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Hepatitis B virus (HBV) infection affects hepatic metabolism. Serum metabolomics studies have suggested that HBV possibly hijacks the glycerol-3-phosphate (G3P) shuttle. In this study, the two glycerol-3-phosphate dehydrogenases (GPD1 and GPD2) in the G3P shuttle were analyzed for determining their role in HBV replication and the findings revealed that GPD2 and not GPD1 inhibited HBV replication. The knockdown of GPD2 expression upregulated HBV replication, while GPD2 overexpression reduced HBV replication. Moreover, the overexpression of GPD2 significantly reduced HBV replication in hydrodynamic injection-based mouse models. Mechanistically, this inhibitory effect is related to the GPD2-mediated degradation of HBx protein by recruiting the E3 ubiquitin ligase TRIM28 and not to the alterations in G3P metabolism. In conclusion, this study revealed GPD2, a key enzyme in the G3P shuttle, as a host restriction factor in HBV replication. IMPORTANCE The glycerol-3-phosphate (G3P) shuttle is important for the delivery of cytosolic reducing equivalents into mitochondria for oxidative phosphorylation. The study analyzed two key components of the G3P shuttle and identified GPD2 as a restriction factor in HBV replication. The findings revealed a novel mechanism of GPD2-mediated inhibition of HBV replication via the recruitment of TRIM28 for degrading HBx, and the HBx-GPD2 interaction could be another potential therapeutic target for anti-HBV drug development.
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Glicerolfosfato Desidrogenase , Hepatite B , Proteína 28 com Motivo Tripartido , Proteínas Virais Reguladoras e Acessórias , Animais , Camundongos , Glicerol/metabolismo , Glicerolfosfato Desidrogenase/metabolismo , Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Mitocôndrias/enzimologia , Fosfatos/metabolismo , Proteína 28 com Motivo Tripartido/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo , Replicação ViralRESUMO
Because of their various reactivities, propargyl acetates are refined chemical intermediates that are extensively applied in pharmaceutical synthesis. Currently, reactions between propargyl acetates and chlorosilanes may be the most effective method for synthesizing silylallenes. Nevertheless, owing to the adaptability and selectivity of substrates, transition metal catalysis is difficult to achieve. Herein, nickel-catalyzed reductive cross-coupling reactions between propargyl acetates and substituted vinyl chlorosilanes for the synthesis of tetrasubstituted silylallenes are described. Therein, metallic zinc is a crucial reductant that effectively enables two electrophilic reagents to selectively construct C(sp2)-Si bonds. Additionally, a Ni-catalyzed reductive mechanism involving a radical process is proposed on the basis of deuteration-labeled experiments.
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A visible-light-driven photoredox dialkylation of styrenes with α-carbonyl alkyl bromides and pyridin-1-ium salts for the synthesis of polysubstituted 1,4-dihydropyridines is reported. This reaction enables the formation of two new C(sp3)-C(sp3) bonds in a single reaction step and provides a strategy that employs pyridin-1-ium salts as the functionalized alkylating reagents via dearomatization to directly trap the resulting alkyl radicals from radical addition of alkenes and then terminate the alkene dialkylation.
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Tick-borne encephalitis virus (TBEV) is the causative agent of a potentially fatal neurological infection in humans. Investigating virus-host interaction is important for understanding the pathogenesis of TBEV and developing effective antiviral drugs against this virus. Here, we report that mammalian ste20-like kinase 3 (MST3) is involved in the regulation of TBEV infection. The knockdown or knockout of MST3, but not other mammalian ste20-like kinase family members, inhibited TBEV replication. The knockdown of MST3 also significantly reduced TBEV replication in mouse primary astrocytes. Life cycle analysis indicated that MST3 remarkably impaired virion assembly efficiency and specific infectivity by respectively 59% and 95% in MST3-knockout cells. We further found that MST3 interacts with the viral proteins NS2A and prM; and MST3 enhances the interaction of NS2A-NS4A. Thus, MST3-NS2A complex plays a major role in recruiting prM-E heterodimers and NS4A and mediates the virion assembly. Additionally, we found that MST3 was biotinylated and combined with other proteins (e.g., ATG5, Sec24A, and SNX4) that are associated with the cellular membrane required for TBEV infection. Overall, our study revealed a novel function for MST3 in TBEV infection and identified as a novel host factor supporting TBEV assembly.
