RESUMEN
Mosquito-borne flaviviruses such as dengue (DENV) and Zika (ZIKV) cause hundreds of millions of infections annually. The single-stranded RNA genome of flaviviruses is translated into a polyprotein, which is cleaved equally into individual functional proteins. While structural proteins are packaged into progeny virions and released, most of the nonstructural proteins remain intracellular and could become cytotoxic if accumulated over time. However, the mechanism by which nonstructural proteins are maintained at the levels optimal for cellular fitness and viral replication remains unknown. Here, we identified that the ubiquitin E3 ligase HRD1 is essential for flaviviruses infections in both mammalian hosts and mosquitoes. HRD1 directly interacts with flavivirus NS4A and ubiquitylates a conserved lysine residue for ER-associated degradation. This mechanism avoids excessive accumulation of NS4A, which otherwise interrupts the expression of processed flavivirus proteins in the ER. Furthermore, a small-molecule inhibitor of HRD1 named LS-102 effectively interrupts DENV2 infection in both mice and Aedes aegypti mosquitoes, and significantly disturbs DENV transmission from the infected hosts to mosquitoes owing to reduced viremia. Taken together, this study demonstrates that flaviviruses have evolved a sophisticated mechanism to exploit the ubiquitination system to balance the homeostasis of viral proteins for their own advantage and provides a potential therapeutic target to interrupt flavivirus infection and transmission.
Asunto(s)
Aedes , Infecciones por Flavivirus , Flavivirus , Infección por el Virus Zika , Virus Zika , Animales , Ratones , Flavivirus/genética , Virus Zika/genética , Ubiquitina/metabolismo , Ligasas/metabolismo , Proteínas Virales/metabolismo , MamíferosRESUMEN
Mitochondria are energy producers in cells, which can affect viral replication by regulating the host innate immune signaling pathways, and the changes in their biological functions are inextricably linked the viral life cycle. In this study, we screened a library of 382 mitochondria-targeted compounds and identified the antiviral inhibitors of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo synthesis pathway of pyrimidine ribonucleotides, against classical swine fever virus (CSFV). Our data showed that the inhibitors interfered with viral RNA synthesis in a dose-dependent manner, with half-maximal effective concentrations (EC50) ranging from 0.975 to 26.635 nM. Remarkably, DHODH inhibitors obstructed CSFV replication by enhancing the innate immune response including the TBK1-IRF3-STAT1 and NF-κB signaling pathways. Furthermore, the data from a series of compound addition and supplementation trials indicated that DHODH inhibitors also inhibited CSFV replication by blocking the de novo pyrimidine synthesis. Remarkably, DHODH knockdown demonstrated that it was essential for CSFV replication. Mechanistically, confocal microscopy and immunoprecipitation assays showed that the non-structural protein 4A (NS4A) recruited and interacted with DHODH in the perinuclear. Notably, NS4A enhanced the DHODH activity and promoted the generation of UMP for efficient viral replication. Structurally, the amino acids 65-229 of DHODH and the amino acids 25-40 of NS4A were pivotal for this interaction. Taken together, our findings highlight the critical role of DHODH in the CSFV life cycle and offer a potential antiviral target for the development of novel therapeutics against CSF. IMPORTANCE: Classical swine fever remains one of the most economically important viral diseases of domestic pigs and wild boar worldwide. dihydroorotate dehydrogenase (DHODH) inhibitors have been shown to suppress the replication of several viruses in vitro and in vivo, but the effects on Pestivirus remain unknown. In this study, three specific DHODH inhibitors, including DHODH-IN-16, BAY-2402234, and Brequinar were found to strongly suppress classical swine fever virus (CSFV) replication. These inhibitors target the host DHODH, depleting the pyrimidine nucleotide pool to exert their antiviral effects. Intriguingly, we observed that the non-structural protein 4A of CSFV induced DHODH to accumulate around the nucleus in conjunction with mitochondria. Moreover, NS4A exhibited a strong interaction with DHODH, enhancing its activity to promote efficient CSFV replication. In conclusion, our findings enhance the understanding of the pyrimidine synthesis in CSFV infection and expand the novel functions of CSFV NS4A in viral replication, providing a reference for further exploration of antiviral targets against CSFV.
