RESUMEN
Flaviviridae is a family of positive-stranded RNA viruses, including human pathogens, such as Japanese encephalitis virus (JEV), dengue virus (DENV), Zika virus (ZIKV), and West Nile virus (WNV). Nuclear localization of the viral core protein is conserved among Flaviviridae, and this feature may be targeted for developing broad-ranging anti-flavivirus drugs. However, the mechanism of core protein translocation to the nucleus and the importance of nuclear translocation in the viral life cycle remain unknown. We aimed to identify the molecular mechanism underlying core protein nuclear translocation. We identified importin-7 (IPO7), an importin-ß family protein, as a nuclear carrier for Flaviviridae core proteins. Nuclear import assays revealed that core protein was transported into the nucleus via IPO7, whereas IPO7 deletion by CRISPR/Cas9 impaired their nuclear translocation. To understand the importance of core protein nuclear translocation, we evaluated the production of infectious virus or single-round-infectious-particles in wild-type or IPO7-deficient cells; both processes were significantly impaired in IPO7-deficient cells, whereas intracellular infectious virus levels were equivalent in wild-type and IPO7-deficient cells. These results suggest that IPO7-mediated nuclear translocation of core proteins is involved in the release of infectious virus particles of flaviviruses.
Asunto(s)
Transporte Activo de Núcleo Celular , Núcleo Celular , Flavivirus , Humanos , Flavivirus/metabolismo , Flavivirus/fisiología , Animales , Núcleo Celular/metabolismo , Núcleo Celular/virología , Replicación Viral/fisiología , Proteínas del Núcleo Viral/metabolismo , Proteínas del Núcleo Viral/genética , Carioferinas/metabolismo , Carioferinas/genética , Infecciones por Flavivirus/metabolismo , Infecciones por Flavivirus/virología , Chlorocebus aethiops , Células HEK293RESUMEN
Viral genetic diversity presents significant challenges in developing antivirals with broad-spectrum activity and high barriers to resistance. Here we report development of proteolysis targeting chimeras (PROTACs) targeting the dengue virus envelope (E) protein through coupling of known E fusion inhibitors to ligands of the CRL4CRBN E3 ubiquitin ligase. The resulting small molecules block viral entry through inhibition of E-mediated membrane fusion and interfere with viral particle production by depleting intracellular E in infected Huh 7.5 cells. This activity is retained in the presence of point mutations previously shown to confer partial resistance to the parental inhibitors due to decreased inhibitor-binding. The E PROTACs also exhibit broadened spectrum of activity compared to the parental E inhibitors against a panel of mosquito-borne flaviviruses. These findings encourage further exploration of targeted protein degradation as a differentiated and potentially advantageous modality for development of broad-spectrum direct-acting antivirals.
Asunto(s)
Antivirales , Virus del Dengue , Flavivirus , Proteolisis , Internalización del Virus , Humanos , Proteolisis/efectos de los fármacos , Animales , Antivirales/farmacología , Flavivirus/efectos de los fármacos , Flavivirus/genética , Flavivirus/metabolismo , Internalización del Virus/efectos de los fármacos , Virus del Dengue/efectos de los fármacos , Virus del Dengue/fisiología , Virus del Dengue/genética , Culicidae/virología , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Línea CelularRESUMEN
Flaviviruses are single-stranded positive-sense RNA (+RNA) viruses that are responsible for several (re)emerging diseases such as yellow, dengue, or West Nile fevers. The Zika epidemic highlighted their dangerousness when a relatively benign virus known since the 1950s turned into a deadly pathogen. The central protein for their replication is NS5 (non-structural protein 5), which is composed of the N-terminal methyltransferase (MTase) domain and the C-terminal RNA-dependent RNA-polymerase (RdRp) domain. It is responsible for both RNA replication and installation of the 5' RNA cap. We structurally and biochemically analyzed the Ntaya virus MTase and RdRp domains and we compared their properties to other flaviviral NS5s. The enzymatic centers are well conserved across Flaviviridae, suggesting that the development of drugs targeting all flaviviruses is feasible. However, the enzymatic activities of the isolated proteins were significantly different for the MTase domains.