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Vírus da Encefalite Transmitidos por Carrapatos , Encefalite Transmitida por Carrapatos , Animais , Camundongos , Humanos , Vírus da Encefalite Transmitidos por Carrapatos/genética , Proteínas Virais/metabolismo , Mamíferos/metabolismo , Proteínas de Transporte VesicularRESUMO
Histone deacetylase 5 (HDAC5) has been reported to have a strong regulatory function in the proinflammatory response, but the mechanism is still unknown. Here, we identified HDAC5 as a positive regulator of NF-κB signaling in vivo. HDAC5-deficient mice exhibited enhanced survival in response to LPS challenge. Using LPS, TNFα, different kinds of viruses, hydrogen peroxide, or ultraviolet stimulation, we demonstrate that HDAC5-mediated regulation of NF-κB occurs in manners both dependent on and independent of IKK, an upstream kinase in the NF-κB signaling pathway. Deficiency in HDAC5 impaired the phosphorylation of IKKß, subsequent phosphorylation of the NF-κB inhibitor protein IκBα and NF-κB subunit p65. We also show that the phosphatase PP2A repressed transcriptional activation of NF-κB by decreasing phosphorylation of IKKß, p65, and IκBα. In vitro deacetylation experiments and site-directed mutagenesis experiments indicated that HDAC5 directly deacetylated PP2Ac at Lys136, which resulted in the deactivation of PP2A. Our data add mechanistic insight into the cross talk between epigenetic and posttranslational modifications regulating NF-κB signaling and protein phosphatase activation that mediate survival in response to inflammatory challenges.
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Histona Desacetilases/metabolismo , Proteína Fosfatase 2/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Acetilação , Animais , Chlorocebus aethiops , Células HEK293 , Histona Desacetilases/genética , Humanos , Camundongos , Camundongos Knockout , Proteína Fosfatase 2/genética , Células RAW 264.7 , Células THP-1 , Fator de Transcrição RelA/genética , Células VeroRESUMO
A novel hypervalent iodine-triggered hydroamination of homopropargyl sulfonamides with copper halides to obtain dihalo-2,3-dihydropyrroles was efficiently developed. The reaction can provide access to chloro/bromo-cyclization extended from iodocyclization to prepare potentially useful halogenated building blocks for drug development with broad functional group tolerance and high yield yet in a short time.
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Site selective functionalization of inert remote C(sp3)-H bonds to increase molecular complexity offers vital potential for chemical synthesis and new drug development, thus it has been attracting ongoing research interest. In particular, typical ß-C(sp3)-H arylation methods using chelation-assisted metal catalysis or metal-catalyzed oxidative/photochemical in situ generated allyl C(sp3)-H bond processes have been well developed. However, radical-mediated direct ß-C(sp3)-H arylation of carbonyls remains elusive. Herein, we describe an iodoarene-directed photoredox ß-C(sp3)-H arylation of 1-(o-iodoaryl)alkan-1-ones with cyanoarenes via halogen atom transfer (XAT) and hydrogen atom transfer (HAT). The method involves diethylaminoethyl radical-mediated generation of an aryl radical intermediate via XAT, then directed 1,5-HAT to form the remote alkyl radical intermediate and radical-radical coupling with cyanoarenes, and is applicable to a broad scope of unactivated remote C(sp3)-H bonds like ß-C(sp3)-H bonds of o-iodoaryl-substituted alkanones and α-C(sp3)-H bonds of o-iodoarylamides. Experimental findings are supported by computational studies (DFT calculations), revealing that this method operates via a radical-relay stepwise mechanism involving multiple SET, XAT, 1,5-HAT and radical-radical coupling processes.
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An electroreductive cross-coupling of prop-2-yn-1-yl acetates with chloro(vinyl)silanes for producing tetrasubstituted silylallenes is developed. The method enables the formation of a new CâSi bond through the cathodic reduction formation of the silyl radical, radical addition across the C≡C bond, the alkenyl anion intermediate formation, and deacetoxylation and represents a mild, practical route to the synthesis of silylallenes. Mechanistic studies reveal that CoCl2 acts as the mediator to promote the formation of the alkenyl anion intermediate via electron transfer.
