RESUMEN

BACKGROUND: Dengue fever has become a significant worldwide health concern, because of its high morbidity rate and the potential for an increase in mortality rates due to lack of adequate treatment. There is an immediate need for the development of effective medication for dengue fever. METHODS: Homology modeling of dengue virus (DENV) non-structural 4B (NS4B) protein was performed by SWISS-MODEL to predict the 3D structure of the protein. Structure validation was conducted using PROSA, PROCHECK, Ramachandran plot, and VERIFY-3D. MOE software was used to find out the in-Silico inhibitory potential of the five triterpenoids against the DENV-NS4B protein. RESULTS: The SWISS-MODEL was employed to predict the three-dimensional protein structure of the NS4B protein. Through molecular docking, it was found that the chosen triterpenoid NS4B protein had a high binding affinity interaction. It was observed that the NS4B protein binding energy for 15-oxoursolic acid, betulinic acid, ursolic acid, lupeol, and 3-o-acetylursolic acid were - 7.18, - 7.02, - 5.71, - 6.67 and - 8.00 kcal/mol, respectively. CONCLUSIONS: NS4B protein could be a promising target which showed good interaction with tested triterpenoids which can be developed as a potential antiviral drug for controlling dengue virus pathogenesis by inhibiting viral replication. However, further investigations are necessary to validate and confirm their efficacy.

Asunto(s)

Antivirales , Virus del Dengue , Simulación del Acoplamiento Molecular , Triterpenos , Proteínas no Estructurales Virales , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Triterpenos/farmacología , Triterpenos/química , Virus del Dengue/efectos de los fármacos , Virus del Dengue/química , Antivirales/farmacología , Antivirales/química , Unión Proteica , Humanos , Dengue/virología , Dengue/tratamiento farmacológico , Conformación Proteica , Proteínas de la Membrana

RESUMEN

Japanese Encephalitis Virus (JEV), the predominant cause of viral encephalitis in many Asian countries, affects approximately 68,000 people annually. Lysosomes are dynamic structures that regulate cellular metabolism by mediating lysosomal biogenesis and autophagy. Here, we showed that lysosome-associated membrane protein 1 (LAMP1) and LAMP2 were downregulated in cells after JEV infection, resulting in a decrease in the quantity of acidified lysosomes and impaired lysosomal catabolism. What's more, JEV nonstructural protein 4B plays key roles in the reduction of LAMP1/2 via the autophagy-lysosome pathway. JEV NS4B also promoted abnormal aggregation of SLA-DR, an important component of the swine MHC-II molecule family involved in antigen presentation and CD4+ cell activation initiation. Mechanistically, NS4B localized to the ER during JEV infection and interacted with GRP78, leading to the activation of ER stress-mediated autophagy. The 131-204 amino acid (aa) region of NS4B is essential for autophagy induction and LAMP1/2 reduction. In summary, our findings reveal a novel pathway by which JEV induces autophagy and disrupts lysosomal function.

Asunto(s)

Autofagia , Regulación hacia Abajo , Virus de la Encefalitis Japonesa (Especie) , Proteína 2 de la Membrana Asociada a los Lisosomas , Lisosomas , Lisosomas/metabolismo , Animales , Virus de la Encefalitis Japonesa (Especie)/fisiología , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Porcinos , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Proteína 1 de la Membrana Asociada a los Lisosomas/genética , Encefalitis Japonesa/virología , Encefalitis Japonesa/veterinaria , Línea Celular , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Proteínas de Membrana de los Lisosomas/genética

