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1.
J Biol Chem ; 300(6): 107307, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38657868

RESUMO

African swine fever, caused by the African swine fever virus (ASFV), is a viral hemorrhagic disease that affects domestic pigs and wild boars. ASFV infection causes extensive tissue damage, and the associated mechanism is poorly understood. Pyroptosis is characterized by the activation of inflammatory caspases and pore formation in the cellular plasma membrane, resulting in the release of inflammatory cytokines and cell damage. How ASFV infection regulates pyroptosis remains unclear. Here, using siRNA assay and overexpression methods, we report that ASFV infection regulated pyroptosis by cleaving the pyroptosis execution protein gasdermin A (GSDMA). ASFV infection activated caspase-3 and caspase-4, which specifically cleaved GSDMA at D75-P76 and D241-V242 to produce GSDMA into five fragments, including GSDMA-N1-75, GSDMA-N1-241, and GSDMA-N76-241 fragments at the N-terminal end of GSDMA. Only GSDMA-N1-241, which was produced in the late stage of ASFV infection, triggered pyroptosis and inhibited ASFV replication. The fragments, GSDMA-N1-75 and GSDMA-N76-241, lose the ability to induce pyroptosis. Overall ASFV infection differentially regulates pyroptosis by GSDMA in the indicated phase, which may be conducive to its own replication. Our findings reveal a novel molecular mechanism for the regulation of pyroptosis.


Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Caspase 3 , Caspases Iniciadoras , Piroptose , Vírus da Febre Suína Africana/metabolismo , Animais , Febre Suína Africana/metabolismo , Febre Suína Africana/virologia , Febre Suína Africana/patologia , Suínos , Caspase 3/metabolismo , Caspase 3/genética , Caspases Iniciadoras/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Ligação a Fosfato/metabolismo , Células HEK293 , Replicação Viral
2.
Virology ; 593: 110014, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38401340

RESUMO

African swine fever (ASF) caused by African swine fever virus (ASFV) is a highly infectious and lethal swine disease. Currently, there is only one novel approved vaccine and no antiviral drugs for ASFV. In the study, a high-throughput screening of an FDA-approved drug library was performed to identify several drugs against ASFV infection in primary porcine alveolar macrophages. Triapine and cytarabine hydrochloride were identified as ASFV infection inhibitors in a dose-dependent manner. The two drugs executed their antiviral activity during the replication stage of ASFV. Furthermore, molecular docking studies showed that triapine might interact with the active center Fe2+ in the small subunit of ASFV ribonucleotide reductase while cytarabine hydrochloride metabolite might interact with three residues (Arg589, Lys593, and Lys631) of ASFV DNA polymerase to block new DNA chain extension. Taken together, our results suggest that triapine and cytarabine hydrochloride displayed significant antiviral activity against ASFV in vitro.


Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Piridinas , Tiossemicarbazonas , Suínos , Animais , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/metabolismo , Febre Suína Africana/prevenção & controle , Simulação de Acoplamento Molecular , Antivirais/farmacologia , Antivirais/metabolismo , Citarabina/metabolismo , Citarabina/farmacologia , Replicação Viral
3.
Proc Natl Acad Sci U S A ; 120(49): e2306390120, 2023 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-38015841

RESUMO

Hepatitis B virus (HBV) remains a major public health threat with nearly 300 million people chronically infected worldwide who are at a high risk of developing hepatocellular carcinoma. Current therapies are effective in suppressing HBV replication but rarely lead to cure. Current therapies do not affect the HBV covalently closed circular DNA (cccDNA), which serves as the template for viral transcription and replication and is highly stable in infected cells to ensure viral persistence. In this study, we aim to identify and elucidate the functional role of cccDNA-associated host factors using affinity purification and protein mass spectrometry in HBV-infected cells. Nucleolin was identified as a key cccDNA-binding protein and shown to play an important role in HBV cccDNA transcription, likely via epigenetic regulation. Targeting nucleolin to silence cccDNA transcription in infected hepatocytes may be a promising therapeutic strategy for a functional cure of HBV.