Asunto(s)
Antivirales , Virus de la Fiebre Porcina Clásica , Dihidroorotato Deshidrogenasa , Proteínas no Estructurales Virales , Replicación Viral , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Línea Celular , Peste Porcina Clásica/tratamiento farmacológico , Peste Porcina Clásica/inmunología , Peste Porcina Clásica/metabolismo , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/efectos de los fármacos , Virus de la Fiebre Porcina Clásica/crecimiento & desarrollo , Virus de la Fiebre Porcina Clásica/inmunología , Virus de la Fiebre Porcina Clásica/metabolismo , Dihidroorotato Deshidrogenasa/metabolismo , Relación Dosis-Respuesta a Droga , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Inmunoprecipitación , Microscopía Confocal , Mitocondrias/enzimología , Mitocondrias/metabolismo , ARN Viral/biosíntesis , Transducción de Señal/efectos de los fármacos , Porcinos/virología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
The non-structural (NS) proteins of the Flaviviridae members play a dual role in genome replication and virion morphogenesis. For pestiviruses, like bovine viral diarrhea virus, the NS2-3 region and its processing by the NS2 autoprotease is of particular importance. While uncleaved NS2-3 in complex with NS4A is essential for virion assembly, it cannot replace free NS3/4A in the viral replicase. Furthermore, surface interactions between NS3 and the C-terminal cytosolic domain of NS4A were shown to serve as a molecular switch between RNA replication and virion morphogenesis. To further characterize the functionality of NS4A, we performed an alanine-scanning mutagenesis of two NS4A regions, a short highly conserved cytoplasmic linker downstream of the transmembrane domain and the C-terminal domain. NS4A residues critical for polyprotein processing, RNA replication, and/or virion morphogenesis were identified. Three double-alanine mutants, two in the linker region and one close to the C-terminus of NS4A, showed a selective effect on virion assembly. All three packaging defective mutants could be rescued by a selected set of two second-site mutations, located in NS2 and NS3, respectively. This phenotype was additionally confirmed by complementation studies providing the NS2-3/4A packaging molecules containing the rescue mutations in trans. This indicates that the linker region and the cytosolic C-terminal part of NS4A are critical for the formation of protein complexes required for virion morphogenesis. The ability of the identified sets of second-site mutations in NS2-3 to compensate for diverse NS4A defects highlights a surprising functional flexibility for pestiviral NS proteins. IMPORTANCE Positive-strand RNA viruses have a limited coding capacity due to their rather small genome size. To overcome this constraint, viral proteins often exhibit multiple functions that come into play at different stages during the viral replication cycle. The molecular basis for this multifunctionality is often unknown. For the bovine viral diarrhea virus, the non-structural protein (NS) 4A functions as an NS3 protease cofactor, a replicase building block, and a component in virion morphogenesis. Here, we identified the critical amino acids of its C-terminal cytosolic region involved in those processes and show that second-site mutations in NS2 and NS3 can compensate for diverse NS4A defects in virion morphogenesis. The ability to evolve alternative functional solutions by gain-of-function mutations highlights the astounding plasticity of the pestiviral system.
Asunto(s)
Virus de la Diarrea Viral Bovina , Proteínas no Estructurales Virales , Replicación Viral , Humanos , Virus de la Diarrea Viral Bovina/genética , Hepacivirus/metabolismo , Mutación , Proteínas no Estructurales Virales/metabolismo , Ensamble de Virus , Línea Celular , AnimalesRESUMEN
RNA interference (RNAi) is a significant posttranscriptional gene silencing mechanism and can function as an antiviral immunity in eukaryotes. However, numerous viruses can evade this antiviral RNAi by encoding viral suppressors of RNA silencing (VSRs). Classical swine fever virus (CSFV), belonging to the genus Pestivirus, is the cause of classical swine fever (CSF), which has an enormous impact on animal health and the pig industry. Notably, little is known about how Pestivirus blocks RNAi in their host. In this paper, we uncovered that CSFV NS4A protein can antagonize RNAi efficiently in mammalian cells by binding to double-stranded RNA and small interfering RNA. In addition, the VSR activity of CSFV NS4A was conserved among Pestivirus. Furthermore, the replication of VSR-deficient CSFV was attenuated but could be restored by the deficiency of RNAi in mammalian cells. In conclusion, our studies uncovered that CSFV NS4A is a novel VSR that suppresses RNAi in mammalian cells and shed new light on knowledge about CSFV and other Pestivirus. IMPORTANCE It is well known that RNAi is an important posttranscriptional gene silencing mechanism that is also involved in the antiviral response in mammalian cells. While numerous viruses have evolved to block this antiviral immunity by encoding VSRs. Our data demonstrated that the NS4A protein of CSFV exhibited a potent VSR activity through binding to dsRNA and siRNA in the context of CSFV infection in mammalian cells, which are a conservative feature among Pestivirus. In addition, the replication of VSR-deficient CSFV was attenuated but could be restored by the deficiency of RNAi, providing a theoretical basis for the development of other important attenuated Pestivirus vaccines.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Pestivirus , Proteínas no Estructurales Virales/metabolismo , Animales , Línea Celular , Peste Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/genética , Mamíferos/virología , Pestivirus/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Porcinos , Replicación ViralRESUMEN