RESUMEN

Duck Tembusu virus (DTMUV) belongs to the Flaviviridae family and mainly infects ducks. Duck Tembusu virus genome encodes one polyprotein that undergoes cleavage to produce 10 proteins. Among these, NS4B, the largest transmembrane protein, plays a crucial role in the viral life cycle. In this study, we investigated the localization of NS4B and found that it is located in the endoplasmic reticulum, where it co-localizes with DTMUV dsRNA. Subsequently, we confirmed 5 different transmembrane domains of NS4B and discovered that only its transmembrane domain 3 (TMD3) can traverse ER membrane. Then mutations were introduced in the conserved amino acids of NS4B TMD3 of DTMUV replicon and infectious clone. The results showed that V111G, V117G, and I118G mutations enhanced viral RNA replication, while Q104A, T106A, A113L, M116A, H120A, Y121A, and A122G mutations reduced viral replication. Recombinant viruses with these mutations were rescued and studied in BHK21 cells. The findings demonstrated that A113L and H120A mutations led to higher viral titers than the wild-type strain, while Q104A, T106A, V111G, V117G, and Y121A mutations attenuated viral proliferation. Additionally, H120A, M116A, and A122G mutations enhanced viral proliferation. Furthermore, Q104A, T106A, V111G, M116A, V117G, Y121A, and A122G mutants showed reduced viral virulence to 10-d duck embryos. Animal experiments further indicated that all mutation viruses resulted in lower genome copy numbers in the spleen compared to the WT group 5 days postinfection. Our data provide insights into the topological model of DTMUV NS4B, highlighting the essential role of NS4B TMD3 in viral replication and proliferation.

Asunto(s)

Patos , Flavivirus , Proteínas no Estructurales Virales , Replicación Viral , Animales , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Flavivirus/fisiología , Flavivirus/genética , Enfermedades de las Aves de Corral/virología , Infecciones por Flavivirus/veterinaria , Infecciones por Flavivirus/virología , Mutación

RESUMEN

BACKGROUND: The Nonstructural Protein (NSP) 4B of Zika virus of 251 amino acids from (ZIKV/Human/POLG_ZIKVF) with accession number (A0A024B7W1), Induces the production of Endoplasmic Reticulum ER-derived membrane vesicles, which are the sites of viral replication. To understand the physical basis of how proteins fold in nature and to solve the challenge of protein structure prediction, Ab-initio and comparative modeling are crucial tools. RESULTS: The systematic in silico technique, ThreaDom, had only predicted one domain (4 - 190) of NSP4B. I-TASSER, and Alphafold were ranked as the best servers for full-length 3-D protein structure predictions of NSP4B, where the predicted models were evaluated quantitatively using benchmarked metrics including C-score (-3.43), TM-score (0.77949), RMSD (2.73), and Z-score (1.561). The functional and protein binding motifs were realized using motif databases, secondary and surface accessibility predictions combined with Post-Translational Modification Sites (PTMs) prediction. Two highly conserved protein-binding motifs (Flavi NS4B and Bacillus papRprotein), together with three (PTMs) (Casein Kinase II, Myristyl site, and ASN-Glycosylation site) were predicted utilizing the Motif scan and Scanprosite servers. These patterns and PTMs were associated with NSP4B's role in triggering the development of the viral replication complex and its participation in the localization of NS3 and NS5 on the membrane. Only one hit from Structural Classification of Protein (SCOP) matched the protein sequence at positions 10 to 397 and was categorized six-hairpin glycosidases superfamily according to CATH (Class, Architecture, Topology, and Homology). Integrating this NSP4B information with the templates' SCOP and CATH annotations achieves it easier to attribute structure-function/evolution links to both previously known and recently discovered protein structures.

RESUMEN

Zika virus (ZIKV) is a mosquito-borne virus that has re-emerged as a significant threat to global health in the recent decade. Whilst infections are primarily asymptomatic, the virus has been associated with the manifestation of severe neurological complications. At present, there is still a lack of approved antivirals for ZIKV infections. In this study, chelerythrine chloride, a benzophenanthridine alkaloid, was identified from a mid-throughput screen conducted on a 502-compound natural products library to be a novel and potent inhibitor of ZIKV infection in both in-vitro and in-vivo assays. Subsequent downstream studies demonstrated that the compound inhibits a post-entry step of the viral replication cycle and is capable of disrupting viral RNA synthesis and protein expression. The successful generation and sequencing of a ZIKV resistant mutant revealed that a single S61T mutation on the viral NS4B allowed ZIKV to overcome chelerythrine chloride inhibition. Further investigation revealed that chelerythrine chloride could directly inhibit ZIKV protein synthesis, and that the NS4B-S61T mutation confers resistance to this inhibition. This study has established chelerythrine chloride as a potential candidate for further development as a therapeutic agent against ZIKV infection.