Assuntos
Hepatite B , Neoplasias Hepáticas , Humanos , Vírus da Hepatite B/fisiologia , Epigênese Genética , Replicação Viral/genética , DNA Viral/metabolismo , DNA Circular/genética , DNA Circular/metabolismo , Neoplasias Hepáticas/genética , Hepatite B/genética , Hepatite B/metabolismo , Nucleolina
4.
J Biol Chem ; 299(7): 104844, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37209818

RESUMO

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs are regulated by different stimulators including viral infection, which is involved in the antiviral activity of host cells to limit viral propagation. To survive, several viruses have been reported to execute various strategies, such as modulating SG formation, to create optimal surroundings for viral replication. African swine fever virus (ASFV) is one of the most notorious pathogens in the global pig industry. However, the interplay between ASFV infection and SG formation remains largely unknown. In this study, we found that ASFV infection inhibited SG formation. Through SG inhibitory screening, we found that several ASFV-encoded proteins are involved in inhibition of SG formation. Among them, an ASFV S273R protein (pS273R), the only cysteine protease encoded by the ASFV genome, significantly affected SG formation. ASFV pS273R interacted with G3BP1 (Ras-GTPase-activating protein [SH3 domain] binding protein 1), a vital nucleating protein of SG formation. Furthermore, we found that ASFV pS273R cleaved G3BP1 at the G140-F141 to produce two fragments (G3BP1-N1-140 and G3BP1-C141-456). Interestingly, both the pS273R-cleaved fragments of G3BP1 lost the ability to induce SG formation and antiviral activity. Taken together, our finding reveals that the proteolytic cleavage of G3BP1 by ASFV pS273R is a novel mechanism by which ASFV counteracts host stress and innate antiviral responses.


Assuntos
Vírus da Febre Suína Africana , Grânulos de Estresse , Proteínas Virais , Animais , Febre Suína Africana/metabolismo , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/enzimologia , Vírus da Febre Suína Africana/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Grânulos de Estresse/metabolismo , Suínos , Replicação Viral/fisiologia , Chlorocebus aethiops , Humanos , Células HEK293 , Células Cultivadas , Macrófagos Alveolares/virologia , Proteínas Virais/metabolismo , Proteólise
5.
J Biol Chem ; 298(1): 101480, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34890644

RESUMO

African swine fever (ASF) is a viral hemorrhagic disease that affects domestic pigs and wild boar and is caused by the African swine fever virus (ASFV). The ASFV virion contains a long double-stranded DNA genome, which encodes more than 150 proteins. However, the immune escape mechanism and pathogenesis of ASFV remain poorly understood. Here, we report that the pyroptosis execution protein gasdermin D (GSDMD) is a new binding partner of ASFV-encoded protein S273R (pS273R), which belongs to the SUMO-1 cysteine protease family. Further experiments demonstrated that ASFV pS273R-cleaved swine GSDMD in a manner dependent on its protease activity. ASFV pS273R specifically cleaved GSDMD at G107-A108 to produce a shorter N-terminal fragment of GSDMD consisting of residues 1 to 107 (GSDMD-N1-107). Interestingly, unlike the effect of GSDMD-N1-279 fragment produced by caspase-1-mediated cleavage, the assay of LDH release, cell viability, and virus replication showed that GSDMD-N1-107 did not trigger pyroptosis or inhibit ASFV replication. Our findings reveal a previously unrecognized mechanism involved in the inhibition of ASFV infection-induced pyroptosis, which highlights an important function of pS273R in inflammatory responses and ASFV replication.


Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Cisteína Proteases , Proteínas de Ligação a Fosfato , Proteínas Citotóxicas Formadoras de Poros , Proteínas Virais , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/enzimologia , Vírus da Febre Suína Africana/metabolismo , Animais , Cisteína Proteases/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptose , Sus scrofa , Suínos , Proteínas Virais/metabolismo
6.
Viruses ; 13(11)2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34835046