Whole-genome phylogenetic analysis, the most suitable strategy for subtyping bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2, is not feasible for many laboratories. Consequently, BVDV isolates/strains have been frequently subtyped based on analysis of single genomic regions, mainly the 5' untranslated region (UTR). This approach, however, may lead to inaccurate and/or poorly statistically supported viral classification. Herein, we describe novel primer sets whose amplicons may be easily sequenced and used for BVDV subtyping. Initially, genomic regions previously described as the most suitable targets for BVDV subtyping were analyzed for design of high-coverage primers. The putative amplicons were analyzed in silico for their suitability to reproduce the phylogenetic classification of 118 BVDV-1 and 88 BVDV-2 complete/near-complete genomes (CNCGs) (GenBank). This analysis was also performed considering the region amplifiable by primers HCV90-368, 324-326 and BP189-389 (5'UTR), which have been used for BVDV diagnosis and/or classification. After confirming the agreement between the analyses of our primers' amplicon versus the CNCGs, we optimized the RT-PCRs and evaluated their performance for amplification of BVDV isolates/strains (n = 35 for BVDV-1; n = 33 for BVDV-2). Among the potential targets for BVDV subtyping, we designed high-coverage primers for NS3-NS4A (BVDV-1) (526 bp amplicon) and NS5B (BVDV-2) (728 bp). The classification based on these regions fully reproduced the subtyping of all CNCGs. On the other hand, subtyping based on the putative amplicons from primers HCV90-368, 324-326 and BP189-389 showed disagreements in relation the CNCG analysis. The NS3-NS4A and NS5B primers also allowed the amplification of all BVDV isolates/strains tested. Finally, we suggest the use of these primers in future phylogenetic and epidemiological studies of BVDVs.
Asunto(s)
Diarrea Mucosa Bovina Viral , Virus de la Diarrea Viral Bovina Tipo 1 , Virus de la Diarrea Viral Bovina Tipo 2 , Virus de la Diarrea Viral Bovina , Animales , Bovinos , Virus de la Diarrea Viral Bovina Tipo 1/genética , Virus de la Diarrea Viral Bovina Tipo 2/genética , Filogenia , Genómica , Regiones no Traducidas 5'/genética , Virus de la Diarrea Viral Bovina/genéticaRESUMEN
Zika virus (ZIKV) is a mosquito-borne pathogen classified by the World Health Organization (WHO) as a public health emergency of international concern in 2016, and it is still identified as a priority disease. Although most infected individuals are asymptomatic or show mild symptoms, a risk of neurologic complications is associated with infection in adults. Additionally, infection during pregnancy is directly linked to microcephaly and other congenital malformations. Since there are no currently available vaccines or approved therapeutics for this virus, there is a critical unmet need in developing treatments to prevent future ZIKV outbreaks. Toward this end, we performed a large-scale cell-based high-content screen of 51,520 chemical compounds to identify potential antiviral drug candidates. The compound (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) was found to inhibit replication of multiple ZIKV strains and in different cell systems. SBI-0090799 did not affect viral entry or RNA translation but suppressed RNA replication by preventing the formation of the membranous replication compartment. Selection of drug-resistant viruses identified single-amino-acid substitutions in the N-terminal region of nonstructural protein NS4A, arguing this is the likely drug target. These resistance mutations rescued viral RNA replication and restored the formation of the membranous replication compartment. This mechanism of action is similar to clinically approved NS5A inhibitors for hepatitis C virus (HCV). Taken together, SBI-0090799 represents a promising lead candidate for the development of an antiviral treatment against ZIKV infection for the mitigation of severe complications and potential resurgent outbreaks of the virus. IMPORTANCE This study describes the elucidation of (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) as a selective and potent inhibitor of Zika virus (ZIKV) replication using a high-throughput screening approach. Mapping and resistance studies, supported by electron microscopy observations, indicate that the small molecule is functioning through inhibition of NS4A-mediated formation of ZIKV replication compartments in the endoplasmic reticulum (ER). Intriguingly, this defines a novel nonenzymatic target and chemical matter for the development of a new class of ZIKV antivirals. Moreover, chemical modulation affecting this nonstructural protein mirrors the identification and development of hepatitis C virus (HCV) NS5A inhibitor daclatasvir and its derivatives, similarly interfering with the formation of the viral replication compartment and also targeting a protein with no enzymatic activity, which have been part of a curative strategy for HCV.