Asunto(s)

Infección por el Virus Zika , Virus Zika , Animales , Chlorocebus aethiops , Infección por el Virus Zika/tratamiento farmacológico , Benzofenantridinas/farmacología , Benzofenantridinas/metabolismo , Benzofenantridinas/uso terapéutico , Células Vero , Proteínas Virales/metabolismo , Replicación Viral , Antivirales/uso terapéutico

RESUMEN

BACKGROUND: During viral infections, nucleic acid sensing by intracellular receptors can trigger type I interferon (IFN-I) production, key mediators in antiviral innate immunity. However, many flaviviruses use non-structural proteins to evade immune sensing favoring their survival. These mechanisms remain poorly characterized. Here, we studied the role of Zika virus (ZIKV) NS4B protein in the inhibition of IFN-I induction pathway and its biophysical interaction with host proteins. METHODS: Using different cell-based assays, we studied the effect of ZIKV NS4B in the activation of interferon regulatory factors (IRFs), NF-κB, cytokines secretion and the expression of interferon-stimulating genes (ISG). We also analyzed the in vitro interaction between recombinant ZIKV NS4B and TANK-binding kinase 1 (TBK1) using surface plasmon resonance (SPR). RESULTS: Transfection assays showed that ZIKV NS4B inhibits IRFs activation involved in different nucleic acid sensing cascades. Cells expressing NS4B secreted lower levels of IFN-ß and IL-6. Furthermore, early induction of ISGs was also restricted by ZIKV NS4B. For the first time, we demonstrate by SPR assays that TBK1, a critical component in IFN-I production pathway, binds directly to ZIKV NS4B (KD of 3.7 × 10-6 M). In addition, we show that the N-terminal region of NS4B is directly involved in this interaction. CONCLUSIONS: Altogether, our results strongly support that ZIKV NS4B affects nucleic acid sensing cascades and disrupts the TBK1/IRF3 axis, leading to an impairment of IFN-ß production. SIGNIFICANCE: This study provides the first biophysical data of the interaction between ZIKV NS4B and TBK1, and highlights the role of ZIKV NS4B in evading the early innate immune response.

Asunto(s)

Interferón Tipo I , Ácidos Nucleicos , Infección por el Virus Zika , Virus Zika , Humanos , Virus Zika/metabolismo , Infección por el Virus Zika/metabolismo , Transducción de Señal , Proteínas Virales/genética

RESUMEN

Japanese encephalitis virus (JEV) is a flavivirus transmitted by mosquitoes, causing epidemics of encephalitis in humans and reproductive disorders in pigs. This virus is predominantly distributed in Asian countries and causes tens of thousands of infections in humans annually. Interferon (IFN) is an essential component of host defense against viral infection. Multiple studies have indicated that multifunctional nonstructural proteins of flaviviruses suppress the host IFN response via various strategies to facilitate viral replication. The flaviviruses encoded nonstructural protein 4B (NS4B) is a multifunctional hydrophobic nonstructural protein widely involved in viral replication, pathogenesis and host immune evasion. In this study, we demonstrated that NS4B of JEV suppressed the induction of IFN-ß production, mainly through targeting the TLR3 and TRIF (a TIR domain-containing linker that induces IFN-ß) proteins in the TLR3 pathway. In a dual-luciferase reporter assay, JEV NS4B significantly inhibited the activation of IFN-ß promoter induced by TLR3 and simultaneously treated with poly (I:C). Moreover, NS4B also inhibited the activation of IFN-ß promoter triggered by interferon regulatory factor 3 (IRF3)/5D or its upstream molecules in TLR3 signaling pathway. Furthermore, NS4B inhibited the phosphorylation of IRF3 under the stimulation of TLR3 and TRIF molecules. Mechanistically, JEV NS4B interacts with TLR3 and TRIF and confirmed by co-localization and co-immunoprecipitation assay, thereby inhibiting the activation of downstream sensors in the TLR3-mediated pathway. Overall, our results provide a novel mechanism by which JEV NS4B interferes with the host's antiviral response through targeting TLR3 receptor signaling pathway.

