RESUMEN
Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.
Asunto(s)
Coronavirus/fisiología , Hemaglutininas Virales/genética , Glicoproteína de la Espiga del Coronavirus/genética , Proteínas Virales de Fusión/genética , Virión/metabolismo , Animales , Evolución Biológica , Línea Celular , Coronavirus/genética , Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/metabolismo , Coronavirus Humano OC43/fisiología , Coronavirus Bovino/genética , Coronavirus Bovino/metabolismo , Coronavirus Bovino/fisiología , Hemaglutininas Virales/química , Hemaglutininas Virales/metabolismo , Humanos , Lectinas/genética , Lectinas/metabolismo , Ratones , Mutación , Unión Proteica , Dominios Proteicos , Receptores Virales/metabolismo , Selección Genética , Ácidos Siálicos/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/metabolismo , Virión/genética , Acoplamiento Viral , Liberación del VirusRESUMEN
Preexisting immune responses toward adenoviral vectors limit the use of a vector based on particular serotypes and its clinical applicability for gene therapy and/or vaccination. Therefore, there is a significant interest in vectorizing novel adenoviral types that have low seroprevalence in the human population. Here, we describe the discovery and vectorization of a chimeric human adenovirus, which we call HAdV-20-42-42. Full-genome sequencing revealed that this virus is closely related to human serotype 42, except for the penton base, which is derived from serotype 20. The HAdV-20-42-42 vector could be propagated stably to high titers on existing E1-complementing packaging cell lines. Receptor-binding studies revealed that the vector utilized both CAR and CD46 as receptors for cell entry. Furthermore, the HAdV-20-42-42 vector was potent in transducing human and murine cardiovascular cells and tissues, irrespective of the presence of blood coagulation factor X. In vivo characterizations demonstrate that when delivered intravenously (i.v.) in mice, HAdV-20-42-42 mainly targeted the lungs, liver, and spleen and triggered robust inflammatory immune responses. Finally, we demonstrate that potent T-cell responses against vector-delivered antigens could be induced upon intramuscular vaccination in mice. In summary, from the data obtained we conclude that HAdV-20-42-42 provides a valuable addition to the portfolio of adenoviral vectors available to develop efficacious products in the fields of gene therapy and vaccination. IMPORTANCE Adenoviral vectors are under investigation for a broad range of therapeutic indications in diverse fields, such as oncology and gene therapy, as well as for vaccination both for human and veterinary use. A wealth of data shows that preexisting immune responses may limit the use of a vector. Particularly in the current climate of global pandemic, there is a need to expand the toolbox with novel adenoviral vectors for vaccine development. Our data demonstrate that we have successfully vectorized a novel adenovirus type candidate with low seroprevalence. The cell transduction data and antigen-specific immune responses induced in vivo demonstrate that this vector is highly promising for the development of gene therapy and vaccine products.
Asunto(s)
Adenovirus Humanos , Terapia Genética/métodos , Vectores Genéticos , Desarrollo de Vacunas/métodos , Células A549 , Adenovirus Humanos/genética , Adenovirus Humanos/inmunología , Animales , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Células HEK293 , Humanos , Masculino , Ratones , Estudios SeroepidemiológicosRESUMEN
The hydrophobic molecules of the metabolome - also named the lipidome - constitute a major part of the entire metabolome. Novel technologies show the existence of a staggering number of individual lipid species, the biological functions of which are, with the exception of only a few lipid species, unknown. Much can be learned from pathogens that have evolved to take advantage of the complexity of the lipidome to escape the immune system of the host organism and to allow their survival and replication. Different types of pathogens target different lipids as shown in interaction maps, allowing visualization of differences between different types of pathogens. Bacterial and viral pathogens target predominantly structural and signaling lipids to alter the cellular phenotype of the host cell. Fungal and parasitic pathogens have complex lipidomes themselves and target predominantly the release of polyunsaturated fatty acids from the host cell lipidome, resulting in the generation of eicosanoids by either the host cell or the pathogen. Thus, whereas viruses and bacteria induce predominantly alterations in lipid metabolites at the host cell level, eukaryotic pathogens focus on interference with lipid metabolites affecting systemic inflammatory reactions that are part of the immune system. A better understanding of the interplay between host-pathogen interactions will not only help elucidate the fundamental role of lipid species in cellular physiology, but will also aid in the generation of novel therapeutic drugs.
