RESUMEN
Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments, ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.
Asunto(s)
Anexina A3 , Hepacivirus , Hepatitis C , Antígeno SS-B , Internalización del Virus , Humanos , Anexina A3/metabolismo , Anexina A3/genética , Autoantígenos/metabolismo , Autoantígenos/genética , Células HEK293 , Hepacivirus/metabolismo , Hepacivirus/fisiología , Hepatitis C/metabolismo , Hepatitis C/virología , Hepatitis C/genética , Interacciones Huésped-Patógeno , Gotas Lipídicas/metabolismo , Gotas Lipídicas/virología , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Proteínas del Núcleo Viral/metabolismo , Proteínas del Núcleo Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/genéticaRESUMEN
LINE-1 (L1) retrotransposons are autonomous transposable elements that can affect gene expression and genome integrity. Potential consequences of exogenous viral infections for L1 activity have not been studied to date. Here, we report that hepatitis C virus (HCV) infection causes a significant increase of endogenous L1-encoded ORF1 protein (L1ORF1p) levels and translocation of L1ORF1p to HCV assembly sites at lipid droplets. HCV replication interferes with retrotransposition of engineered L1 reporter elements, which correlates with HCV RNA-induced formation of stress granules and can be partially rescued by knockdown of the stress granule protein G3BP1. Upon HCV infection, L1ORF1p localizes to stress granules, associates with HCV core in an RNA-dependent manner and translocates to lipid droplets. While HCV infection has a negative effect on L1 mobilization, L1ORF1p neither restricts nor promotes HCV infection. In summary, our data demonstrate that HCV infection causes an increase of endogenous L1 protein levels and that the observed restriction of retrotransposition of engineered L1 reporter elements is caused by sequestration of L1ORF1p in HCV-induced stress granules.
Asunto(s)
Carcinoma Hepatocelular/virología , ADN Helicasas/metabolismo , Hepacivirus/fisiología , Hepatitis C/virología , Neoplasias Hepáticas/virología , Elementos de Nucleótido Esparcido Largo/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Ribonucleoproteínas/metabolismo , Línea Celular Tumoral , Gránulos Citoplasmáticos/virología , ADN Helicasas/genética , Humanos , Gotas Lipídicas/virología , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genética , Ribonucleoproteínas/genéticaRESUMEN
Lipid droplets (LDs) are now recognized as omnipresent and dynamic subcellular organelles of amazing morphological and functional diversity. Beyond the obvious benefit of having molecules full of chemical energy stored in a dedicated structural entity, LDs may also be viewed as a safe harbor for potentially damaging metabolites. This protective function might in many cases even supersede the relevance of lipid storage for eventual energy gain and membrane biogenesis. Furthermore, the LD surface constitutes a unique membrane environment, creating a platform for hosting specific proteins and thus enabling their interactions. These metabolic hotspots would contribute decisively to compartmentalized metabolism in the cytosol. LDs are also communicating extensively with other subcellular organelles in directing and regulating lipid metabolism. Deciphering the relevance of LD storage and regulation at the organismic level will be essential for the understanding of widespread and serious metabolic complications in humans. Increasing attention is also devoted to pathogens appropriating LDs for their own benefit. LD biology is still considered an emerging research area in rapid and vibrant development, attracting scientists from all disciplines of the life sciences and beyond, which is mirrored by the accompanying review collection. Here, we present our personal views on areas we believe are especially exciting and hold great potential for future developments. Particularly, we address issues relating to LD biogenesis and heterogeneity, required technological advances, and the complexity of human physiology.