Asunto(s)
Metiltransferasas , Modelos Moleculares , ARN Polimerasa Dependiente del ARN , Proteínas no Estructurales Virales , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Polimerasa Dependiente del ARN/química , Metiltransferasas/metabolismo , Metiltransferasas/química , Cristalografía por Rayos X , Flavivirus/enzimología , Flavivirus/metabolismo , Unión Proteica , Secuencia de Aminoácidos , Dominios Proteicos , Virus de la Encefalitis Transmitidos por Garrapatas/metabolismoRESUMEN
A high level of disorder in many viral proteins is a direct consequence of their small genomes, which makes interaction with multiple binding partners a necessity for infection and pathogenicity. A segment of the flaviviral capsid protein (C), also known as the molecular recognition feature (MoRF), undergoes a disorder-toorder transition upon binding to several protein partners. To understand their role in pathogenesis, MoRFs were identified and their occurrence across different flaviviral capsids were studied. Despite lack of sequence similarities, docking studies of Cs with the host proteins indicate conserved interactions involving MoRFs across members of phylogenetic subclades. Additionally, it was observed from the protein-protein networks that some MoRFs preferentially bind proteins that are involved in specialized functions such as ribosome biogenesis. The findings point to the importance of MoRFs in the flaviviral life cycle, with important consequences for disease progression and suppression of the host immune system. Potentially, they might have impacted the way flaviviruses evolved to infect varied hosts using multiple vectors.
Asunto(s)
Proteínas de la Cápside , Flavivirus , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/química , Flavivirus/patogenicidad , Flavivirus/genética , Flavivirus/fisiología , Flavivirus/metabolismo , Filogenia , Humanos , Unión Proteica , Cápside/metabolismo , Cápside/química , Infecciones por Flavivirus/virología , Infecciones por Flavivirus/metabolismo , Simulación del Acoplamiento Molecular , Secuencia de AminoácidosRESUMEN
Flaviviruses encode a conserved, membrane-associated nonstructural protein 1 (NS1) with replication and immune evasion functions. The current knowledge of secreted NS1 (sNS1) oligomers is based on several low-resolution structures, thus hindering the development of drugs and vaccines against flaviviruses. Here, we revealed that recombinant sNS1 from flaviviruses exists in a dynamic equilibrium of dimer-tetramer-hexamer states. Two DENV4 hexameric NS1 structures and several tetrameric NS1 structures from multiple flaviviruses were solved at atomic resolution by cryo-EM. The stacking of the tetrameric NS1 and hexameric NS1 is facilitated by the hydrophobic ß-roll and connector domains. Additionally, a triacylglycerol molecule located within the central cavity may play a role in stabilizing the hexamer. Based on differentiated interactions between the dimeric NS1, two distinct hexamer models (head-to-head and side-to-side hexamer) and the step-by-step assembly mechanisms of NS1 dimer into hexamer were proposed. We believe that our study sheds light on the understanding of the NS1 oligomerization and contributes to NS1-based therapies.
Asunto(s)
Microscopía por Crioelectrón , Flavivirus , Modelos Moleculares , Multimerización de Proteína , Proteínas no Estructurales Virales , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Flavivirus/metabolismo , Flavivirus/química , Conformación ProteicaRESUMEN
Alongshan virus (ALSV), a newly discovered member of unclassified Flaviviridae family, is able to infect humans. ALSV has a multi-segmented genome organization and is evolutionarily distant from canonical mono-segmented flaviviruses. The virus-encoded methyltransferase (MTase) plays an important role in viral replication. Here we show that ALSV MTase readily binds S-adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) but exhibits significantly lower affinities than canonical flaviviral MTases. Structures of ALSV MTase in the free and SAM/SAH-bound forms reveal that the viral enzyme possesses a unique loop-element lining side-wall of the SAM/SAH-binding pocket. While the equivalent loop in flaviviral MTases half-covers SAM/SAH, contributing multiple hydrogen-bond interactions; the pocket-lining loop of ALSV MTase is of short-length and high-flexibility, devoid of any physical contacts with SAM/SAH. Subsequent mutagenesis data further corroborate such structural difference affecting SAM/SAH-binding. Finally, we also report the structure of ALSV MTase bound with sinefungin, an SAM-analogue MTase inhibitor. These data have delineated the basis for the low-affinity interaction between ALSV MTase and SAM/SAH and should inform on antiviral drug design.