Asunto(s)

Virus de la Encefalitis Japonesa (Especie) , Animales , Proteínas Adaptadoras del Transporte Vesicular/genética , Virus de la Encefalitis Japonesa (Especie)/genética , Interferón beta/genética , Interferones , Porcinos , Receptor Toll-Like 3/genética

RESUMEN

Zika virus (ZIKV) evolves non-structural proteins to evade immune response and ensure efficient replication in the host cells. Cholesterol metabolic enzyme 7-dehydrocholesterol reductase (DHCR7) was recently reported to impact innate immune responses in ZIKV infection. However, the vital non-structural protein and mechanisms involved in DHCR7-mediated viral evasion are not well elucidated. In this study, we demonstrated that ZIKV infection facilitated DHCR7 expression. Notably, the upregulated DHCR7 in turn facilitated ZIKV infection and blocking DHCR7 suppressed ZIKV infection. Mechanically, ZIKV non-structural protein 4B (NS4B) interacted with DHCR7 to induce DHCR7 expression. Moreover, DHCR7 inhibited TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) phosphorylation, which resulted in the reduction of interferon-beta (IFN-ß) and interferon-stimulated genes (ISGs) productions. Therefore, we propose that ZIKV NS4B binds to DHCR7 to repress TBK1 and IRF3 activation, which in turn inhibits IFN-ß and ISGs, and thereby facilitating ZIKV evasion. This study broadens the insights on how viral non-structural proteins antagonize innate immunity to facilitate viral infection via cholesterol metabolic enzymes and intermediates.

Asunto(s)

Infección por el Virus Zika , Virus Zika , Humanos , Transducción de Señal , Proteínas no Estructurales Virales/metabolismo , Interferón beta/genética , Interferón beta/metabolismo , Inmunidad Innata , Colesterol , Replicación Viral

RESUMEN

Dengue virus is an important pathogen affecting global population while no specific treatment is available against this virus. Effort has been made to develop inhibitors through targeting viral nonstructural proteins such as NS3 and NS5 with enzymatic activities. No potent inhibitors entering clinical studies have been developed so far due to many challenges. The genome of dengue virus encodes four membrane-bound nonstructural proteins which do not possess any enzymatic activities. Studies have shown that the membrane protein-NS4B is a validated target for drug discovery and several NS4B inhibitors exhibited antiviral activities in various assays and entered preclinical studies.. Here, we summarize the recent studies on dengue NS4B protein. The structure and membrane topology of dengue NS4B derived from biochemical and biophysical studies are described. Function of NS4B through protein-protein interactions and some available NS4B inhibitors are summarized. Accumulated studies demonstrated that cell-based assays play important roles in developing NS4B inhibitors. Although the atomic structure of NS4B is not obtained, target-based drug discovery approach become feasible to develop NS4B inhibitors as recombinant NS4B protein is available.

Asunto(s)

Virus del Dengue , Dengue , Antivirales/metabolismo , Antivirales/farmacología , Dengue/tratamiento farmacológico , Virus del Dengue/genética , Humanos , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas no Estructurales Virales/genética , Replicación Viral

RESUMEN

Dengue virus replicates its single-stranded RNA genome in membrane-bound complexes formed on the endoplasmic reticulum, where viral non-structural proteins (NS) and RNA co-localize. The NS proteins interact with one another and with the host proteins. The interaction of the viral helicase and protease, NS3, with the RNA-dependent RNA polymerase, NS5, and NS4b proteins is critical for replication. In vitro, NS3 helicase activity is enhanced by interaction with NS4b. We characterized the interaction between NS3 and NS4b and explained a possible mechanism for helicase activity modulation by NS4b. Our bacterial two-hybrid assay results showed that the N-terminal 57 residues region of NS4b is enough to interact with NS3. The molecular docking of the predicted NS4b structure onto the NS3 structure revealed that the N-terminal disordered region of NS4b wraps around the C-terminal subdomain (CTD) of the helicase. Further, NS3 helicase activity is enhanced upon interaction with NS4b. Molecular dynamics simulations on the NS4b-docked NS3 crystal structure and intrinsic tryptophan fluorescence studies suggest that the interaction results in NS3 CTD domain motions. Based on the interpretation of our results in light of the mechanism explained for NS3 helicase, NS4b-NS3 interaction modulating CTD dynamics is a plausible explanation for the helicase activity enhancement.