Asunto(s)
Gotas Lipídicas/metabolismo , Animales , Humanos , Espacio Intracelular/metabolismo , FenotipoRESUMEN
BACKGROUND & AIMS: Hepatitis E virus (HEV) infections are prevalent worldwide. Various viruses have been detected in the ejaculate and can outlast the duration of viremia, indicating replication beyond the blood-testis barrier. HEV replication in diverse organs, however, is still widely misunderstood. We aimed to determine the occurrence, features and morphology of HEV in the ejaculate. METHODS: The presence of HEV in testis was assessed in 12 experimentally HEV-genotype 3-infected pigs. We further tested ejaculate, urine, stool and blood from 3 chronically HEV genotype 3-infected patients and 6 immunocompetent patients with acute HEV infection by HEV-PCR. Morphology and genomic characterization of HEV particles from various human compartments were determined by HEV-PCR, density gradient measurement, immune-electron microscopy and genomic sequencing. RESULTS: In 2 of the 3 chronically HEV-infected patients, we observed HEV-RNA (genotype 3c) in seminal plasma and semen with viral loads >2 logs higher than in the serum. Genomic sequencing showed significant differences between viral strains in the ejaculate compared to stool. Under ribavirin-treatment, HEV shedding in the ejaculate continued for >9 months following the end of viremia. Density gradient measurement and immune-electron microscopy characterized (enveloped) HEV particles in the ejaculate as intact. CONCLUSIONS: The male reproductive system was shown to be a niche of HEV persistence in chronic HEV infection. Surprisingly, sequence analysis revealed distinct genetic HEV variants in the stool and serum, originating from the liver, compared to variants in the ejaculate originating from the male reproductive system. Enveloped HEV particles in the ejaculate did not morphologically differ from serum-derived HEV particles. LAY SUMMARY: Enveloped hepatitis E virus particles could be identified by PCR and electron microscopy in the ejaculate of immunosuppressed chronically infected patients, but not in immunocompetent experimentally infected pigs or in patients with acute self-limiting hepatitis E.
Asunto(s)
Heces/virología , Virus de la Hepatitis E , Hepatitis E , Inmunocompetencia , Infección Persistente , Semen/virología , Animales , Eyaculación , Genoma Viral , Pruebas Hematológicas/métodos , Hepatitis E/sangre , Hepatitis E/inmunología , Hepatitis E/virología , Virus de la Hepatitis E/genética , Virus de la Hepatitis E/aislamiento & purificación , Humanos , Huésped Inmunocomprometido , Masculino , Infección Persistente/inmunología , Infección Persistente/virología , Análisis de Semen/métodos , Porcinos , Urinálisis/métodos , Envoltura Viral , Compartimentos de Replicación ViralRESUMEN
In hepatocytes, PLIN2 is the major protein coating lipid droplets (LDs), an organelle the hepatitis C virus (HCV) hijacks for virion morphogenesis. We investigated the consequences of PLIN2 deficiency on LDs and on HCV infection. Knockdown of PLIN2 did not affect LD homeostasis, likely due to compensation by PLIN3, but severely impaired HCV particle production. PLIN2-knockdown cells had slightly larger LDs with altered protein composition, enhanced local lipase activity and higher ß-oxidation capacity. Electron micrographs showed that, after PLIN2 knockdown, LDs and HCV-induced vesicular structures were tightly surrounded by ER-derived double-membrane sacs. Strikingly, the LD access for HCV core and NS5A proteins was restricted in PLIN2-deficient cells, which correlated with reduced formation of intracellular HCV particles that were less infectious and of higher density, indicating defects in maturation. PLIN2 depletion also reduced protein levels and secretion of ApoE due to lysosomal degradation, but did not affect the density of ApoE-containing lipoproteins. However, ApoE overexpression in PLIN2-deficient cells did not restore HCV spreading. Thus, PLIN2 expression is required for trafficking of core and NS5A proteins to LDs, and for formation of functional low-density HCV particles prior to ApoE incorporation.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Hepacivirus/patogenicidad , Hepatitis C/virología , Hepatocitos/virología , Gotas Lipídicas/virología , Lipoproteínas/metabolismo , Perilipina-2/metabolismo , Virión/fisiología , Células HEK293 , Hepatitis C/metabolismo , Hepatocitos/metabolismo , Humanos , Gotas Lipídicas/metabolismo , Perilipina-2/genética , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
Lysine acetylation regulates transcription by targeting histones and nonhistone proteins. Here we report that the central regulator of transcription, RNA polymerase II, is subject to acetylation in mammalian cells. Acetylation occurs at eight lysines within the C-terminal domain (CTD) of the largest polymerase subunit and is mediated by p300/KAT3B. CTD acetylation is specifically enriched downstream of the transcription start sites of polymerase-occupied genes genome-wide, indicating a role in early stages of transcription initiation or elongation. Mutation of lysines or p300 inhibitor treatment causes the loss of epidermal growth-factor-induced expression of c-Fos and Egr2, immediate-early genes with promoter-proximally paused polymerases, but does not affect expression or polymerase occupancy at housekeeping genes. Our studies identify acetylation as a new modification of the mammalian RNA polymerase II required for the induction of growth factor response genes.