Asunto(s)
Fenómenos Fisiológicos Bacterianos , Hongos/fisiología , Interacciones Huésped-Patógeno/fisiología , Metabolismo de los Lípidos , Metaboloma , Fenómenos Fisiológicos de los Virus , Fenómenos Fisiológicos Bacterianos/genética , Enfermedades Transmisibles/inmunología , Enfermedades Transmisibles/microbiología , Enfermedades Transmisibles/virología , Hongos/genética , Interacciones Huésped-Patógeno/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunidad Innata , Metabolismo de los Lípidos/fisiología , Metaboloma/fisiología , Fenómenos Fisiológicos de los Virus/genéticaRESUMEN
Cardioviruses, including encephalomyocarditis virus (EMCV) and the human Saffold virus, are small non-enveloped viruses belonging to the Picornaviridae, a large family of positive-sense RNA [(+)RNA] viruses. All (+)RNA viruses remodel intracellular membranes into unique structures for viral genome replication. Accumulating evidence suggests that picornaviruses from different genera use different strategies to generate viral replication organelles (ROs). For instance, enteroviruses (e.g. poliovirus, coxsackievirus, rhinovirus) rely on the Golgi-localized phosphatidylinositol 4-kinase III beta (PI4KB), while cardioviruses replicate independently of the kinase. By which mechanisms cardioviruses develop their ROs is currently unknown. Here we show that cardioviruses manipulate another PI4K, namely the ER-localized phosphatidylinositol 4-kinase III alpha (PI4KA), to generate PI4P-enriched ROs. By siRNA-mediated knockdown and pharmacological inhibition, we demonstrate that PI4KA is an essential host factor for EMCV genome replication. We reveal that the EMCV nonstructural protein 3A interacts with and is responsible for PI4KA recruitment to viral ROs. The ensuing phosphatidylinositol 4-phosphate (PI4P) proved important for the recruitment of oxysterol-binding protein (OSBP), which delivers cholesterol to EMCV ROs in a PI4P-dependent manner. PI4P lipids and cholesterol are shown to be required for the global organization of the ROs and for viral genome replication. Consistently, inhibition of OSBP expression or function efficiently blocked EMCV RNA replication. In conclusion, we describe for the first time a cellular pathway involved in the biogenesis of cardiovirus ROs. Remarkably, the same pathway was reported to promote formation of the replication sites of hepatitis C virus, a member of the Flaviviridae family, but not other picornaviruses or flaviviruses. Thus, our results highlight the convergent recruitment by distantly related (+)RNA viruses of a host lipid-modifying pathway underlying formation of viral replication sites.
Asunto(s)
Infecciones por Cardiovirus/metabolismo , Virus de la Encefalomiocarditis/fisiología , Interacciones Huésped-Parásitos/fisiología , Metabolismo de los Lípidos/fisiología , Replicación Viral/fisiología , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Animales , Western Blotting , Hepacivirus/fisiología , Humanos , Inmunoprecipitación , Microscopía Fluorescente , Fosfatos de Fosfatidilinositol/metabolismo , Picornaviridae , Virus ARN , ARN Interferente Pequeño , Receptores de Esteroides/metabolismo , TransfecciónRESUMEN
Encephalomyocarditis virus (EMCV), like hepatitis C virus (HCV), requires phosphatidylinositol 4-kinase IIIα (PI4KA) for genome replication. Here, we demonstrate that tyrphostin AG1478, a known epidermal growth factor receptor (EGFR) inhibitor, also inhibits PI4KA activity, both in vitro and in cells. AG1478 impaired replication of EMCV and HCV but not that of an EMCV mutant previously shown to escape PI4KA inhibition. This work uncovers novel cellular and antiviral properties of AG1478, a compound previously regarded only as a cancer chemotherapy agent.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/antagonistas & inhibidores , Antivirales/farmacología , Virus de la Encefalomiocarditis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hepacivirus/efectos de los fármacos , Quinazolinas/farmacología , Tirfostinos/farmacología , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Relación Dosis-Respuesta a Droga , Virus de la Encefalomiocarditis/genética , Virus de la Encefalomiocarditis/fisiología , Células HeLa/efectos de los fármacos , Células HeLa/virología , Hepacivirus/fisiología , Humanos , Terapia Molecular Dirigida/métodos , Mutación , Replicación Viral/efectos de los fármacosRESUMEN