Asunto(s)

Virus del Dengue , ADN Helicasas/metabolismo , Virus del Dengue/genética , Simulación del Acoplamiento Molecular , ARN/metabolismo , ARN Helicasas/metabolismo , Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/genética , Replicación Viral

RESUMEN

Classical swine fever virus (CSFV) is an ancient and economically important flavivirus that utilizes a Rab5-dependent endocytic pathway to enter host cells. Rab22a is a small GTPase that cooperates with Rab5 in the regulation of early endosome dynamics. Until now, the role of Rab22a in the flavivirus life cycle has been poorly defined. In this study, we systematically analyzed the role of Rab22a in CSFV proliferation and internalization using multiple viral replication analyses in combination with the overexpression, knockdown, and mutation of Rab22a, and found that Rab22a is involved in the entry process of CSFV. Confocal microscopy results showed that Rab22a colocalized with virus particles during the early phase of infection. Furthermore, by using glutathione S-transferase pull-down and co-immunoprecipitation assays, we verified the interaction between Rab22a and CSFV non-structural protein NS4B, and determined that NS4B can only bind to wild-type Rab22a, but not to the mutants Q64L and S19N. In addition, we explored the relationship between Rab22a, Rab5 and NS4B in CSFV internalization, and found out that these three proteins bind in early endosomes, and then through a Rab22a-Rab5-NS4B cascade allows the entry of CSFV. Taken together, our findings highlight the role of Rab proteins in CSFV internalization, and extend the understanding of the life cycle of flaviviruses.

Asunto(s)

Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Enfermedades de los Porcinos , Animales , Virus de la Fiebre Porcina Clásica/fisiología , Inmunoprecipitación/veterinaria , Porcinos , Replicación Viral

RESUMEN

Classical swine fever virus (CSFV), the etiological agent of classical swine fever (CSF), causes serious financial losses to the pig industry. Using yeast two-hybrid screening, we have previously identified ribosomal protein RPLP1 as a potential binding partner of CSFV NS4B. In this study, the interaction between host RPLP1 and CSFV NS4B was further characterized by co-immunoprecipitation (co-IP), glutathione S-transferase (GST) pulldown, and confocal microscopy. In addition, lentivirus-mediated shRNA knockdown of RPLP1 drastically attenuated CSFV growth, while stable overexpression of RPLP1 markedly enhanced CSFV production. Moreover, cellular RPLP1 expression was found to be significantly up-regulated along with CSFV infection. Dual-luciferase reporter assay showed that depletion of RPLP1 had no effects on the activity of CSFV internal ribosome entry site (IRES). In the first life cycle of CSFV, further studies revealed that RPLP1 depletion did not influence the intracellular viral RNA abundance but diminished the intracellular and extracellular progeny virus titers as well as the viral E2 protein expression, which indicates that RPLP1 is crucial for CSFV genome translation. In summary, this study demonstrated that RPLP1 interacts with CSFV NS4B and enhances virus production via promoting translation of viral genome.

Asunto(s)

Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Animales , Peste Porcina Clásica/metabolismo , Virus de la Fiebre Porcina Clásica/genética , Genoma Viral , Unión Proteica , Porcinos , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral

RESUMEN

Flaviviruses are human pathogens that can cause severe diseases, such as dengue fever and Japanese encephalitis, which can lead to death. Valosin-containing protein (VCP)/p97, a cellular ATPase associated with diverse cellular activities (AAA-ATPase), is reported to have multiple roles in flavivirus replication. Nevertheless, the importance of each role still has not been addressed. In this study, the functions of 17 VCP mutants that are reportedly unable to interact with the VCP cofactors were validated using the short-interfering RNA rescue experiments. Our findings of this study suggested that VCP exerts its functions in replication of the Japanese encephalitis virus by interacting with the VCP cofactor nuclear protein localization 4 (NPL4). We show that the depletion of NPL4 impaired the early stage of viral genome replication. In addition, we demonstrate that the direct interaction between NPL4 and viral nonstructural protein (NS4B) is critical for the translocation of NS4B to the sites of viral replication. Finally, we found that Japanese encephalitis virus and dengue virus promoted stress granule formation only in VCP inhibitor-treated cells and the expression of NS4B or VCP attenuated stress granule formation mediated by protein kinase R, which is generally known to be activated by type I interferon and viral genome RNA. These results suggest that the NS4B-mediated recruitment of VCP to the virus replication site inhibits cellular stress responses and consequently facilitates viral protein synthesis in the flavivirus-infected cells.