Asunto(s)
Histonas/genética , Lisina/genética , ARN Polimerasa II/metabolismo , Transcripción Genética , Acetilación , Animales , Proteína 2 de la Respuesta de Crecimiento Precoz/biosíntesis , Células Madre Embrionarias/citología , Regulación de la Expresión Génica , Genes fos/genética , Histonas/metabolismo , Humanos , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Replication of the hepatitis C virus (HCV) strongly relies on various lipid metabolic processes in different steps of the viral life cycle. In general, HCV changes the cells' lipidomic profile by differentially regulating key pathways of lipid synthesis, remodeling, and utilization. In this review, we sum up the latest data mainly from the past five years, emphasizing the role of lipids in HCV RNA replication, assembly, and egress. In detail, we highlight changes in the fatty acid content as well as alterations of the membrane lipid composition during replication vesicle formation. We address the role of lipid droplets as a lipid provider during replication and as an essential hub for HCV assembly. Finally, we depict different ideas of HCV maturation and egress including lipoprotein association and potential secretory routes.
Asunto(s)
Hepacivirus/fisiología , Hepatitis C/metabolismo , Hepatitis C/virología , Metabolismo de los Lípidos , ARN Viral/genética , Transcripción Genética , Virión , Autofagosomas/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Interacciones Huésped-Patógeno , Humanos , Lípidos , Virión/metabolismoRESUMEN
Killer-cell immunoglobulin-like receptors (KIRs) are transmembrane glycoproteins expressed by natural killer (NK) cells. Binding of KIR3DS1 to its recently discovered ligand, HLA-F, activates NK cells and has been associated with resolution of hepatitis C virus (HCV) infection. We investigated the mechanisms by which KIR3DS1 contributes to the antiviral immune response. Using cell culture systems, mice with humanized livers, and primary liver tissue from HCV-infected individuals, we found that the KIR3DS1 ligand HLA-F is up-regulated on HCV-infected cells, and that interactions between KIR3DS1 and HLA-F contribute to NK cell-mediated control of HCV. Strategies to promote interaction between KIR3DS1 and HLA-F might be developed for treatment of infectious diseases and cancer.
Asunto(s)
Hepacivirus/fisiología , Antígenos de Histocompatibilidad Clase I/fisiología , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Receptores KIR3DS1/fisiología , Replicación Viral , Células Cultivadas , Hepatitis C/tratamiento farmacológico , HumanosRESUMEN
The hepatitis C virus (HCV) life cycle is tightly linked to the host cell lipid metabolism with the endoplasmic reticulum-derived membranous web harboring viral RNA replication complexes and lipid droplets as virion assembly sites. To investigate HCV-induced changes in the lipid composition, we performed quantitative shotgun lipidomic studies of whole cell extracts and subcellular compartments. Our results indicate that HCV infection reduces the ratio of neutral to membrane lipids. While the amount of neutral lipids and lipid droplet morphology were unchanged, membrane lipids, especially cholesterol and phospholipids, accumulated in the microsomal fraction in HCV-infected cells. In addition, HCV-infected cells had a higher relative abundance of phosphatidylcholines and triglycerides with longer fatty acyl chains and a strikingly increased utilization of C18 fatty acids, most prominently oleic acid (FA [18:1]). Accordingly, depletion of fatty acid elongases and desaturases impaired HCV replication. Moreover, the analysis of free fatty acids revealed increased levels of polyunsaturated fatty acids (PUFAs) caused by HCV infection. Interestingly, inhibition of the PUFA synthesis pathway via knockdown of the rate-limiting Δ6-desaturase enzyme or by treatment with a high dose of a small-molecule inhibitor impaired viral progeny production, indicating that elevated PUFAs are needed for virion morphogenesis. In contrast, pretreatment with low inhibitor concentrations promoted HCV translation and/or early RNA replication. Taken together our results demonstrate the complex remodeling of the host cell lipid metabolism induced by HCV to enhance both virus replication and progeny production.