PI4KIIIß recruitment to Golgi membranes relies on GBF1/Arf and ACBD3. Enteroviruses such as poliovirus and coxsackievirus recruit PI4KIIIß to their replication sites via their 3A proteins. Here, we show that human rhinovirus (HRV) 3A also recruited PI4KIIIß to replication sites. Unlike other enterovirus 3A proteins, HRV 3A failed to bind GBF1. Although HRV 3A was previously shown to interact with ACBD3, our data suggest that PI4KIIIß recruitment occurred independently of both GBF1 and ACBD3.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Interacciones Huésped-Patógeno , Proteínas de la Membrana/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Rhinovirus/fisiología , Proteínas del Núcleo Viral/metabolismo , Humanos , Unión ProteicaRESUMEN
Coxsackieviruses require phosphatidylinositol-4-kinase IIIß (PI4KIIIß) for replication but can bypass this need by an H57Y mutation in protein 3A (3A-H57Y). We show that mutant coxsackievirus is not outcompeted by wild-type virus during 10 passages in vitro. In mice, the mutant virus proved as virulent as wild-type virus, even when mice were treated with a PI4KIIIß inhibitor. Our data suggest that upon emergence, the 3A-H57Y mutant has the fitness to establish a resistant population with a virulence similar to that of wild-type virus.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/metabolismo , Enterovirus/fisiología , Aptitud Genética , Mutación , 1-Fosfatidilinositol 4-Quinasa/genética , Animales , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/virología , Enterovirus/patogenicidad , Interacciones Huésped-Patógeno , Ratones , Proteínas Virales/genética , Proteínas Virales/metabolismo , Virulencia/genética , Replicación ViralRESUMEN
UNLABELLED: Members of the Enterovirus (poliovirus [PV], coxsackieviruses, and human rhinoviruses) and Kobuvirus (Aichi virus) genera in the Picornaviridae family rely on PI4KIIIß (phosphatidylinositol-4-kinase IIIß) for efficient replication. The small membrane-anchored enteroviral protein 3A recruits PI4KIIIß to replication organelles, yet the underlying mechanism has remained elusive. Recently, it was shown that kobuviruses recruit PI4KIIIß through interaction with ACBD3 (acyl coenzyme A [acyl-CoA]-binding protein domain 3), a novel interaction partner of PI4KIIIß. Therefore, we investigated a possible role for ACBD3 in recruiting PI4KIIIß to enterovirus replication organelles. Although ACBD3 interacted directly with coxsackievirus B3 (CVB3) 3A, its depletion from cells by RNA interference did not affect PI4KIIIß recruitment to replication organelles and did not impair CVB3 RNA replication. Enterovirus 3A was previously also proposed to recruit PI4KIIIß via GBF1/Arf1, based on the known interaction of 3A with GBF1, an important regulator of secretory pathway transport and a guanine nucleotide exchange factor (GEF) of Arf1. However, our results demonstrate that inhibition of GBF1 or Arf1 either by pharmacological inhibition or depletion with small interfering RNA (siRNA) treatment did not affect the ability of 3A to recruit PI4KIIIß. Furthermore, we show that a 3A mutant that no longer binds GBF1 was capable of recruiting PI4KIIIß, even in ACBD3-depleted cells. Together, our findings indicate that unlike originally envisaged, coxsackievirus recruits PI4KIIIß to replication organelles independently of ACBD3 and GBF1/Arf1. IMPORTANCE: A hallmark of enteroviral infection is the generation of new membranous structures to support viral RNA replication. The functionality of these "replication organelles" depends on the concerted actions of both viral nonstructural proteins and co-opted host factors. It is thus essential to understand how these structures are formed and which cellular components are key players in this process. GBF1/Arf1 and ACBD3 have been proposed to contribute to the recruitment of the essential lipid-modifying enzyme PI4KIIIß to enterovirus replication organelles. Here we show that the enterovirus CVB3 recruits PI4KIIIß by a mechanism independent of both GBF1/Arf1 and ACBD3. This study shows that the strategy employed by coxsackievirus to recruit PI4KIIIß to replication organelles is far more complex than initially anticipated.
Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Enterovirus Humano B/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de la Membrana/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Replicación Viral , Animales , Línea Celular , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/virología , Humanos , Unión Proteica , ARN Viral/genética , ARN Viral/metabolismo , Proteínas Virales/metabolismoRESUMEN
Despite their high clinical and socioeconomic impacts, there is currently no approved antiviral therapy for the prophylaxis or treatment of enterovirus infections. Here we report on a novel inhibitor of enterovirus replication, compound 1, 2-fluoro-4-(2-methyl-8-(3-(methylsulfonyl)benzylamino)imidazo[1,2-a]pyrazin-3-yl)phenol. This compound exhibited a broad spectrum of antiviral activity, as it inhibited all tested species of enteroviruses and rhinoviruses, with 50% effective concentrations ranging between 4 and 71 nM. After a lengthy resistance selection process, coxsackievirus mutants resistant to compound 1 were isolated that carried substitutions in their 3A protein. Remarkably, the same substitutions were recently shown to provide resistance to inhibitors of phosphatidylinositol 4-kinase IIIß (PI4KIIIß), a lipid kinase that is essential for enterovirus replication, suggesting that compound 1 may also target this host factor. Accordingly, compound 1 directly inhibited PI4KIIIß in an in vitro kinase activity assay. Furthermore, the compound strongly reduced the PI 4-phosphate levels of the Golgi complex in cells. Rescue of coxsackievirus replication in the presence of compound 1 by a mutant PI4KIIIß carrying a substitution in its ATP-binding pocket revealed that the compound directly binds the kinase at this site. Finally, we determined that an analogue of compound 1, 3-(3-fluoro-4-methoxyphenyl)-2-methyl-N-(pyridin-4-ylmethyl)imidazo[1,2-a]pyrazin-8-amine, is well tolerated in mice and has a dose-dependent protective activity in a coxsackievirus serotype B4-induced pancreatitis model.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/metabolismo , Antivirales/farmacología , Antivirales/uso terapéutico , Enterovirus/efectos de los fármacos , Enterovirus/metabolismo , Animales , Enterovirus/patogenicidad , Activación Enzimática/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Masculino , Ratones , Estructura Molecular , Pancreatitis/tratamiento farmacológico , Pancreatitis/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Cells infected with dengue virus release a high proportion of immature prM-containing virions. In accordance, substantial levels of prM antibodies are found in sera of infected humans. Furthermore, it has been recently described that the rates of prM antibody responses are significantly higher in patients with secondary infection compared to those with primary infection. This suggests that immature dengue virus may play a role in disease pathogenesis. Interestingly, however, numerous functional studies have revealed that immature particles lack the ability to infect cells. In this report, we show that fully immature dengue particles become highly infectious upon interaction with prM antibodies. We demonstrate that prM antibodies facilitate efficient binding and cell entry of immature particles into Fc-receptor-expressing cells. In addition, enzymatic activity of furin is critical to render the internalized immature virus infectious. Together, these data suggest that during a secondary infection or primary infection of infants born to dengue-immune mothers, immature particles have the potential to be highly infectious and hence may contribute to the development of severe disease.