Asunto(s)

Virus de la Encefalitis Japonesa (Especie) , Flavivirus , Proteínas Nucleares , Gránulos de Estrés , Proteína que Contiene Valosina , Proteínas no Estructurales Virales , Replicación Viral , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis Japonesa (Especie)/metabolismo , Virus de la Encefalitis Japonesa (Especie)/fisiología , Flavivirus/genética , Flavivirus/metabolismo , Flavivirus/fisiología , Genoma Viral , Humanos , Proteínas Nucleares/metabolismo , ARN Viral/genética , Gránulos de Estrés/genética , Gránulos de Estrés/metabolismo , Proteína que Contiene Valosina/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiología

RESUMEN

The hepatitis C virus is a communicable disease that gradually harms the liver leading to cirrhosis and hepatocellular carcinoma. Important therapeutic interventions have been reached since the discovery of the disease. However, its resurgence urges the need for new approaches against this malady. The NS4B receptor is one of the important proteins for Hepatitis C Virus RNA replication that acts by mediating different viral properties. In this work, we opt to explore the relationships between the molecular structures of biologically tested NS4B inhibitors and their corresponding inhibitory activities to assist the design of novel and potent NS4B inhibitors. For that, a set of 115 indol-2-ylpyridine-3-sulfonamides (IPSA) compounds with inhibitory activity against NS4B is used. A hybrid genetic algorithm combined with multiple linear regressions (GA-MLR) was implemented to construct a predictive model. This model was further used and applied to a set of compounds that were generated based on a pharmacophore modeling study combined with virtual screening to identify structurally similar lead compounds. Multiple filtrations were implemented for selecting potent hits. The selected hits exhibited advantageous molecular features, allowing for favorable inhibitory activity against HCV. The results showed that 7 out of 1285 screened compounds, were selected as potent candidate hits where Zinc14822482 exhibits the best predicted potency and pharmacophore features. The predictive pharmacokinetic analysis further justified the compounds as potential hit molecules, prompting their recommendation for a confirmatory biological evaluation. We believe that our strategy could help in the design and screening of potential inhibitors in drug discovery.Communicated by Ramaswamy H. Sarma.

Asunto(s)

Hepacivirus , Hepatitis C , Descubrimiento de Drogas , Hepacivirus/genética , Hepatitis C/tratamiento farmacológico , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Relación Estructura-Actividad Cuantitativa

RESUMEN

Classical swine fever virus (CSFV) infection causes a severe disease of pigs, resulting in significant economic losses. The CSFV NS4B protein is crucial for viral replication and pathogenicity. Interleukin 8 (IL-8), a main chemokine, is induced by multiple cell types and plays an essential role in host defense mechanisms against numerous viruses. It has been reported that NS4A of CSFV is involved in the induction of IL-8 expression in swine umbilical vein endothelial cells. However, the effect of CSFV NS4B on IL-8 expression is unknown. In this study, we showed that CSFV NS4B inhibited IL-8 expression in porcine alveolar macrophages (PAMs), and NS4B inhibited mitochondrial antiviral signaling protein (MAVS)-induced IL-8 expression. Moreover, CSFV NS4B interacted with MAVS. However, NS4B did not alter MAVS expression. Subsequently, we demonstrated that IRF3 knockdown or NF-κB inhibition reduced MAVS-induced IL-8 expression. Furthermore, the IRF3 and NF-κB pathways were activated by MAVS expression. However, CSFV NS4B inhibited MAVS-mediated NF-κB activation and IRF3 expression. Finally, CSFV NS4B inhibited IRF3 expression. Our findings reveal that CSFV NS4B interacts with MAVS and inhibits IL-8 expression by blocking the activation of IRF3 and NF-κB. Taken together, this study provides insights into the mechanism of NS4B-inhibited IL-8 expression.

Asunto(s)

Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Animales , Virus de la Fiebre Porcina Clásica/fisiología , Células Endoteliales/metabolismo , Interacciones Huésped-Patógeno , Interleucina-8/genética , Macrófagos Alveolares , FN-kappa B/metabolismo , Porcinos , Replicación Viral/fisiología