Asunto(s)
Flavivirus , Metiltransferasas , Humanos , Metiltransferasas/genética , Flavivirus/genética , Flavivirus/metabolismo , S-Adenosilmetionina/metabolismo , MutagénesisRESUMEN
In the case of the Japanese encephalitis virus (JEV), the envelope protein (E), a major component of viral particles, contains a highly conserved N-linked glycosylation site (E: N154). Glycosylation of the E protein is thought to play an important role in the ability of the virus to attach to target cells during transmission; however, its role in viral particle formation and release remains poorly understood. In this study, we investigated the role of N-glycosylation of flaviviral structural proteins in viral particle formation and secretion by introducing mutations in viral structural proteins or cellular factors involved in glycoprotein transport and processing. The number of secreted subviral particles (SVPs) was significantly reduced in N154A, a glycosylation-null mutant, but increased in D67N, a mutant containing additional glycosylation sites, indicating that the amount of E glycosylation regulates the release of SVPs. SVP secretion was reduced in cells deficient in galactose, sialic acid, and N-acetylglucosamine modifications in the Golgi apparatus; however, these reductions were not significant, suggesting that glycosylation mainly plays a role in pre-Golgi transport. Fluorescent labeling of SVPs using a split green fluorescent protein (GFP) system and time-lapse imaging by retention using selective hooks (RUSH) system revealed that the glycosylation-deficient mutant was arrested before endoplasmic reticulum (ER)- Golgi transport. However, the absence of ERGIC-53 and ERGIC-L, ER-Golgi transport cargo receptors that recognize sugar chains on cargo proteins, does not impair SVP secretion. In contrast, the solubility of the N154A mutant of E or the N15A/T17A mutant of prM in cells was markedly lower than that of the wild type, and proteasome-mediated rapid degradation of these mutants was observed, indicating the significance of glycosylation of both prM and E in proper protein folding and assembly of viral particles in the ER.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie) , Flavivirus , Glicosilación , Flavivirus/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Virus de la Encefalitis Japonesa (Especie)/metabolismo , Virión/metabolismoRESUMEN
Flavivirus remodels the host endoplasmic reticulum (ER) to generate replication compartments (RCs) as the fundamental structures to accommodate viral replication. Here, a centralized replication mode of flavivirus is reported, i.e., flavivirus concentrates host ER in perinuclear main replication compartments (MRCs) for efficient replication. Superresolution live-cell imaging demonstrated that flavivirus MRCs formed via a series of events, including multisite ER clustering, growth and merging of ER clusters, directional movement, and convergence in the perinuclear region. The dynamic activities of viral RCs are driven by nonstructural (NS) proteins and are independent of microtubules and actin. Moreover, disrupting MRCs formation by small molecule compounds inhibited flavivirus replication. Overall, the findings reveal unprecedented insight into dynamic ER reorganization by flavivirus and identify a new inhibition strategy.
Asunto(s)
Flavivirus , Flavivirus/metabolismo , Retículo Endoplásmico/metabolismo , Replicación ViralRESUMEN
Flaviviruses encompass many important human pathogens, including Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as well as several emerging viruses that affect millions of people worldwide. They enter cells by endocytosis, fusing their membrane with the late endosomal one in a pH-dependent manner, so membrane fusion is one of the main targets for obtaining new antiviral inhibitors. The envelope E protein, a class II membrane fusion protein, is responsible for fusion and contains different domains involved in the fusion mechanism, including the fusion peptide. However, other segments, apart from the fusion peptide, have been implicated in the mechanism of membrane fusion, in particular a segment containing a His residue supposed to act as a specific pH sensor. We have used atomistic molecular dynamics to study the binding of the envelope E protein segment containing the conserved His residue in its three different tautomer forms with a complex membrane mimicking the late-endosomal one. We show that this His-containing segment is capable of spontaneous membrane binding, preferentially binds electronegatively charged phospholipids and does not bind cholesterol. Since Flaviviruses have caused epidemics in the past, continue to do so and will undoubtedly continue to do so, this specific segment could characterise a new target that would allow finding effective antiviral molecules against DENV virus in particular and Flaviviruses in general.