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A palladium(0)-catalyzed ß-C(sp3)-H arylation of silyl prop-1-en-1-ol ethers with aryl halides for the synthesis of α,ß-unsaturated ketones is presented. In contrast to the reported ß-C(sp3)-H arylation of ketones, the chemoselectivity of this current method relies on the Pd(0) catalytic systems and reaction temperatures: While using the Pd(dba)2/DavePhos/KF system at 80 °C resulted in ß-C(sp3)-H monoarylation to produce ß-monoarylated α,ß-unsaturated ketones, harnessing the Pd(OAc)2/t-Bu XPhos/K2HPO4 system at 110 °C induced ß-C(sp3)-H diarylation to afford ß,ß-diarylated α,ß-unsaturated ketones. The method provides a versatile route that uses readily available ketone-derivatized α-nonsubstituted silyl prop-1-en-1-ol ethers as the alkene sources and is characterized by a good functional group compatibility, a broad substrate scope, and an excellent selectivity.
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Tick-borne encephalitis virus (TBEV) is an important tick-borne pathogen that poses as a serious public health concern. The coverage and immunogenicity of the currently available vaccines against TBEV are relatively low; therefore, it is crucial to develop novel and effective vaccines against TBEV. The present study describes a novel strategy for the assembly of virus-like particles (VLPs) by co-expressing the structural (core/prM/E) and non-structural (NS2B/NS3Pro) proteins of TBEV. The efficacy of the VLPs was subsequently evaluated in C57BL/6 mice, and the resultant IgG serum could neutralize both Far-Eastern and European subtypes of TBEV. These findings indicated that the VLP-based vaccine elicited the production of cross-subtype reactive antibodies. The VLPs provided protection to mice lacking the type I interferon receptor (IFNAR-/-) against lethal TBEV challenge, with undetectable viral load in brain and intestinal tissues. Furthermore, the group that received the VLP vaccine did not exhibit significant pathological changes and the inflammatory factors were significantly suppressed compared to the control group. Immunization with the VLP vaccine induced the production of multiple-cytokine-producing antiviral CD4+ T cells in vivo, including TNF-α+, IL-2+, and IFN-γ+ T cells. Altogether, the findings suggest that noninfectious VLPs can serve as a potentially safe and effective vaccine candidate against diverse subtypes of TBEV.
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Vírus da Encefalite Transmitidos por Carrapatos , Encefalite Transmitida por Carrapatos , Vacinas de Partículas Semelhantes a Vírus , Animais , Camundongos , Vírus da Encefalite Transmitidos por Carrapatos/genética , Vacinas de Partículas Semelhantes a Vírus/genética , Anticorpos Antivirais , Encefalite Transmitida por Carrapatos/prevenção & controle , Camundongos Endogâmicos C57BLRESUMO
Several variants of concern (VOCs) have emerged since the WIV04 strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first isolated in January 2020. Due to mutations in the spike (S) protein, these VOCs have evolved to enhance viral infectivity and immune evasion. However, whether mutations of the other viral proteins lead to altered viral propagation and drug resistance remains obscure. The replicon is a noninfectious viral surrogate capable of recapitulating certain steps of the viral life cycle. Although several SARS-CoV-2 replicons have been developed, none of them were derived from emerging VOCs and could only recapitulate viral genome replication and subgenomic RNA (sgRNA) transcription. In this study, SARS-CoV-2 replicons derived from the WIV04 strain and two VOCs (the Beta and Delta variants) were prepared by removing the S gene from their genomes, while other structural genes remained untouched. These replicons not only recapitulate viral genome replication and sgRNA transcription but also support the assembly and release of viral-like particles, as manifested by electron microscopic assays. Thus, the S-deletion replicon could recapitulate virtually all the post-entry steps of the viral life cycle and provides a versatile tool for measuring viral intracellular propagation and screening novel antiviral drugs, including inhibitors of virion assembly and release. Through the quantification of replicon RNA released into the supernatant, we demonstrate that viral intracellular propagation and drug response to remdesivir have not yet substantially changed during the evolution of SARS-CoV-2 from the WIV04 strain to the Beta and Delta VOCs.