RESUMEN

The evasion of the Interferon response has important implications in Zika virus (ZIKV) disease. Mutations in ZIKV viral protein NS4B, associated with modulation of the interferon (IFN) system, have been linked to increased pathogenicity in animal models. In this study, we unravel ZIKV NS4B as antagonist of the IFN signaling cascade. Firstly, we reported the genomic characterization of NS4B isolated from a strain of the 2016 outbreak, ZIKV Brazil/2016/INMI1, and we predicted its membrane topology. Secondly, we analyzed its phylogenetic correlation with other flaviviruses, finding a high similarity with dengue virus 2 (DEN2) strains; in particular, the highest conservation was found when NS4B was aligned with the IFN inhibitory domain of DEN2 NS4B. Hence, we asked whether ZIKV NS4B was also able to inhibit the IFN signaling cascade, as reported for DEN2 NS4B. Our results showed that ZIKV NS4B was able to strongly inhibit the IFN stimulated response element and the IFN-γ-activated site transcription, blocking IFN-I/-II responses. mRNA expression levels of the IFN stimulated genes ISG15 and OAS1 were also strongly reduced in presence of NS4B. We found that the viral protein was acting by suppressing the STAT1 phosphorylation and consequently blocking the nuclear transport of both STAT1 and STAT2.

Asunto(s)

Interferón Tipo I/metabolismo , Interferón gamma/metabolismo , Factor de Transcripción STAT1/metabolismo , Proteínas no Estructurales Virales/metabolismo , Infección por el Virus Zika/virología , Virus Zika/metabolismo , 2',5'-Oligoadenilato Sintetasa/genética , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citocinas/genética , Células HEK293 , Humanos , Interferón Tipo I/antagonistas & inhibidores , Interferón Tipo I/inmunología , Interferón beta/biosíntesis , Interferón gamma/antagonistas & inhibidores , Interferón gamma/inmunología , Fosforilación , Filogenia , Conformación Proteica , Elementos de Respuesta , Transducción de Señal , Ubiquitinas/genética , Células Vero , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Virus Zika/química , Virus Zika/aislamiento & purificación , Virus Zika/patogenicidad

RESUMEN

The dengue virus (DENV) causes the most prevalent arthropod-borne viral disease worldwide. While its incidence is increasing in many countries, there is no approved antiviral therapy currently available. In infected cells, the DENV induces extensive morphological alterations of the endoplasmic reticulum (ER) to generate viral replication organelles (vRO), which include convoluted membranes (CM) and vesicle packets (VP) hosting viral RNA replication. The viral non-structural protein NS4B localizes to vROs and is absolutely required for viral replication through poorly defined mechanisms, which might involve cellular protein partners. Previous interactomic studies identified the ATPase valosin-containing protein (VCP) as a DENV NS4B-interacting host factor in infected cells. Using both pharmacological and dominant-negative inhibition approaches, we show, in this study, that VCP ATPase activity is required for efficient DENV replication. VCP associates with NS4B when expressed in the absence of other viral proteins while in infected cells, both proteins colocalize within large DENV-induced cytoplasmic structures previously demonstrated to be CMs. Consistently, VCP inhibition dramatically reduces the abundance of DENV CMs in infected cells. Most importantly, using a recently reported replication-independent plasmid-based vRO induction system, we show that de novo VP biogenesis is dependent on VCP ATPase activity. Overall, our data demonstrate that VCP ATPase activity is required for vRO morphogenesis and/or stability. Considering that VCP was shown to be required for the replication of other flaviviruses, our results argue that VCP is a pan-flaviviral host dependency factor. Given that new generation VCP-targeting drugs are currently evaluated in clinical trials for cancer treatment, VCP may constitute an attractive broad-spectrum antiviral target in drug repurposing approaches.

Asunto(s)

Virus del Dengue/metabolismo , Proteína que Contiene Valosina/metabolismo , Compartimentos de Replicación Viral/fisiología , Adenosina Trifosfatasas/genética , Línea Celular , Dengue/virología , Virus del Dengue/genética , Virus del Dengue/patogenicidad , Retículo Endoplásmico/virología , Humanos , ARN Viral/genética , Proteína que Contiene Valosina/genética , Proteínas no Estructurales Virales/genética , Replicación Viral/fisiología

RESUMEN

Hepatitis C virus (HCV) nonstructural protein NS4B is necessary for HCV replication. Our previous research found that NS4B-associated cellular proteins PREB and Surfeit 4 are involved in HCV replication. However, the molecular mechanism of HCV replication is not fully understood. Here we identified cellular ovarian cancer immunoreactive antigen domain containing 2 (OCIAD2) protein as a novel NS4B-associated HCV host cofactor by screening with small interfering RNA. Knockdown of OCIAD2 reduced significantly the HCV replication in a dose-dependent and genotype-independent manner. Further research showed that OCIAD2 was recruited into the HCV RNA replication complex by the interaction with NS4B. Interestingly, HCV replication induced OCIAD2 expression. In turn, overexpression of wild OCIAD2 also promoted virus replication whereas that of OCIAD2 mutant lacking the ability to bind NS4B exerted no effect on HCV replication. We also examined whether OCIAD2 interacted with other proteins participating in the HCV RNA replication complex including viral proteins NS5A, NS5B, and cellular proteins PREB, Surfeit 4. The results showed that OCIAD2 interacted with PREB and NS5A, but not NS5B or Surfeit 4. Our findings provide new insights into the function of OCIAD2 and HCV replication mechanism.