Asunto(s)
Anticuerpos Antivirales/inmunología , Virus del Dengue/patogenicidad , Furina/metabolismo , Virión/inmunología , Línea Celular , Dengue/inmunología , Virus del Dengue/inmunología , Humanos , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The genus Enterovirus, belonging to the family Picornaviridae, includes well-known pathogens, such as poliovirus, coxsackievirus, and rhinovirus. Brefeldin A (BFA) impedes replication of several enteroviruses through inhibition of Golgi-specific BFA resistance factor 1 (GBF1), a regulator of secretory pathway integrity and transport. GBF1 mediates the GTP exchange of Arf1, which in activated form recruits coatomer protein complex I (COP-I) to Golgi vesicles, a process important in transport between the endoplasmic reticulum and Golgi vesicles. Recently, the drugs AG1478 and Golgicide A (GCA) were put forward as new inhibitors of GBF1. In this study, we investigated the effects of these putative GBF1 inhibitors on secretory pathway function and enterovirus replication. We show that both drugs induced fragmentation of the Golgi vesicles and caused dissociation of Arf1 and COP-I from Golgi membranes, yet they differed in their effect on GBF1 localization. The effects of AG1478, but not those of GCA, could be countered by overexpression of Arf1, indicating a difference in their molecular mechanism of action. Consistent with this idea, we observed that GCA drastically reduced replication of coxsackievirus B3 (CVB3) and other human enterovirus species, whereas AG1478 had no effect at all on enterovirus replication. Time-of-addition studies and analysis of RNA replication using a subgenomic replicon both showed that GCA suppresses RNA replication of CVB3, which could be countered by overexpression of GBF1. These results indicate that, in contrast to AG1478, GCA inhibits CVB3 RNA replication by targeting GBF1. AG1478 and GCA may be valuable tools to further dissect enterovirus replication.
Asunto(s)
Enterovirus Humano B/efectos de los fármacos , Enterovirus Humano B/fisiología , Inhibidores Enzimáticos/farmacología , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Piridinas/farmacología , Quinolinas/farmacología , Tirfostinos/farmacología , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , Cricetinae , Aparato de Golgi/efectos de los fármacos , Humanos , QuinazolinasRESUMEN
The replication of enteroviruses is sensitive to brefeldin A (BFA), an inhibitor of endoplasmic reticulum-to-Golgi network transport that blocks activation of guanine exchange factors (GEFs) of the Arf GTPases. Mammalian cells contain three BFA-sensitive Arf GEFs: GBF1, BIG1, and BIG2. Here, we show that coxsackievirus B3 (CVB3) RNA replication is insensitive to BFA in MDCK cells, which contain a BFA-resistant GBF1 due to mutation M832L. Further evidence for a critical role of GBF1 stems from the observations that viral RNA replication is inhibited upon knockdown of GBF1 by RNA interference and that replication in the presence of BFA is rescued upon overexpression of active, but not inactive, GBF1. Overexpression of Arf proteins or Rab1B, a GTPase that induces GBF1 recruitment to membranes, failed to rescue RNA replication in the presence of BFA. Additionally, the importance of the interaction between enterovirus protein 3A and GBF1 for viral RNA replication was investigated. For this, the rescue from BFA inhibition of wild-type (wt) replicons and that of mutant replicons of both CVB3 and poliovirus (PV) carrying a 3A protein that is impaired in binding GBF1 were compared. The BFA-resistant GBF1-M832L protein efficiently rescued RNA replication of both wt and mutant CVB3 and PV replicons in the presence of BFA. However, another BFA-resistant GBF1 protein, GBF1-A795E, also efficiently rescued RNA replication of the wt replicons, but not that of mutant replicons, in the presence of BFA. In conclusion, this study identifies a critical role for GBF1 in CVB3 RNA replication, but the importance of the 3A-GBF1 interaction requires further study.
Asunto(s)
Enterovirus Humano B/fisiología , Factores de Intercambio de Guanina Nucleótido/fisiología , ARN Viral/biosíntesis , Replicación Viral/fisiología , Factor 1 de Ribosilacion-ADP/fisiología , Animales , Brefeldino A/farmacología , Línea Celular , Enterovirus Humano B/efectos de los fármacos , GTP Fosfohidrolasas/fisiología , Células HeLa , Humanos , Replicación Viral/efectos de los fármacosRESUMEN
Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments.