Asunto(s)
Dengue , Flavivirus , Infección por el Virus Zika , Virus Zika , Humanos , Envoltura Viral/metabolismo , Proteínas del Envoltorio Viral/química , Flavivirus/química , Flavivirus/metabolismo , Virus Zika/metabolismo , Péptidos , Antivirales , FosfolípidosRESUMEN
Infection by many (+)RNA viruses is accompanied by ER-expansion and membrane remodelling to form viral replication organelles, followed by assembly and secretion of viral progenies. We previously identified that virus-triggered lipophagy was critical for flaviviral assembly, and is driven by the lipid droplet associated protein Ancient ubiquitin protein 1 (Aup1). A ubiquitin conjugating protein Ube2g2 that functions as a co-factor for Aup1 was identified as a host dependency factor in our study. Here we characterized its function: Ube2g2-deficient cells displayed a dramatic reduction in virus production, which could be rescued by reconstituting the wild-type but not the catalytically deficient (C89K) mutant of Ube2g2, suggesting that its enzymatic activity is necessary. Ube2g2 deficiency did not affect entry of virus particles but resulted in a profound loss in formation of replication organelles, and production of infectious progenies. This phenomenon resulted from its dual activity in (i) triggering lipophagy in conjunction with Aup1, and (ii) degradation of ER chaperones such as Herpud1, SEL1L, Hrd1, along with Sec62 to restrict ER-phagy upon Xbp1-IRE1 triggered ER expansion. Our results therefore underscore an exquisite fine-tuning of selective autophagy by flaviviruses that drive host membrane reorganization during infection to enable biogenesis of viral replication organelles.
Asunto(s)
Flavivirus , Proteínas , Proteínas/metabolismo , Flavivirus/metabolismo , Autofagia/genética , Gotas Lipídicas/metabolismo , Replicación Viral/genética , Ubiquitinas/metabolismoRESUMEN
INTRODUCTION: Flaviviruses are emerging or reemerging pathogens that have caused several outbreaks throughout the world and pose serious threats on human health and economic development. RNA-based therapeutics are developing rapidly, and hold promise in the fight against flaviviruses. However, to develop efficient and safe therapeutics for flaviviruses, many challenges remain unsolved. AREAS COVERED: In this review, the authors briefly introduced the biology of flaviviruses and the current advances in RNA-based therapeutics for them. Furthermore, the authors list the challenges and possible solutions in this area. Finally, the authors give their opinion on the development and future of RNA-based therapeutics for flaviviruses. EXPERT OPINION: With the rapid development of structural biology, the crystal structures of flavivirus proteins may lay the foundation for future rational drug design. Studies regarding the interactions between the flavivirus and the host will also be invaluable to inhibitor design. Researchers should maintain the current momentum to bring about safe and effective anti-flavivirus drugs to licensure through joint efforts of academia, government, and industry.
Asunto(s)
Infecciones por Flavivirus , Flavivirus , Humanos , Flavivirus/genética , Flavivirus/metabolismo , ARN/metabolismo , ARN/farmacología , Infecciones por Flavivirus/tratamiento farmacológicoRESUMEN
Zika virus (ZIKV) is an RNA-enveloped virus that belongs to the Flavivirus genus, and ZIKV infections potentially induce severe neurodegenerative diseases and impair male fertility. Palmitoylation is an important post-translational modification of proteins that is mediated by a series of DHHC-palmitoyl transferases, which are implicated in various biological processes and viral infections. However, it remains to be investigated whether palmitoylation regulates ZIKV infections. In this study, we initially observed that the inhibition of palmitoylation by 2-bromopalmitate (2-BP) enhanced ZIKV infections, and determined that the envelope protein of ZIKV is palmitoylated at Cys308. ZDHHC11 was identified as the predominant enzyme that interacts with the ZIKV envelope protein and catalyzes its palmitoylation. Notably, ZDHHC11 suppressed ZIKV infections in an enzymatic activity-dependent manner and ZDHHC11 knockdown promoted ZIKV infection. In conclusion, we proposed that the envelope protein of ZIKV undergoes a novel post-translational modification and identified a distinct mechanism in which ZDHHC11 suppresses ZIKV infections via palmitoylation of the ZIKV envelope protein.