Asunto(s)
Hepacivirus/metabolismo , Hepatocitos/metabolismo , Interacciones Huésped-Patógeno , Metabolismo de los Lípidos/genética , Metaboloma , Virión/metabolismo , Replicación Viral/fisiología , Acetiltransferasas/antagonistas & inhibidores , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Línea Celular Tumoral , Colesterol/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Ácido Graso Desaturasas/antagonistas & inhibidores , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Elongasas de Ácidos Grasos , Ácidos Grasos Insaturados/metabolismo , Regulación de la Expresión Génica , Hepacivirus/crecimiento & desarrollo , Hepatocitos/química , Hepatocitos/virología , Humanos , Gotas Lipídicas/metabolismo , Gotas Lipídicas/virología , Microsomas/metabolismo , Microsomas/virología , Ácido Oléico/metabolismo , Fosfatidilcolinas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Viral/biosíntesis , ARN Viral/genética , Triglicéridos/metabolismo , Virión/crecimiento & desarrollo , Ensamble de Virus/fisiologíaRESUMEN
BACKGROUND & AIMS: Hepatitis E virus (HEV) is a major cause of acute hepatitis as well as chronic infection in immunocompromised individuals; however, in vivo infection models are limited. The aim of this study was to establish a small animal model to improve our understanding of HEV replication mechanisms and permit the development of effective therapeutics. METHODS: UPA/SCID/beige mice repopulated with primary human hepatocytes were used for infection experiments with HEV genotype (GT) 1 and 3. Virological parameters were determined at the serological and intrahepatic level by real time PCR, immunohistochemistry and RNA in situ hybridization. RESULTS: Establishment of HEV infection was achieved after intravenous injection of stool-derived virions and following co-housing with HEV-infected animals but not via inoculation of serum-derived HEV. GT 1 infection resulted in a rapid rise of viremia and high stable titres in serum, liver, bile and faeces of infected mice for more than 25 weeks. In contrast, viremia in GT 3 infected mice developed more slowly and displayed lower titres in all analysed tissues as compared to GT 1. HEV-infected human hepatocytes could be visualized using HEV ORF2 and ORF3 specific antibodies and HEV RNA in situ hybridization probes. Finally, six-week administration of ribavirin led to a strong reduction of viral replication in the serum and liver of GT 1 infected mice. CONCLUSION: We established an efficient model of HEV infection to test the efficacy of antiviral agents and to exploit mechanisms of HEV replication and interaction with human hepatocytes in vivo.