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COVID-19 , SARS-CoV-2 , Humanos , Antivirais/farmacologia , Replicon , RNA , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus , Proteínas Virais , Vírion/genéticaRESUMO
Hepatitis viruses are primary causative agents of hepatitis and represent a major source of public health problems in the world. The host innate immune system forms the first line of defense against hepatitis viruses. Hepatitis viruses are sensed by specific pathogen recognition receptors (PRRs) that subsequently trigger the innate immune response and interferon (IFN) production. However, hepatitis viruses evade host immune surveillance via multiple strategies, which help compromise the innate immune response and create a favorable environment for viral replication. Therefore, this article reviews published findings regarding host innate immune sensing and response against hepatitis viruses. Furthermore, we also focus on how hepatitis viruses abrogate the antiviral effects of the host innate immune system.
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Merging photoredox/nickel catalysis enabling the cross-electrophile coupling of aziridines with pyridin-1-ium salts involving dearomatization for the synthesis of ß-(1,4-dihydropyridin-4-yl)-ethylamines, especially including bioactive motif-based analogues, is described. This method allows incorporation of a 1,4-dihydropyridin-4-yl group and formation a N-H amino group to construct highly valuable ß-(1,4-dihydropyridin-4-yl)-ethylamine frameworks in a single step through the C2-N bond regioselective cleavage and dearomatization alkylation cascades with precise regioselectivity and excellent functional group tolerance, and represents an appealing cross-electrophile coupling strategy to accomplish transformations between two electrophiles, including aziridines and pyridin-1-ium salts, by avoiding prefunctionalization.
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TANK-binding kinase 1 (TBK1), a core kinase of antiviral pathways, activates the production of interferons (IFNs). It has been reported that deacetylation activates TBK1; however, the precise mechanism still remains to be uncovered. We show here that during the early stage of viral infection, the acetylation of TBK1 was increased, and the acetylation of TBK1 at Lys241 enhanced the recruitment of IRF3 to TBK1. HDAC3 directly deacetylated TBK1 at Lys241 and Lys692, which resulted in the activation of TBK1. Deacetylation at Lys241 and Lys692 was critical for the kinase activity and dimerization of TBK1 respectively. Using knockout cell lines and transgenic mice, we confirmed that a HDAC3 null mutant exhibited enhanced susceptibility to viral challenge via impaired production of type I IFNs. Furthermore, activated TBK1 phosphorylated HDAC3, which promoted the deacetylation activity of HDAC3 and formed a feedback loop. In this study, we illustrated the roles the acetylated and deacetylated forms of TBK1 play in antiviral innate responses and clarified the post-translational modulations involved in the interaction between TBK1 and HDAC3.
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Histona Desacetilases/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Viroses/imunologia , Animais , Chlorocebus aethiops , Células HEK293 , Histona Desacetilases/genética , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/genética , Células RAW 264.7 , Células THP-1 , Células Vero , Viroses/genéticaRESUMO
A three-component alkene alkylazidation using sodium azide as the azido resource and heteroarenium salts as functionalized alkyl reagents for producing highly valuable 2-azido-1-(1,4-dihydropyridin-4-yl)-ethanes is described. This reaction allows the incorporation of both an azido group and a 1,4-dihydropyridin-4-yl group across C[double bond, length as m-dash]C bonds to construct two new bonds in a single reaction step, and represents a practical and mechanistically distinct alternative that harnesses an electrophilic heteroarenium ion to accomplish the alkene difunctionalization reaction.