Asunto(s)
Virus del Dengue/fisiología , Transducción de Señal , Vesículas Transportadoras/ultraestructura , Virión/metabolismo , Internalización del Virus , Aedes , Animales , Células Cultivadas , Vesículas Cubiertas por Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/virología , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Dengue/virología , Difusión , Células HeLa , Humanos , Microscopía Fluorescente/métodos , Transducción de Señal/fisiología , Vesículas Transportadoras/patología , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
The enterovirus genus of the picornavirus family includes a large number of important human pathogens such as poliovirus, coxsackievirus, enterovirus A71, and rhinoviruses. Like all other positive-strand RNA viruses, genome replication of enteroviruses occurs on rearranged membranous structures called replication organelles (ROs). Phosphatidylinositol 4-kinase IIIß (PI4KB) is required by all enteroviruses for RO formation. The enteroviral 3A protein recruits PI4KB to ROs, but the exact mechanism remains elusive. Here, we investigated the role of acyl-coenzyme A binding domain containing 3 (ACBD3) in PI4KB recruitment upon enterovirus replication using ACBD3 knockout (ACBD3KO) cells. ACBD3 knockout impaired replication of representative viruses from four enterovirus species and two rhinovirus species. PI4KB recruitment was not observed in the absence of ACBD3. The lack of ACBD3 also affected the localization of individually expressed 3A, causing 3A to localize to the endoplasmic reticulum instead of the Golgi. Reconstitution of wild-type (wt) ACBD3 restored PI4KB recruitment and 3A localization, while an ACBD3 mutant that cannot bind to PI4KB restored 3A localization, but not virus replication. Consistently, reconstitution of a PI4KB mutant that cannot bind ACBD3 failed to restore virus replication in PI4KBKO cells. Finally, by reconstituting ACBD3 mutants lacking specific domains in ACBD3KO cells, we show that acyl-coenzyme A binding (ACB) and charged-amino-acid region (CAR) domains are dispensable for 3A-mediated PI4KB recruitment and efficient enterovirus replication. Altogether, our data provide new insight into the central role of ACBD3 in recruiting PI4KB by enterovirus 3A and reveal the minimal domains of ACBD3 involved in recruiting PI4KB and supporting enterovirus replication.IMPORTANCE Similar to all other positive-strand RNA viruses, enteroviruses reorganize host cellular membranes for efficient genome replication. A host lipid kinase, PI4KB, plays an important role in this membrane rearrangement. The exact mechanism of how enteroviruses recruit PI4KB was unclear. Here, we revealed a role of a Golgi-residing protein, ACBD3, as a mediator of PI4KB recruitment upon enterovirus replication. ACBD3 is responsible for proper localization of enteroviral 3A proteins in host cells, which is important for 3A to recruit PI4KB. By testing ACBD3 and PI4KB mutants that abrogate the ACBD3-PI4KB interaction, we showed that this interaction is crucial for enterovirus replication. The importance of specific domains of ACBD3 was evaluated for the first time, and the domains that are essential for enterovirus replication were identified. Our findings open up a possibility for targeting ACBD3 or its interaction with enteroviruses as a novel strategy for the development of broad-spectrum antienteroviral drugs.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Enterovirus Humano A/fisiología , Interacciones Huésped-Patógeno , Proteínas de la Membrana/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Humanos , Proteínas de la Membrana/genética , Unión ProteicaRESUMEN
Enterovirus genome replication occurs at virus-induced structures derived from cellular membranes and lipids. However, the origin of these replication organelles (ROs) remains uncertain. Ultrastructural evidence of the membrane donor is lacking, suggesting that the sites of its transition into ROs are rare or fleeting. To overcome this challenge, we combined live-cell imaging and serial block-face scanning electron microscopy of whole cells to capture emerging enterovirus ROs. The first foci of fluorescently labeled viral protein correlated with ROs connected to the endoplasmic reticulum (ER) and preceded the appearance of ROs stemming from the trans-Golgi network. Whole-cell data sets further revealed striking contact regions between ROs and lipid droplets that may represent a route for lipid shuttling to facilitate RO proliferation and genome replication. Our data provide direct evidence that enteroviruses use ER and then Golgi membranes to initiate RO formation, demonstrating the remarkable flexibility with which enteroviruses usurp cellular organelles.IMPORTANCE Enteroviruses are causative agents of a range of human diseases. The replication of these viruses within cells relies on specialized membranous structures termed replication organelles (ROs) that form during infection but whose origin remains elusive. To capture the emergence of enterovirus ROs, we use correlative light and serial block-face scanning electron microscopy, a powerful method to pinpoint rare events in their whole-cell ultrastructural context. RO biogenesis was found to occur first at ER and then at Golgi membranes. Extensive contacts were found between early ROs and lipid droplets (LDs), which likely serve to provide LD-derived lipids required for replication. Together, these data establish the dual origin of enterovirus ROs and the chronology of their biogenesis at different supporting cellular membranes.