Asunto(s)
Antivirales/uso terapéutico , Virus de la Hepatitis E/genética , Hepatitis E/tratamiento farmacológico , Hígado/virología , ARN Viral/análisis , Replicación Viral/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Hepatitis E/virología , Humanos , Hibridación in Situ , Hígado/patología , Ratones , Ratones SCID , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Liver steatosis is a common health problem associated with hepatitis C virus (HCV) and an important risk factor for the development of liver fibrosis and cancer. Steatosis is caused by triglycerides (TG) accumulating in lipid droplets (LDs), cellular organelles composed of neutral lipids surrounded by a monolayer of phospholipids. The HCV nucleocapsid core localizes to the surface of LDs and induces steatosis in cultured cells and mouse livers by decreasing intracellular TG degradation (lipolysis). Here we report that core at the surface of LDs interferes with the activity of adipose triglyceride lipase (ATGL), the key lipolytic enzyme in the first step of TG breakdown. Expressing core in livers or mouse embryonic fibroblasts of ATGL(-/-) mice no longer decreases TG degradation as observed in LDs from wild-type mice, supporting the model that core reduces lipolysis by engaging ATGL. Core must localize at LDs to inhibit lipolysis, as ex vivo TG hydrolysis is impaired in purified LDs coated with core but not when free core is added to LDs. Coimmunoprecipitation experiments revealed that core does not directly interact with the ATGL complex but, unexpectedly, increased the interaction between ATGL and its activator CGI-58 as well as the recruitment of both proteins to LDs. These data link the anti-lipolytic activity of the HCV core protein with altered ATGL binding to CGI-58 and the enhanced association of both proteins with LDs.
Asunto(s)
1-Acilglicerol-3-Fosfato O-Aciltransferasa/metabolismo , Lipasa/metabolismo , Gotas Lipídicas/enzimología , Proteínas del Núcleo Viral/fisiología , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Humanos , Hidrólisis , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , Triglicéridos/metabolismoRESUMEN
The nonstructural protein NS5A has emerged as a new drug target in antiviral therapies for Hepatitis C Virus (HCV) infection. NS5A is critically involved in viral RNA replication that takes place at newly formed membranes within the endoplasmic reticulum (membranous web) and assists viral assembly in the close vicinity of lipid droplets (LDs). To identify host proteins that interact with NS5A, we performed a yeast two-hybrid screen with the N-terminus of NS5A (amino acids 1-31), a well-studied α-helical domain important for the membrane tethering of NS5A. Our studies identified the LD-associated host protein, Tail-Interacting Protein 47 (TIP47) as a novel NS5A interaction partner. Coimmunoprecipitation experiments in Huh7 hepatoma cells confirmed the interaction of TIP47 with full-length NS5A. shRNA-mediated knockdown of TIP47 caused a more than 10-fold decrease in the propagation of full-length infectious HCV in Huh7.5 hepatoma cells. A similar reduction was observed when TIP47 was knocked down in cells harboring an autonomously replicating HCV RNA (subgenomic replicon), indicating that TIP47 is required for efficient HCV RNA replication. A single point mutation (W9A) in NS5A that disrupts the interaction with TIP47 but preserves proper subcellular localization severely decreased HCV RNA replication. In biochemical membrane flotation assays, TIP47 cofractionated with HCV NS3, NS5A, NS5B proteins, and viral RNA, and together with nonstructural viral proteins was uniquely distributed to lower-density LD-rich membrane fractions in cells actively replicating HCV RNA. Collectively, our data support a model where TIP47--via its interaction with NS5A--serves as a novel cofactor for HCV infection possibly by integrating LD membranes into the membranous web.