RESUMO

African swine fever (ASF) is a severe hemorrhagic disease in swine characterized by massive lymphocyte depletion and cell death, with apoptosis and necrosis in infected lymphoid tissues. However, the molecular mechanism regarding ASFV-induced cell death remains largely unknown. In this study, 94 ASFV-encoded proteins were screened to determine the viral proteins involved in cell death in vitro, and pE199L showed the most significant effect. Ectopic expression of pE199L in porcine cells (CRL-2843) and human cells (HEK293T and HeLa cells) induced cell death remarkably, showing obvious shrinking, blistering, apoptotic bodies, and nuclear DNA fragments. Meanwhile, cell death was markedly alleviated when the expression of pE199L was knocked down during ASFV infection. Additionally, the expression of pE199L caused a loss of mitochondrial membrane potential, release of cytochrome C, and caspase-9 and -3/7 activation, indicating that the mitochondrial apoptotic pathway was involved in pE199L-induced apoptosis. Further investigations showed that pE199L interacted with several anti-apoptotic BCL-2 subfamily members (such as BCL-XL, MCL-1, BCL-W, and BCL-2A1) and competed with BAK for BCL-XL, which promoted BAK and BAX activation. Taken together, ASFV pE199L induces the mitochondrial-dependent apoptosis, which may provide clues for a comprehensive understanding of ASFV pathogenesis.


Assuntos
Vírus da Febre Suína Africana , Apoptose , Mitocôndrias , Proteínas Virais , Animais , Humanos , Febre Suína Africana/patologia , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/crescimento & desenvolvimento , Vírus da Febre Suína Africana/metabolismo , Vírus da Febre Suína Africana/patogenicidade , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína bcl-X/metabolismo , Caspases/metabolismo , Linhagem Celular , Citocromos c/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Suínos , Proteínas Virais/genética , Proteínas Virais/metabolismo
7.
J Virol Methods ; 285: 113958, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32827600

RESUMO

Inflammasome plays a major role in innate immune responses by activating caspase-1, resulting in secretion of interleukin-1ß (IL-1ß) and inflammatory pathologic responses. IL-1ß release is widely used as an indirect readout to study inflammasome activation. Here we report an iGLuc reporter (pro-IL-1ß-Gluc) of pig origin to monitor cytosolic pro-IL-1ß cleavage and mature IL-1ß release. Based on the iGLuc reporter, we reconstructed the inflammasome system in vitro and screened PRRSV- and ASFV-encoded proteins involved in regulating inflammasome activation. We found that three non-structural proteins (nsps) of PRRSV, nsp1ß, nsp2 and nsp5, activate the NLRP3 inflammasome, and four nsps of PRRSV, nsp1ɑ, nsp7, nsp10 and nsp11, inhibit NLRP3 inflammasome activation, of which nsp10 and nsp11 have a highly significant inhibitory effect. In addition, we also found that four ASFV-encoded proteins, S183L, E199L, O61R and I7L activate the inflammatory response and four ASFV-encoded proteins, I226L, A151R, NP419L and QP383R, inhibit the inflammatory response. Our results provide a highly sensitive and high-throughput tool to screen for proteins that regulate inflammasome activation in vitro.


Assuntos
Febre Suína Africana , Asfarviridae/imunologia , Imunidade Inata , Inflamassomos/imunologia , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Febre Suína Africana/imunologia , Febre Suína Africana/virologia , Animais , Caspase 1/imunologia , Células HEK293 , Humanos , Interleucina-1beta/imunologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Suínos , Proteínas Virais/imunologia
8.
J Food Sci Technol ; 50(3): 561-6, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24425953

RESUMO

Starch was phosphorylated through dry-heating in the presence of pyrophosphate at various conditions, and the characteristics of phosphorylated starch (PS) were examined. Starch phosphorylation increases as the pH increases from 3 to 6, but diminishes at pH 7. Increased temperatures enhance phosphorylation. Data from (31)P NMR suggests that starch phosphorylation occurs mainly at the C3-OH and C6-OH of the glucose residue. The phosphate linkage is mainly due to monostarch monophosphate. Although starch had almost no calcium phosphate-solubilising capacity, this capacity was markedly enhanced by phosphorylation. X-ray diffraction analysis indicates that the crystal structure of hydroxyapatite was not present in the calcium phosphate-PS complex.

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