Asunto(s)
Retículo Endoplásmico/ultraestructura , Enterovirus/fisiología , Aparato de Golgi/ultraestructura , Microscopía Electrónica de Rastreo , Replicación Viral , Animales , Chlorocebus aethiops , Retículo Endoplásmico/virología , Infecciones por Enterovirus , Aparato de Golgi/virología , Procesamiento de Imagen Asistido por Computador , Gotas Lipídicas/ultraestructura , Células VeroRESUMEN
Enteroviruses (e.g., poliovirus, enterovirus-A71, coxsackievirus, enterovirus-D68, rhinovirus) include many human pathogens causative of various mild and more severe diseases, especially in young children. Unfortunately, antiviral drugs to treat enterovirus infections have not been approved yet. Over the past decades, several direct-acting inhibitors have been developed, including capsid binders, which block virus entry, and inhibitors of viral enzymes required for genome replication. Capsid binders and protease inhibitors have been clinically evaluated, but failed due to limited efficacy or toxicity issues. As an alternative approach, host-targeting inhibitors with potential broad-spectrum activity have been identified. Furthermore, drug repurposing screens have recently uncovered promising new inhibitors with disparate viral and host targets. Together, these findings raise hope for the development of (broad-range) anti-enteroviral drugs.
Asunto(s)
Antivirales/uso terapéutico , Infecciones por Enterovirus/tratamiento farmacológico , Enterovirus/efectos de los fármacos , Animales , Antivirales/administración & dosificación , Cápside/efectos de los fármacos , Ciclofilinas/uso terapéutico , Reposicionamiento de Medicamentos , Humanos , Ratones , Poliovirus/efectos de los fármacos , Inhibidores de Proteasas/uso terapéutico , Rhinovirus/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Enteroviruses (e.g. poliovirus, coxsackievirus, and rhinovirus) require several host factors for genome replication. Among these host factors are phosphatidylinositol-4-kinase IIIß (PI4KB) and oxysterol binding protein (OSBP). Enterovirus mutants resistant to inhibitors of PI4KB and OSBP were previously isolated, which demonstrated a role of single substitutions in the non-structural 3A protein in conferring resistance. Besides the 3A substitutions (i.e., 3A-I54F and 3A-H57Y) in coxsackievirus B3 (CVB3), substitution N2D in 2C was identified in each of the PI4KB-inhibitor resistant CVB3 pools, but its possible benefit has not been investigated yet. In this study, we set out to investigate the possible role of 2C-N2D in the resistance to PI4KB and OSBP inhibition. We show that 2C-N2D by itself did not confer any resistance to inhibitors of PI4KB and OSBP. However, the double mutant (i.e., 2C-N2D/3A-H57Y) showed better replication than the 3A-H57Y single mutant in the presence of inhibitors. Growing evidence suggests that alterations in lipid homeostasis affect the proteolytic processing of the poliovirus polyprotein. Therefore, we studied the effect of PI4KB or OSBP inhibition on proteolytic processing of the CVB3 polyprotein during infection as well as in a replication-independent system. We show that both PI4KB and OSBP inhibitors specifically affected the cleavage at the 3A-3B junction, and that mutation 3A-H57Y recovered impaired proteolytic processing at this junction. Although 2C-N2D enhanced replication of the 3A-H57Y single mutant, we did not detect additional effects of this substitution on polyprotein processing, which leaves the mechanism of how 2C-N2D contributes to the resistance to be revealed.