Asunto(s)
Hepacivirus/fisiología , ARN Viral/biosíntesis , Proteínas de Transporte Vesicular/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Línea Celular Tumoral , Retículo Endoplásmico/virología , Células HEK293 , Hepacivirus/genética , Hepatitis C/metabolismo , Hepatitis C/virología , Humanos , Lípidos , Perilipina-3 , Mutación Puntual , Interferencia de ARN , ARN Interferente Pequeño , ARN Viral/genética , ARN Viral/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas no Estructurales Virales/genética , Ensamble de Virus , Replicación Viral/genéticaRESUMEN
The triglyceride-synthesizing enzyme acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) plays a critical role in hepatitis C virus (HCV) infection by recruiting the HCV capsid protein core onto the surface of cellular lipid droplets (LDs). Here we find a new interaction between the non-structural protein NS5A and DGAT1 and show that the trafficking of NS5A to LDs depends on DGAT1 activity. DGAT1 forms a complex with NS5A and core and facilitates the interaction between both viral proteins. A catalytically inactive mutant of DGAT1 (H426A) blocks the localization of NS5A, but not core, to LDs in a dominant-negative manner and impairs the release of infectious viral particles, underscoring the importance of DGAT1-mediated translocation of NS5A to LDs in viral particle production. We propose a model whereby DGAT1 serves as a cellular hub for HCV core and NS5A proteins, guiding both onto the surface of the same subset of LDs, those generated by DGAT1. These results highlight the critical role of DGAT1 as a host factor for HCV infection and as a potential drug target for antiviral therapy.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/química , Diacilglicerol O-Acetiltransferasa/fisiología , Regulación Viral de la Expresión Génica , Hepacivirus/metabolismo , Proteínas no Estructurales Virales/química , Animales , Antivirales/farmacología , Cápside/química , Línea Celular , Genes Dominantes , Células HEK293 , Hepatitis C/virología , Humanos , Lentivirus/genética , Lípidos/química , Ratones , Microscopía Fluorescente/métodos , Mutación , Plásmidos/metabolismo , Unión Proteica , Triglicéridos/química , Triglicéridos/metabolismo , Proteínas no Estructurales Virales/fisiologíaRESUMEN
X-ray microscopy is a commonly used method especially in material science application, where the large penetration depth of X-rays is necessary for three-dimensional structural studies of thick specimens with high-Z elements. In this paper it is shown that full-field X-ray microscopy at 6.2â keV can be utilized for imaging of biological specimens with high resolution. A full-field Zernike phase-contrast microscope based on diffractive optics is used to study lipid droplet formation in hepatoma cells. It is shown that the contrast of the images is comparable with that of electron microscopy, and even better contrast at tender X-ray energies between 2.5â keV and 4â keV is expected.
Asunto(s)
Microscopía de Contraste de Fase/métodos , Intensificación de Imagen Radiográfica/métodos , Interpretación de Imagen Radiográfica Asistida por Computador/métodos , Tomografía Computarizada por Rayos X/métodos , Difracción de Rayos X/métodos , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Cells infected with hepatitis C virus (HCV) become refractory to further infection by HCV (T. Schaller et al., J. Virol. 81:4591-4603, 2007; D. M. Tscherne et al., J. Virol. 81:3693-3703, 2007). This process, termed superinfection exclusion, does not involve downregulation of surface viral receptors but instead occurs inside the cell at the level of RNA replication. The originally infecting virus may occupy replication niches or sequester host factors necessary for viral growth, preventing effective growth of viruses that enter the cell later. However, there appears to be an additional level of intracellular competition between viral genomes that occurs at or shortly following mitosis. In the setting of cellular division, when two viral replicons of equivalent fitness are present within a cell, each has an equal opportunity to exclude the other. In a population of dividing cells, the competition between viral genomes proceeds apace, randomly clearing one or the other genome from cells in the span of 9 to 12 days. These findings demonstrate a new mechanism of intracellular competition between HCV strains, which may act to further limit HCV's genetic diversity and ability to recombine in vivo.
Asunto(s)
Hepacivirus/fisiología , Hepatocitos/virología , Interacciones Huésped-Patógeno , Mitosis , Interferencia Viral , Línea Celular , Hepacivirus/crecimiento & desarrollo , HumanosRESUMEN
Intracellular pathogens rely on host metabolic networks for multiplication. Enveloped viruses need lipids for formation of the viral envelope and positive sense RNA viruses that replicate in membranous inclusions require lipids for formation of the replication compartments. In addition, all intracellular pathogens need energy for their replicative cycle. As triglycerides in lipid droplets are the main energy storage unit of cells and major source of membrane lipids, it is not surprising that viruses have evolved various strategies to exploit different aspects of lipid droplet biology.
