913

RESUMEN

Infection of healthy individuals with human cytomegalovirus (HCMV) is usually unnoticed and results in life-long latency, whereas HCMV reactivation as well as infection of newborns or immunocompromised patients can cause life-threatening disease. To better understand HCMV pathogenesis we studied mechanisms that restrict HCMV spread. We discovered that HCMV-infected cells can directly trigger plasmacytoid dendritic cells (pDC) to mount antiviral type I interferon (IFN-I) responses, even in the absence of cell-free virus. In contrast, monocyte-derived cells only expressed IFN-I when stimulated by cell-free HCMV, or upon encounter of HCMV-infected cells that already produced cell-free virus. Nevertheless, also in the absence of cell-free virus, i.e., upon co-culture of infected epithelial/endothelial cells and monocyte-derived macrophages (moMΦ) or dendritic cells (moDC), antiviral responses were induced that limited HCMV spread. The induction of this antiviral effect was dependent on cell-cell contact, whereas cell-free supernatants from co-culture experiments also inhibited virus spread, implying that soluble factors were critically needed. Interestingly, the antiviral effect was independent of IFN-γ, TNF-α, and IFN-I as indicated by cytokine inhibition experiments using neutralizing antibodies or the vaccinia virus-derived soluble IFN-I binding protein B18R, which traps human IFN-α and IFN-ß. In conclusion, our results indicate that human macrophages and dendritic cells can limit HCMV spread by IFN-I dependent as well as independent mechanisms, whereas the latter ones might be particularly relevant for the restriction of HCMV transmission via cell-to-cell spread.


Asunto(s)
Citocinas/inmunología , Citomegalovirus , Macrófagos/inmunología , Anticuerpos Neutralizantes/inmunología , Técnicas de Cocultivo , Medios de Cultivo , Citocinas/antagonistas & inhibidores , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Humanos , Interferón Tipo I/inmunología , Interferón beta/inmunología , Macrófagos/virología , Factor de Necrosis Tumoral alfa/inmunología , Replicación Viral/efectos de los fármacos
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...