Asunto(s)
Flavivirus , Infección por el Virus Zika , Virus Zika , Humanos , Masculino , Anticuerpos Antivirales/metabolismo , Flavivirus/metabolismo , Proteínas/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Virus Zika/fisiologíaRESUMEN
Viruses from the Flavivirus genus infect millions of people worldwide and cause severe diseases, including recent epidemics of dengue virus (DENV), and Zika virus (ZIKV). There is currently no antiviral treatment against flavivirus infections, despite considerable efforts to develop inhibitors against essential viral enzymes including NS2B/NS3 protease. Targeting the flavivirus NS2B/NS3 protease proved to be challenging because of the conformational dynamics, topology, and electrostatic properties of the active site. Here, we report the identification of quinoxaline-based allosteric inhibitors by fragment-based drug discovery approach as a promising new drug-like scaffold to target the NS2B/NS3 protease. Enzymatic assays and mutational analysis of the allosteric site in ZIKV NS2B/NS3 protease support noncompetitive inhibition mechanism as well as engineered DENV protease construct indicating the compounds likely compete with the NS2B cofactor for binding to the protease domain. Furthermore, antiviral activity confirmed the therapeutic potential of this new inhibitor scaffold.
Asunto(s)
Flavivirus , Infección por el Virus Zika , Virus Zika , Humanos , Flavivirus/química , Flavivirus/metabolismo , Virus Zika/metabolismo , Péptido Hidrolasas , Quinoxalinas/farmacología , Proteínas no Estructurales Virales , Serina Endopeptidasas/metabolismo , Inhibidores de Proteasas/farmacología , Antivirales/químicaRESUMEN
Usutu virus (USUV), is a mosquito-borne flavivirus currently spreading outside the African continent producing substantial avian mortality. In contrast, infected humans could exhibit mild neurological symptoms or remain asymptomatic. As in other flaviviruses, the capped USUV genome encodes three structural and seven non-structural (NS) proteins. Among the NS proteins, NS5 plays crucial roles in virus replication, harbouring the capping and methyltransferase (MTase) activities in its N-terminal domain and the RNA-dependent RNA polymerase (RdRP) activity at the C-terminus. In this work, we present the first structural and functional characterization of the USUV MTase domain. The first structure of the USUV MTase has been determined in complex with its natural ligands (S-adenosyl-L-methionine [SAM]) and S-adenosyl-L-homocysteine [SAH]) at 2.2 Å resolution, showing a molecular dimer in the crystal asymmetric unit. One molecule is bound to the methyl donor SAM while the second is bound to the reaction by-product SAH. Both molecules are almost identical and also show a high structural similarity to the MTase domains of other flaviviruses. The structure of the USUV MTase bound to the inhibitor sinefungin at 1.8 Å resolution is also described. Careful comparisons of the interactions in the SAM-binding cavity prompt us to hypothesize about the strength and weakness of the structure-based design of antivirals directed to the SAM/SAH binding site that could be effective to deal with this threat.