Asunto(s)

Hepacivirus/genética , Hepatitis C/genética , Proteínas de Neoplasias/genética , Replicación Viral/genética , Línea Celular , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/genética , Genotipo , Factores de Intercambio de Guanina Nucleótido/genética , Hepacivirus/patogenicidad , Hepatitis C/patología , Hepatitis C/virología , Interacciones Huésped-Patógeno/genética , Humanos , Proteínas de la Membrana/genética , Provirus/genética , ARN Interferente Pequeño/genética , Factores de Transcripción/genética , Proteínas no Estructurales Virales/genética

RESUMEN

The endoplasmic reticulum (ER) of eukaryotic cells is a dynamic organelle, which undergoes continuous remodeling. At the three-way tubular junctions of the ER, the lunapark (LNP) protein acts as a membrane remodeling factor to stabilize these highly curved membrane junctions. In addition, during flavivirus infection, the ER membrane is invaginated to form vesicles (Ve) for virus replication. Thus, LNP may have roles in the generation or maintenance of the Ve during flavivirus infection. In this study, our aim was to characterize the functions of LNP during flavivirus infection and investigate the underlying mechanisms of these functions. To specifically study virus replication, we generated cell lines expressing replicons of West Nile virus (Kunjin strain) or Langat virus. By using these replicon platforms and electron microscopy, we showed that depletion of LNP resulted in reduced virus replication, which is due to its role in the generation of the Ve. By using biochemical assays and high-resolution microscopy, we found that LNP is recruited to the Ve and the protein interacts with the nonstructural protein (NS) 4B. Therefore, these data shed new light on the interactions between flavivirus and host factors during viral replication.

Asunto(s)

Flavivirus/química , Flavivirus/fisiología , Proteínas de la Membrana/genética , Replicación Viral/genética , Células A549 , Animales , Línea Celular , Cricetinae , Virus de la Encefalitis Transmitidos por Garrapatas/genética , Virus de la Encefalitis Transmitidos por Garrapatas/fisiología , Retículo Endoplásmico/virología , Flavivirus/clasificación , Flavivirus/genética , Células HEK293 , Humanos , Proteínas de la Membrana/metabolismo , ARN Viral/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiología , Virus del Nilo Occidental/genética , Virus del Nilo Occidental/fisiología

RESUMEN

Bovine viral diarrhea virus (BVDV) is an important member of the family Flaviviridae and often causes immunosuppression. Previous studies have suggested that BVDV envelope protein Erns and the nonstructural autoprotease Npro can inhibit host innate immune responses. Herein, we found that BVDV NS4B, as a nonstructural protein necessary for replication, is involved in antagonizing the main RNA virus sensing pathway. Overexpression of BVDV NS4B protein significantly inhibited Sendai virus (SeV)-induced interferon-ß promoter activity, IFN-ß mRNA and IFN regulatory factor 3 (IRF3) phosphorylation levels. We also discovered that BVDV NS4B protein significantly inhibited RIG-I like receptor (RLRs)-mediated interferon-ß (IFN-ß) promoter activity and endogenous MDA5 mRNA levels. In addition, the BVDV NS4B protein directly interacts with N-terminal CARDs of MDA5, and co-localized with MDA5 or MDA5-2CARD in the cytoplasm. In summary, the results of this study indicate that the BVDV NS4B protein acts as an interferon-ß antagonist through inhibiting the MDA5-mediated signal transduction pathway. Our study provides an in-depth understanding of the molecular mechanisms of BVDV evading the host's natural immune response.

Asunto(s)

Virus de la Diarrea Viral Bovina , Interferón beta , Diarrea , Virus de la Diarrea Viral Bovina/genética , Humanos , Inmunidad Innata , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , ARN Helicasas/metabolismo , ARN Mensajero