Asunto(s)
Antivirales/farmacología , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Replicación Viral/efectos de los fármacos , Infección por el Virus Zika/tratamiento farmacológico , Virus Zika/efectos de los fármacos , Animales , Astrocitos , Chlorocebus aethiops , Células Dendríticas , Células HEK293 , Humanos , Cultivo Primario de Células , Células Vero , Compartimentos de Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Classical swine fever (CSF) is a severe disease of pigs that results in huge economic losses worldwide and is caused by classical swine fever virus (CSFV). CSFV nonstructural protein 4 A (NS4A) plays a crucial role in infectious CSFV particle formation. However, the function of NS4A during CSFV infection is not well understood. RESULTS: In this study, we used RNA-seq to investigate the functional role of CSFV NS4A in PK-15 cells. A total of 3893 differentially expressed genes (DEGs) were identified in PK-15 cells expressing NS4A compared to cells expressing the empty vector (NC). Twelve DEGs were selected and further verified by RTâqPCR. GO and KEGG enrichment analyses revealed that these DEGs were associated with multiple biological functions, including cell adhesion, apoptosis, host defence response, the inflammatory response, the immune response, and autophagy. Interestingly, some genes associated with host immune defence and inflammatory response were downregulated, and some genes associated with host apoptosis and autophagy were upregulated. CONCLUSION: CSFV NS4A inhibits the innate immune response, and suppresses the expression of important genes associated with defence response to viruses and inflammatory response, and regulates cell adhesion, apoptosis and autophagy.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Enfermedades de los Porcinos , Porcinos , Animales , Virus de la Fiebre Porcina Clásica/genética , Replicación Viral/fisiología , Línea Celular , Perfilación de la Expresión Génica/veterinariaRESUMEN
The dengue virus (DENV) replication complex is made up of its non-structural (NS) proteins and yet-to-be identified host proteins, but the molecular interactions between these proteins are not fully elucidated. In this work, we sought to uncover the interactions between DENV NS1 and its fellow NS proteins using a yeast two-hybrid (Y2H) approach, and found that domain II of NS1 binds to an N-terminal cytoplasmic fragment of NS4A. Mutations in amino acid residues 41 and 43 in this cytoplasmic region of NS4A disrupted the interaction between NS1 and the NS4A-2K-4B precursor protein. When the NS4A Y41F mutation was introduced into the context of the virus via a DENV2 infectious clone, this mutant virus exhibited impaired viral fitness and decreased infectious virus production. The NS4A Y41F mutant virus triggered a significantly muted transcriptional activation of interferon-stimulated genes compared to wild-type virus that is independent of NS4A's ability to antagonize type I interferon signalling. Taken together, we have identified a link between DENV NS1 and the cytoplasmic domain in NS4A that is important for its cellular and viral functions.