Asunto(s)
Flavivirus , Metiltransferasas , Flavivirus/genética , Flavivirus/metabolismo , Metiltransferasas/química , ARN Polimerasa Dependiente del ARN/genética , S-Adenosilmetionina/metabolismo , Proteínas no Estructurales Virales/químicaRESUMEN
Dengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, its true receptor(s) for internalization into live cells has not yet been identified, and no successful antivirals or treatments have been isolated to this date. This study aims to improve our understanding of virus entry routes by exploring the sialic acid-based cell surface molecule GM1a and its role in DENV infection. We studied the interaction of the virus with GM1a using fluorescence correlation spectroscopy, fluorescence crosscorrelation spectroscopy, imaging fluorescence correlation spectroscopy, amide hydrogen/deuterium exchange mass spectrometry, and isothermal titration calorimetry. Additionally, we explored the effect of this interaction on infectivity and movement of the virus during infection was explored using plaque assay and fluorescence-based imaging and single particle tracking. GM1a was deemed to interact with DENV at domain I (DI) and domain II (DII) of the E protein of the protein coat at quaternary contacts of a fully assembled virus, leading to a 10-fold and 7-fold increase in infectivity for DENV1 and DENV2 in mammalian cell systems, respectively. We determined that the interaction of the virus with GM1a triggers a speeding up of virus movement on live cell surfaces, possibly resulting from a reduction in rigidity of cellular rafts during infection. Collectively, our results suggest that GM1a functions as a coreceptor/attachment factor for DENV during infection in mammalian systems.
Asunto(s)
Virus del Dengue , Dengue , Flavivirus , Animales , Humanos , Virus del Dengue/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Gangliósidos/metabolismo , Flavivirus/metabolismo , Mamíferos/metabolismoRESUMEN
Members of the mosquito-borne flavivirus genus such as dengue (DENV), West Nile (WNV), and Zika (ZIKV) viruses cause distinct diseases and affect different tissues. We previously found that the secreted flaviviral nonstructural protein 1 (NS1) interacts with endothelial cells and disrupts endothelial barrier function in a tissue-specific manner consistent with the disease tropism of the respective viruses. However, the underlying molecular mechanism of this tissue-specific NS1-endothelial cell interaction is not well understood. To elucidate the distinct role(s) that the wing and ß-ladder domains of NS1 play in NS1 interactions with endothelial cells, we constructed flavivirus NS1 chimeras that exchanged the wing and ß-ladder domains in a pairwise manner between DENV, WNV, and ZIKV NS1. We found that both the NS1 wing and ß-ladder domains conferred NS1 tissue-specific endothelial dysfunction, with the wing conferring cell binding and the ß-ladder involved in inducing endothelial hyperpermeability as measured by transendothelial electrical resistance. To narrow down the amino acids dictating cell binding specificity, we utilized the DENV-WNV NS1 chimera and identified residues 91 to 93 (GDI) of DENV NS1 as a molecular motif determining binding specificity. Further, using an in vivo mouse model of localized leak, we found that the GDI motif of the wing domain was essential for triggering DENV NS1-induced vascular leak in mouse dermis. Taken together, we identify molecular determinants of flavivirus NS1 that confer NS1 binding and vascular leak and highlight the importance of the NS1 wing domain for flavivirus pathogenesis. IMPORTANCE Flavivirus NS1 is secreted into the bloodstream from infected cells during a viral infection. Dengue virus NS1 contributes to severe dengue pathology such as endothelial dysfunction and vascular leak independently of the virus. We have shown that multiple flavivirus NS1 proteins result in endothelial dysfunction in a tissue-specific manner consistent with their respective viral tropism. Here, we aimed to identify the molecular determinants that make some, but not other, flavivirus NS1 proteins bind to select endothelial cells in vitro and cause vascular leak in a mouse model. We identified the wing domain of NS1 as a primary determinant conferring differential endothelial dysfunction and vascular leak and narrowed the contributing amino acid residues to a three-residue motif within the wing domain. The insights from this study pave the way for future studies on the effects of flavivirus NS1 on viral dissemination and pathogenesis and offer potential new avenues for antiviral therapies.