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Protein modification by small ubiquitin-like modifier (SUMO) is an important regulatory mechanism for multiple cellular processes. Although the canonical pathway involving the ubiquitylation or phosphorylation of IκBα has been well characterized, little is known about the sumoylation of IκBα in the control of NF-κB activity. Here, we find that histone deacetylase 4 (HDAC4) negatively regulates tumor necrosis factor-alpha- or lipopolysaccharide-triggered NF-κB activation. HDAC4 belongs to the SUMO E3 ligase family and can directly sumoylate IκBα. The cytoplasm location of HDAC4 is essential for IκBα sumoylation. The Cys292 of HDAC4 is a key site for its SUMO E3 ligase activity. The sumoylation of IκBα prevents its polyubiquitination and degradation because these two modifications occur both at the Lys21. Our findings reveal a previously undiscovered role for HDAC4 in the inflammatory response as a SUMO E3 ligase for IκBα sumoylation. Our work provides insight into mechanisms ensuring optimal mediation of the NF-κB pathway.
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Histona Desacetilases/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/genética , Sumoilação/genética , Animais , Células HEK293 , Histona Desacetilases/genética , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Células RAW 264.7 , Proteínas Recombinantes/farmacologia , Proteínas Repressoras/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Class II HDACs, such as HDAC4, are critical regulators of the immune response in various immune cells; however, its role in innate immunity remains largely unknown. Here, we report that the overexpression of HDAC4 suppresses the production of type I interferons triggered by pattern-recognition receptors (PRRs). HDAC4 repressed the translocation of transcription factor IRF3 to the nucleus, thereby decreasing IRF3-mediated IFN-ß expression. In particular, we also determined that HDAC4 can be phosphorylated and simultaneously block the phosphorylation of IRF3 at Ser386 and Ser396 by TBK1 and IKKε, respectively, by interacting with the kinase domain of TBK1 and IKKε. Furthermore, IFN-ß may stimulate the expression of HDAC4. Our findings suggest that HDAC4 acts as a regulator of PRR signaling and is a novel mechanism of negative feedback regulation for preventing an over-reactive innate immune response.
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Histona Desacetilases/metabolismo , Quinase I-kappa B/metabolismo , Imunidade Inata/fisiologia , Fator Regulador 3 de Interferon/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Animais , Chlorocebus aethiops , Células HEK293 , Humanos , Interferon Tipo I/metabolismo , Camundongos , Fosforilação , Transporte Proteico , Células RAW 264.7 , Transdução de Sinais , Fatores de Transcrição , Células Vero , Vesiculovirus , VirosesRESUMO
Histone deacetylase (HDAC) inhibitors show clinical promise for the treatment of cancers, including hepatocellular carcinoma (HCC). In this study, we investigated the effect of HDAC inhibitor treatment on hepatitis C virus (HCV) replication in Huh7 human liver cells and in a mouse model of HCV infection. Viral replication was markedly suppressed by the HDAC3 inhibitor at concentrations below 1 mmol/L, with no cellular toxicity. This was accompanied by upregulation of liver-expressed antimicrobial peptide 1(LEAP-1) and downregulation of apolipoprotein-A1 (Apo-A1), as determined by microarray and quantitative RT-PCR analyses. Moreover, HDAC3 was found to modulate the binding of CCAAT-enhancer-binding protein α (C/EBPα), hypoxia-inducible factor 1α (HIF1α), and signal transducer and activator of transcription 3 (STAT3) to the LEAP-1 promoter. HDAC3 inhibitor treatment also blocked HCV replication in a mouse model of HCV infection. These results indicate that epigenetic therapy with HDAC3 inhibitor may be a potential treatment for diseases associated with HCV infection such as HCC.
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Apolipoproteína A-I/metabolismo , Hepacivirus/efeitos dos fármacos , Hepcidinas/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Replicação Viral/efeitos dos fármacos , Acrilamidas/farmacologia , Animais , Apolipoproteína A-I/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Hepacivirus/fisiologia , Hepcidinas/genética , Humanos , Ácidos Hidroxâmicos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Indóis/farmacologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Camundongos , Camundongos Transgênicos , Fenilenodiaminas/farmacologia , Ligação Proteica , Fator de Transcrição STAT3/metabolismoRESUMO
A novel copper/silver cocatalyzed oxidative coupling of vinylarenes with ICH2CF3 or ICH2CHF2 through a radical process has been developed. The transformation provides an attractive approach to ß-CF3/CHF2-substituted ketones, with the advantages of easily available starting materials and operational simplicity.