Asunto(s)
Gotas Lipídicas , Replicación Viral , Gotas Lipídicas/metabolismo , Gotas Lipídicas/virología , Humanos , Animales , Envoltura Viral/metabolismo , Virus ARN/fisiología , Virus ARN/metabolismo , Virus ARN/genética , Metabolismo de los Lípidos , Triglicéridos/metabolismoRESUMEN
Three-dimensional culture models of the brain enable the study of neuroinfection in the context of a complex interconnected cell matrix. Depending on the differentiation status of the neural cells, two models exist: 3D spheroids also called neurospheres and cerebral organoids. Here, we describe the preparation of 3D spheroids and cerebral organoids and give an outlook on their usage to study Rift Valley fever virus and other neurotropic viruses.
Asunto(s)
Organoides , Esferoides Celulares , Organoides/virología , Organoides/citología , Esferoides Celulares/virología , Humanos , Animales , Virus ARN/fisiología , Encéfalo/virología , Encéfalo/citología , Infecciones por Virus ARN/virología , Técnicas de Cultivo de Célula/métodos , Técnicas de Cultivo Tridimensional de Células/métodosRESUMEN
Some viruses are rarely transmitted orally or sexually despite their presence in saliva, breast milk, or semen. We previously identified that extracellular vesicles (EVs) in semen and saliva inhibit Zika virus infection. However, the antiviral spectrum and underlying mechanism remained unclear. Here we applied lipidomics and flow cytometry to show that these EVs expose phosphatidylserine (PS). By blocking PS receptors, targeted by Zika virus in the process of apoptotic mimicry, they interfere with viral attachment and entry. Consequently, physiological concentrations of EVs applied in vitro efficiently inhibited infection by apoptotic mimicry dengue, West Nile, Chikungunya, Ebola and vesicular stomatitis viruses, but not severe acute respiratory syndrome coronavirus 2, human immunodeficiency virus 1, hepatitis C virus and herpesviruses that use other entry receptors. Our results identify the role of PS-rich EVs in body fluids in innate defence against infection via viral apoptotic mimicries, explaining why these viruses are primarily transmitted via PS-EV-deficient blood or blood-ingesting arthropods rather than direct human-to-human contact.
Asunto(s)
Líquidos Corporales , Vesículas Extracelulares , Virus , Infección por el Virus Zika , Virus Zika , Femenino , Humanos , Fosfatidilserinas , Acoplamiento ViralRESUMEN
Lipid droplets (LDs) are highly dynamic cell organelles involved in energy homeostasis and membrane trafficking. Here, we review how select pathogens interact with LDs. Several RNA viruses use host LDs at different steps of their life cycle. Some intracellular bacteria and parasites usurp host LDs or encode their own lipid biosynthesis machinery, thus allowing production of LDs independently of their host. Although many mechanistic details of host/pathogen LD interactions are unknown, a picture emerges in which the unique cellular architecture and energy stored in LDs are important in the replication of diverse pathogens.
Asunto(s)
Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos , Metabolismo Energético , Interacciones Huésped-Patógeno/fisiología , Metabolismo de los Lípidos , Virus ARN/fisiología , Animales , Transporte Biológico Activo , Humanos , Membranas Intracelulares/metabolismoRESUMEN
The elongation competence of the RNA polymerase II complex is critically dependent on the positive transcription elongation factor b (P-TEFb). P-TEFb exists in two forms in cells, an active form composed of cyclin T1 and CDK9 and an inactive form, in which cyclin T1/CDK9 is sequestered by Hexim1 and 7SK snRNA. Here, we report that partitioning of active and inactive P-TEFb is regulated by acetylation of cyclin T1. Cyclin T1 acetylation triggers dissociation of Hexim1 and 7SK snRNA from cyclin T1/CDK9 and activates the transcriptional activity of P-TEFb. This activation is lost in P-TEFb complexes containing cyclin T1 that can no longer be acetylated. An acetylation-deficient cyclin T1 mutant dominantly suppresses NF-kappaB-mediated activation of the interleukin-8 promoter but continues to synergize normally with the HIV Tat protein to transactivate the HIV long terminal repeat. These findings support the model that acetylation of cyclin T1 serves as a physiological switch that liberates P-TEFb from its endogenous inhibitors Hexim1 and 7SK snRNA, but is not required for the cooperative action with HIV Tat.