Asunto(s)
Células Endoteliales , Flavivirus , Proteínas no Estructurales Virales , Tropismo Viral , Aminoácidos/metabolismo , Animales , Antivirales/metabolismo , Comunicación Celular , Virus del Dengue/genética , Células Endoteliales/virología , Flavivirus/metabolismo , Flavivirus/patogenicidad , Infecciones por Flavivirus , Ratones , Proteínas no Estructurales Virales/metabolismo , Virus del Nilo Occidental , Virus ZikaRESUMEN
Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that mainly causes a decrease in egg production in infected waterfowl. Similar to other members of the Flaviviridae family, it can proliferate in most mammalian cells and may also pose a potential threat to nonavian animals. In previous studies, we found that DTMUV infection can upregulate suppressor of cytokine signaling 1 (SOCS1) to inhibit type I interferon (IFN) production and promote virus replication, but the specific mechanism is unclear. Furthermore, little is known about the regulatory role of ubiquitination during flavivirus infection. In this study, we found that activation of Toll-like receptor 3 (TLR3) signaling rather than type I IFN stimulation led to the upregulation of SOCS1 during DTMUV infection. Further studies revealed that JOSD1 stabilized SOCS1 expression by binding to the SH2 domain of SOCS1 and mediating its deubiquitination. In addition, JOSD1 also inhibited type I IFN production through SOCS1. Finally, SOCS1 acts as an E3 ubiquitin ligase that binds to IFN regulatory factor 7 (IRF7) through its SH2 domain and mediates K48-linked ubiquitination and proteasomal degradation of IRF7, ultimately inhibiting type I IFN production mediated by IRF7 and promoting viral proliferation. These results will enrich and deepen our understanding of the mechanism by which DTMUV antagonizes the host interferon system. IMPORTANCE DTMUV is a newly discovered flavivirus that seriously harms the poultry industry. In recent years, there have been numerous studies on the involvement of ubiquitination in the regulation of innate immunity. However, little is known about the involvement of ubiquitination in the regulation of flavivirus-induced type I IFN signaling. In this study, we found that SOCS1 was induced by TLR3 signaling during DTMUV infection. Furthermore, we found for the first time that duck SOCS1 protein was also modified by K48-linked polyubiquitination, whereas our previous study found that SOCS1 was upregulated during DTMUV infection. Further studies showed that JOSD1 stabilized SOCS1 expression by mediating the deubiquitination of SOCS1. While SOCS1 acts as a negative regulator of cytokines, we found that DTMUV utilized SOCS1 to mediate the ubiquitination and proteasomal degradation of IRF7 and ultimately inhibit type I IFN production, thereby promoting its proliferation.
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Infecciones por Flavivirus , Flavivirus , Interacciones Microbiota-Huesped , Interferón Tipo I , Enfermedades de las Aves de Corral , Animales , Patos , Endopeptidasas/genética , Endopeptidasas/metabolismo , Retroalimentación Fisiológica , Flavivirus/metabolismo , Infecciones por Flavivirus/inmunología , Infecciones por Flavivirus/virología , Interacciones Microbiota-Huesped/inmunología , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Interferón Tipo I/inmunología , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/virología , Transducción de Señal/genética , Transducción de Señal/inmunología , Proteína 1 Supresora de la Señalización de Citocinas/genética , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Receptor Toll-Like 3/metabolismo , Ubiquitina-Proteína Ligasas , Regulación hacia ArribaRESUMEN
Dengue virus, a flavivirus that causes dengue shock syndrome and dengue hemorrhagic fever, is currently prevalent worldwide. A two-component protease (NS2B-NS3) is essential for maturation, representing an important target for designing anti-flavivirus drugs. Previously, consideration has been centered on developing active-site inhibitors of NS2B-NS3pro. However, the flat and charged nature of its active site renders difficulties in developing inhibitors, suggesting an alternative strategy for identifying allosteric inhibitors. The allosterically sensitive site of the dengue protease is located near Ala125, between the 120s loop and 150s loop. Using atomistic molecular dynamics simulations, we have explored the protease's conformational dynamics upon binding of an allosteric inhibitor. Furthermore, characterization of the inherent flexible loops (71-75s loop, 120s loop, and 150s loop) is carried out for allosteric-inhibitor-bound wild-type and mutant A125C variants and a comparison is performed with its unbound state to extract the structural changes describing the inactive state of the protease. Our study reveals that compared to the unliganded system, the inhibitor-bound system shows large structural changes in the 120s loop and 150s loop in contrast to the rigid 71-75s loop. The unliganded system shows a closed-state pocket in contrast to the open state for the wild-type complex that locks the protease into the open and inactive-state conformations. However, the mutant complex fluctuates between open and closed states. Also, we tried to see how mutation and binding of an allosteric inhibitor perturb the connectivity in a protein structure network (PSN) at contact levels. Altogether, our study reveals the mechanism of conformational rearrangements of loops at the molecular level, locking the protein in an inactive conformation, which may be useful for developing allosteric inhibitors.