Asunto(s)
Virus del Dengue/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Virus del Dengue/fisiología , Aptitud Genética , Humanos , Interferón Tipo I/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Técnicas del Sistema de Dos Híbridos , Proteínas no Estructurales Virales/química , Virión/metabolismo , Replicación ViralRESUMEN
The hepatitis C virus (HCV) NS3-NS4A protease complex is required for viral replication and is the major viral innate immune evasion factor. NS3-NS4A evades antiviral innate immunity by inactivating several proteins, including MAVS, the signaling adaptor for RIG-I and MDA5, and Riplet, an E3 ubiquitin ligase that activates RIG-I. Here, we identified a Tyr-16-Phe (Y16F) change in the NS4A transmembrane domain that prevents NS3-NS4A targeting of Riplet but not MAVS. This Y16F substitution reduces HCV replication in Huh7 cells, but not in Huh-7.5 cells, known to lack RIG-I signaling. Surprisingly, deletion of RIG-I in Huh7 cells did not restore Y16F viral replication. Rather, we found that Huh-7.5 cells lack Riplet expression and that the addition of Riplet to these cells reduced HCV Y16F replication, whereas the addition of Riplet lacking the RING domain restored HCV Y16F replication. In addition, TBK1 inhibition or IRF3 deletion in Huh7 cells was sufficient to restore HCV Y16F replication, and the Y16F protease lacked the ability to prevent IRF3 activation or interferon induction. Taken together, these data reveal that the NS4A Y16 residue regulates a noncanonical Riplet-TBK1-IRF3-dependent, but RIG-I-MAVS-independent, signaling pathway that limits HCV infection.IMPORTANCE The HCV NS3-NS4A protease complex facilitates viral replication by cleaving and inactivating the antiviral innate immune signaling proteins MAVS and Riplet, which are essential for RIG-I activation. NS3-NS4A therefore prevents IRF3 activation and interferon induction during HCV infection. Here, we uncover an amino acid residue within the NS4A transmembrane domain that is essential for inactivation of Riplet but does not affect MAVS cleavage by NS3-NS4A. Our study reveals that Riplet is involved in a RIG-I- and MAVS-independent signaling pathway that activates IRF3 and that this pathway is normally inactivated by NS3-NS4A during HCV infection. Our study selectively uncouples these distinct regulatory mechanisms within NS3-NS4A and defines a new role for Riplet in the antiviral response to HCV. Since Riplet is known to be inhibited by other RNA viruses, such as such influenza A virus, this innate immune signaling pathway may also be important in controlling other RNA virus infections.
Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepatitis C/virología , Serina Proteasas/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas no Estructurales Virales/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Línea Celular Tumoral , Proteína 58 DEAD Box/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Hepatocitos/virología , Humanos , Evasión Inmune , Inmunidad Innata , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Helicasa Inducida por Interferón IFIH1/metabolismo , Proteínas Serina-Treonina Quinasas , Receptores Inmunológicos , Replicación ViralRESUMEN
Among Flaviviridae, in West Nile virus (WNV) and Hepatitis C virus (HCV), the non-structural protein NS4A modulates the NTPase activity of viral helicases during nucleic acid unwinding through its N-terminal disordered residues (1-50). In HCV, the acidic NS4A also serves as a cofactor for regulating the NS3 protease activity. However, in case of Zika virus (ZIKV), the role of NS4A and its impact on activities of NS3 helicase and protease is not known. In order to elucidate the role of NS4A, we checked the NTPase activity of NS3 helicase and protease activity of NS3 protease in presence of NS4A N-terminal region (residues 1-48) peptide. Our enzyme kinetics results together with binding experiment clearly demonstrate that NS3 helicase in presence of NS4A peptide increased the rate of ATP hydrolysis whereas the protease activity of NS3 protease was not affected. Therefore, like WNV and HCV, our results establish a role of ZIKV NS4A being a cofactor for modulating the NTPase activity of ZIKV NS3 helicase.
Asunto(s)
Nucleósido-Trifosfatasa/química , ARN Helicasas/química , Serina Endopeptidasas/química , Proteínas Virales/química , Virus Zika/enzimología , Coenzimas , Nucleósido-Trifosfatasa/genética , Dominios Proteicos , ARN Helicasas/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Virus Zika/genéticaRESUMEN
The NS4A protein of dengue virus (DENV) has a cytosolic N terminus and four transmembrane domains. NS4A participates in RNA replication and the host antiviral response. However, the roles of amino acid residues within the N-terminus of NS4A during the life cycle of DENV are not clear. Here we explore the function of DENV NS4A by introducing a series of alanine substitutions into the N-terminus of NS4A in the context of a DENV infectious clone or subgenomic replicon. Nine of 17 NS4A mutants displayed a lethal phenotype due to the impairment of RNA replication. M2 and M14 displayed a more than 10â000-fold reduction in viral yields and moderate defects in viral replication by a replicon assay. Sequencing analyses of pseudorevertant viruses derived from M2 and M14 viruses revealed one consensus reversion mutation, A21V, within NS4A. The A21V mutation apparently rescued viral RNA replication in the M2 and M14 mutants although not to wild-type (WT) levels but resulted in 100- and 1000-fold lower titres than that of the WT, respectively. M2 Rev1 (M2+A21V) and M14 Rev1 (M14+A21V) mutants displayed phenotypes of smaller plaque size and WT-like assembly/secretion by a transpackaging assay. A defect in the virus-induced cytopathic effect (CPE) was observed in HEK-293 cells infected with either M2 Rev1 or M14 Rev1 mutant virus by MitoCapture staining, cell proliferation and lactate dehydrogenase release assays. In conclusion, the results revealed the essential roles of the N-terminal NS4A in both RNA replication and virus-induced CPE. Intramolecular interactions in the N-terminus of NS4A were implicated.