Asunto(s)
Antivirales/farmacología , Infecciones por Coxsackievirus/virología , Farmacorresistencia Viral/genética , Enterovirus Humano B/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptores de Esteroides/antagonistas & inhibidores , Sustitución de Aminoácidos , Infecciones por Coxsackievirus/tratamiento farmacológico , Infecciones por Coxsackievirus/enzimología , Farmacorresistencia Viral/efectos de los fármacos , Enterovirus Humano B/genética , Humanos , Poliproteínas/metabolismo , ARN Viral/biosíntesis , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
The genus Enterovirus (e.g. poliovirus, coxsackievirus, rhinovirus) of the Picornaviridae family of positive-strand RNA viruses includes many important pathogens linked to a range of acute and chronic diseases for which no approved antiviral therapy is available. Targeting a step in the life cycle that is highly conserved provides an attractive strategy for developing broad-range inhibitors of enterovirus infection. A step that is currently explored as a target for the development of antivirals is the formation of replication organelles, which support replication of the viral genome. To build replication organelles, enteroviruses rewire cellular machinery and hijack lipid homeostasis pathways. For example, enteroviruses exploit the PI4KIIIß-PI4P-OSBP pathway to direct cholesterol to replication organelles. Here, we uncover that TTP-8307, a known enterovirus replication inhibitor, acts through the PI4KIIIß-PI4P-OSBP pathway by directly inhibiting OSBP activity. However, despite a shared mechanism of TTP-8307 with established OSBP inhibitors (itraconazole and OSW-1), we identify a number of notable differences between these compounds. The antiviral activity of TTP-8307 extends to other viruses that require OSBP, namely the picornavirus encephalomyocarditis virus and the flavivirus hepatitis C virus.
Asunto(s)
Antivirales/farmacología , Benzamidas/farmacología , Enterovirus/efectos de los fármacos , Imidazoles/farmacología , Receptores de Esteroides/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Colestenonas/farmacología , Inhibidores del Citocromo P-450 CYP3A/farmacología , Genoma Viral/efectos de los fármacos , Células HeLa , Humanos , Itraconazol/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/efectos de los fármacos , Poliovirus/efectos de los fármacos , Receptores de Esteroides/metabolismo , Rhinovirus/efectos de los fármacos , Saponinas/farmacologíaRESUMEN
Enteroviruses reorganize cellular endomembranes into replication organelles (ROs) for genome replication. Although enterovirus replication depends on phosphatidylinositol 4-kinase type IIIß (PI4KB), its role, and that of its product, phosphatidylinositol 4-phosphate (PI4P), is only partially understood. Exploiting a mutant coxsackievirus resistant to PI4KB inhibition, we show that PI4KB activity has distinct functions both in proteolytic processing of the viral polyprotein and in RO biogenesis. The escape mutation rectifies a proteolytic processing defect imposed by PI4KB inhibition, pointing to a possible escape mechanism. Remarkably, under PI4KB inhibition, the mutant virus could replicate its genome in the absence of ROs, using instead the Golgi apparatus. This impaired RO biogenesis provided an opportunity to investigate the proposed role of ROs in shielding enteroviral RNA from cellular sensors. Neither accelerated sensing of viral RNA nor enhanced innate immune responses was observed. Together, our findings challenge the notion that ROs are indispensable for enterovirus genome replication and immune evasion.
Asunto(s)
Enterovirus/genética , Enterovirus/fisiología , Genoma Viral/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Orgánulos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , ARN Viral/biosíntesis , ARN Viral/genética , Replicación Viral , Antivirales/metabolismo , Enterovirus/crecimiento & desarrollo , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Células HeLa , Humanos , Proteolisis , Proteínas Virales/metabolismoRESUMEN
All viruses that carry a positive-sense RNA genome (+RNA), such as picornaviruses, hepatitis C virus, dengue virus, and SARS- and MERS-coronavirus, confiscate intracellular membranes of the host cell to generate new compartments (i.e., replication organelles) for amplification of their genome. Replication organelles (ROs) are membranous structures that not only harbor viral proteins but also contain a specific array of hijacked host factors that create a unique lipid microenvironment optimal for genome replication. While some lipids may be locally synthesized de novo, other lipids are shuttled towards ROs. In picornavirus-infected cells, lipids are exchanged at membrane contact sites between ROs and other organelles. In this paper, we review recent advances in our understanding of how picornaviruses exploit host membrane contact site machinery to generate ROs, a mechanism that is used by some other +RNA viruses as well.