Asunto(s)
Virus del Dengue , Dengue , Flavivirus , Proteínas no Estructurales Virales , Dominio Catalítico , Dengue/metabolismo , Virus del Dengue/metabolismo , Flavivirus/metabolismo , Humanos , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , ARN Helicasas/química , Serina Endopeptidasas/química , Proteínas no Estructurales Virales/químicaRESUMEN
Zika virus (ZIKV) NS4B protein is a membranotropic multifunctional protein. Despite its versatile functioning, its topology and dynamics are not entirely understood. There is no X-ray or cryo-EM structure available for any flaviviral NS4B full-length protein. In this study, we have investigated the structural dynamics of full-length ZIKV NS4B protein through 3D structure models using molecular dynamics simulations and experimental techniques. Also, we employed a reductionist approach to understand the dynamics of NS4B protein where we studied its N-terminal (residues 1-38), C-terminal (residues 194-251), and cytosolic (residues 131-169) regions in isolation in addition to the full-length protein. Further, using a series of circular dichroism spectroscopic experiments, we validate the cytosolic region as an intrinsically disordered protein region. The microsecond-long all atoms molecular dynamics and replica-exchange simulations complement the experimental observations. Furthermore, we have also studied the NS4B proteins C-terminal regions of four other flaviviruses viz. DENV2, JEV, WNV, and YFV through microsecond simulations to characterize their behaviour in presence and absence of lipid membranes. There are significant differences observed in the conformations of other flavivirus NS4B C-terminal regions in comparison to ZIKV NS4B. Lastly, we have proposed a ZIKV NS4B protein model illustrating its putative topology consisting of various membrane-spanning and non-membranous regions.
Asunto(s)
Flavivirus , Proteínas Intrínsecamente Desordenadas , Proteínas no Estructurales Virales/química , Infección por el Virus Zika , Virus Zika , Flavivirus/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/metabolismo , Lípidos , Virus Zika/químicaRESUMEN
Diseases caused by flaviviruses such as dengue virus (DENV) and West Nile Virus (WNV), are a serious threat to public health. The flavivirus single-stranded RNA genome is translated into a polyprotein which is cleaved into three structural proteins and seven non-structural proteins by the viral and cellular proteases. Non-structural (NS) protein 3 is a multifunctional protein that has N-terminal protease and C-terminal helicase domains. The NS3 protease requires co-factor NS2B for enzymatic activity and folding. Due to its essential role in viral replication, NS2B-NS3 protease is an attractive target for antiviral drugs. Despite the availability of crystal structures, dynamic interactions of the N- and C-termini of NS2B co-factor have been elusive due to their flexible fold. In this study, we employ integrative structural approaches combined with biochemical assays to elucidate the dynamic interactions of the flexible DENV4 NS2B and NS3 N- and C-termini. We captured the crystal structure of self-cleaved DENV4 NS2B47NS3 protease in post cleavage state. The intermediate conformation adopted in the reported structure can be targeted by allosteric inhibitors. Comparison of our new findings from DENV4 against previously studied ZIKV NS2B-NS3 proteins reveals differences in NS2B-NS3 function between the two viruses. No inhibition of protease activity was observed for unlinked DENV NS2B-NS3 in presence of the cleavage site while ZIKV NS2B-NS3 cleavage inhibits protease activity. Another difference is that binding of the NS2B C-terminus to DENV4 eNS2B47NS3Pro active site is mediated via interactions with P4-P6 residues while for ZIKV, the binding of NS2B C-terminus to active site is mediated by P1-P3 residues. The mapping of NS2B N- and C-termini with NS3 indicates that these intermolecular interactions occur mainly on the beta-barrel 2 of the NS3 protease domain. Our integrative approach enables a comprehensive understanding of the folding and dynamic interactions of DENV NS3 protease and its cofactor NS2B.