Asunto(s)
Efecto Citopatogénico Viral , Virus del Dengue/metabolismo , Dengue/virología , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Virus del Dengue/genética , Virus del Dengue/fisiología , Células HEK293 , Humanos , Mutagénesis , Dominios Proteicos , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
Dengue virus (DENV) non-structural (NS) 4A is a membrane protein essential for viral replication. The N-terminal region of NS4A contains several helices interacting with the cell membrane and the C-terminal region consists of three potential transmembrane regions. The secondary structure of the intact NS4A is not known as the previous structural studies were carried out on its fragments. In this study, we purified the full-length NS4A of DENV serotype 4 into dodecylphosphocholine (DPC) micelles. Solution NMR studies reveal that NS4A contains six helices in DPC micelles. The N-terminal three helices are amphipathic and interact with the membrane. The C-terminal three helices are embedded in micelles. Our results suggest that NS4A contains three transmembrane helices. Our studies provide for the first time structural information of the intact NS4A of DENV and will be useful for further understanding its role in viral replication.
Asunto(s)
Proteínas de la Membrana/química , Micelas , Fosforilcolina/análogos & derivados , Estructura Secundaria de Proteína , Proteínas no Estructurales Virales/química , Secuencia de Aminoácidos , Virus del Dengue/genética , Virus del Dengue/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Espectroscopía de Resonancia Magnética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Fosforilcolina/química , Fosforilcolina/metabolismo , Unión Proteica , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Producing virus at high yield is critically important for development of whole virion inactivated vaccines or live attenuated vaccines. Most dengue virus (DENV) clinical isolates, however, replicate at low levels in cultured cells, which limits their use for vaccine development. The present study examined differences between low-replicating DENV clinical isolates and high-replicating laboratory strains with the aim of engineering high-yield DENV clinical isolates. Construction of a series of recombinant chimeric viruses derived from a high-replicating laboratory DENV type 4 (DENV-4) H241 strain and a clinical isolate revealed that the NS3-NS4B region of H241 conferred a replication advantage in cultured cells. Furthermore, northern blot analysis revealed that this advantage was due to more efficient synthesis of viral RNA. Importantly, replacement of the NS3-NS4B region of H241 did not increase virulence in mice, suggesting that viral production can be increased safely. This study provided information that will facilitate engineering of safe and high-yield viruses that can be used for vaccine development.
Asunto(s)
Técnicas de Cultivo Celular por Lotes/métodos , Virus del Dengue/crecimiento & desarrollo , Virus del Dengue/genética , Mejoramiento Genético/métodos , Carga Viral/genética , Proteínas no Estructurales Virales/metabolismo , Virulencia/fisiología , Recombinación Genética/genética , Carga Viral/fisiología , Proteínas no Estructurales Virales/genéticaRESUMEN
Zika virus (ZIKV), a member of the Flaviviridae family, has recently emerged as an important human pathogen with increasing economic and health impact worldwide. Because of its teratogenic nature and association with the serious neurological condition Guillain-Barré syndrome, a tremendous amount of effort has focused on understanding ZIKV pathogenesis. To gain further insights into ZIKV interaction with host cells, we investigated how this pathogen affects stress response pathways. While ZIKV infection induces stress signaling that leads to phosphorylation of eIF2α and cellular translational arrest, stress granule (SG) formation was inhibited. Further analysis revealed that the viral proteins NS3 and NS4A are linked to translational repression, whereas expression of the capsid protein, NS3/NS2B-3, and NS4A interfered with SG formation. Some, but not all, flavivirus capsid proteins also blocked SG assembly, indicating differential interactions between flaviviruses and SG biogenesis pathways. Depletion of the SG components G3BP1, TIAR, and Caprin-1, but not TIA-1, reduced ZIKV replication. Both G3BP1 and Caprin-1 formed complexes with capsid, whereas viral genomic RNA stably interacted with G3BP1 during ZIKV infection. Taken together, these results are consistent with a scenario in which ZIKV uses multiple viral components to hijack key SG proteins to benefit viral replication.IMPORTANCE There is a pressing need to understand ZIKV pathogenesis in order to advance the development of vaccines and therapeutics. The cellular stress response constitutes one of the first lines of defense against viral infection; therefore, understanding how ZIKV evades this antiviral system will provide key insights into ZIKV biology and potentially pathogenesis. Here, we show that ZIKV induces the stress response through activation of the UPR (unfolded protein response) and PKR (protein kinase R), leading to host translational arrest, a process likely mediated by the viral proteins NS3 and NS4A. Despite the activation of translational shutoff, formation of SG is strongly inhibited by the virus. Specifically, ZIKV hijacks the core SG proteins G3BP1, TIAR, and Caprin-1 to facilitate viral replication, resulting in impaired SG assembly. This process is potentially facilitated by the interactions of the viral RNA with G3BP1 as well as the viral capsid protein with G3BP1 and Caprin-1. Interestingly, expression of capsid proteins from several other flaviviruses also inhibited SG formation. Taken together, the present study provides novel insights into how ZIKV modulates cellular stress response pathways during replication.
RESUMEN
Dengue virus (DENV) infection is a growing public health threat with more than one-third of the world's population at risk. Non-structural protein 4A (NS4A), one of the least characterized viral proteins, is a highly hydrophobic transmembrane protein thought to induce the membrane alterations that harbor the viral replication complex. The NS4A N-terminal (amino acids 1-48), has been proposed to contain an amphipathic α-helix (AH). Mutations (L6E; M10E) designed to reduce the amphipathic character of the predicted AH, abolished viral replication and reduced NS4A oligomerization. Nuclear magnetic resonance (NMR) spectroscopy was used to characterize the N-terminal cytoplasmic region (amino acids 1-48) of both wild type and mutant NS4A in the presence of SDS micelles. Binding of the two N-terminal NS4A peptides to liposomes was studied as a function of membrane curvature and lipid composition. The NS4A N-terminal was found to contain two AHs separated by a non-helical linker. The above mentioned mutations did not significantly affect the helical secondary structure of this domain. However, they reduced the affinity of the N-terminal NS4A domain for lipid membranes. Binding of wild type NS4A(1-48) to liposomes is highly dependent on membrane curvature.
Asunto(s)
Virus del Dengue/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas no Estructurales Virales/metabolismo , Dicroismo Circular , Virus del Dengue/crecimiento & desarrollo , Liposomas , Lípidos de la Membrana/química , Micelas , Mutación , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
Dengue virus (DENV) is a mosquito-borne flavivirus that causes the most prevalent diseases in tropical and subtropical regions. DENV utilizes host factors to facilitate its replication, while host cells intend to restrain virus replication. NS4A of DENV has been implicated to play a crucial role during viral replication. To identify more cellular proteins that are recruited by NS4A, we carried out a tandem affinity purification assay. The mass spectrometry data revealed that human eukaryotic initiation factor 4AI (eIF4AI) was one of potential NS4A-interacting partners. Co-immunoprecipitation data confirmed the interaction between NS4A and eIF4AI, and both the N-terminal ATP-binding domain and C-terminal helicase domain of eIF4AI were involved in their association. Functionally, silencing of eIF4AI by RNAi significantly increased the replication level of DENV1, DENV2 and DENV3. And knockdown of eIF4AI markedly attenuated the phosphorylation of protein kinase regulated by dsRNA-activated (PKR) and eIF2É induced by DENV2 infection. Collectively, these data suggested that a potential role of NS4A in antagonizing host antiviral defense is by recruiting eIF4AI and escaping the translation inhibition mediated by PKR.
Asunto(s)
Virus del Dengue/fisiología , Factor 4A Eucariótico de Iniciación/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/virología , Proteínas no Estructurales Virales/metabolismo , Activación Viral/fisiología , Replicación Viral/fisiología , Línea Celular Tumoral , Clonación Molecular , HumanosRESUMEN
Hepatitis C virus (HCV) chronically infects almost 2% of world's population. Chronic infection can lead to liver failure and hepatocellular carcinoma (HCC). Approximately 10% of the Pakistani population is infected with HCV and type 3 is the most prevalent genotype with 75-90% prevalence. In this study we have developed transiently expressing cell culture based system for the expression of HCV non-structural NS3, NS3-4A and NS4A proteins of genotype 3a. HCV non-structural genes NS3, NS3-4A and NS4A were cloned in to pFLAG-CMV2 and pEGFP-C1vectors. All vectors were transfected separately to Huh-7 cells and their protein expression was analyzed by Western blot and immunofluorescence. All proteins were expressed correctly and in the transfection we have obtained 42-70% efficiency for all clones. This system can be used for the development of novel antiviral strategies to inhibit the viral replication, to study apoptosis pathways induced by HCV, for the evaluation of vaccine candidates and also to study the role of HCV different signaling pathways.
Asunto(s)
Proteínas no Estructurales Virales/genética , Western Blotting , Fluorescencia , Técnica del Anticuerpo Fluorescente , Genotipo , Hepacivirus/clasificación , Hepacivirus/genética , Proteínas no Estructurales Virales/análisisRESUMEN
IMPORTANCE: CSFV infection in pigs causes persistent high fever, hemorrhagic necrotizing multi-organ inflammation, and high mortality, which seriously threatens the global swine industry. Cell death is an essential immune response of the host against pathogen invasion, and lymphopenia is the most typical clinical feature in the acute phase of CSFV infection, which affects the initial host antiviral immunity. As an "old" virus, CSFV has evolved mechanisms to evade host immune response after a long genetic evolution. Here, we show that necroptosis is a limiting host factor for CSFV infection and that CSFV-induced autophagy can subvert this host defense mechanism to promote its sustained replication. Our findings reveal a complex link between necroptosis and autophagy in the process of cell death, provide evidence supporting the important role for CSFV in counteracting host cell necrosis, and enrich our knowledge of pathogens that may subvert and evade this host defense.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Porcinos , Animales , Peste Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/fisiología , Mitofagia , Transducción de Señal , Necroptosis , AutofagiaRESUMEN
Usutu virus (USUV) is an emerging flavivirus that can infect birds and mammals. In humans, in severe cases, it may cause neuroinvasive disease. The innate immune system, and in particular the interferon response, functions as the important first line of defense against invading pathogens such as USUV. Many, if not all, viruses have developed mechanisms to suppress and/or evade the interferon response in order to facilitate their replication. The ability of USUV to antagonize the interferon response has so far remained largely unexplored. Using dual-luciferase reporter assays we observed that multiple of the USUV nonstructural (NS) proteins were involved in suppressing IFN-ß production and signaling. In particular NS4A was very effective at suppressing IFN-ß production. We found that NS4A interacted with the mitochondrial antiviral signaling protein (MAVS) and thereby blocked its interaction with melanoma differentiation-associated protein 5 (MDA5), resulting in reduced IFN-ß production. The TM1 domain of NS4A was found to be essential for binding to MAVS. By screening a panel of flavivirus NS4A proteins we found that the interaction of NS4A with MAVS is conserved among flaviviruses. The increased understanding of the role of NS4A in flavivirus immune evasion could aid the development of vaccines and therapeutic strategies.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Flavivirus , Helicasa Inducida por Interferón IFIH1 , Interferón beta , Transducción de Señal , Proteínas no Estructurales Virales , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Flavivirus/inmunología , Flavivirus/genética , Flavivirus/fisiología , Interferón beta/genética , Interferón beta/inmunología , Interferón beta/metabolismo , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/metabolismo , Helicasa Inducida por Interferón IFIH1/inmunología , Células HEK293 , Evasión Inmune , Infecciones por Flavivirus/inmunología , Infecciones por Flavivirus/virología , Interacciones Huésped-Patógeno/inmunología , Unión Proteica , Inmunidad Innata , AnimalesRESUMEN
Zika virus (ZIKV) remains a global public health threat with the potential risk of a future outbreak. Since viral infections are known to exploit mitochondria-mediated cellular processes, we investigated the effects of ZIKV infection in trophoblast cells in terms of the different mitochondrial quality control pathways that govern mitochondrial integrity and function. Here we demonstrate that ZIKV (PRVABC59) infection of JEG-3 trophoblast cells manipulates mitochondrial dynamics, mitophagy, and formation of mitochondria-derived vesicles (MDVs). Specifically, ZIKV nonstructural protein 4A (NS4A) translocates to the mitochondria, triggers mitochondrial fission and mitophagy, and suppresses mitochondrial associated antiviral protein (MAVS)-mediated type I interferon (IFN) response. Furthermore, proteomics profiling of small extracellular vesicles (sEVs) revealed an enrichment of mitochondrial proteins in sEVs secreted by ZIKV-infected JEG-3 cells, suggesting that MDV formation may also be another mitochondrial quality control mechanism manipulated during placental ZIKV infection. Altogether, our findings highlight the different mitochondrial quality control mechanisms manipulated by ZIKV during infection of placental cells as host immune evasion mechanisms utilized by ZIKV at the placenta to suppress the host antiviral response and facilitate viral infection.