RESUMEN
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 Deshidrogenasa , Hepatitis B , Proteína 28 que Contiene Motivos Tripartito , Proteínas Reguladoras y Accesorias Virales , Animales , Ratones , Glicerol/metabolismo , Glicerolfosfato Deshidrogenasa/metabolismo , Hepatitis B/metabolismo , Virus de la Hepatitis B/fisiología , Mitocondrias/enzimología , Fosfatos/metabolismo , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación ViralRESUMEN
The biogenesis of covalently closed circular DNA (cccDNA) from relaxed circular DNA (rcDNA) is essential for chronic hepatitis B virus (HBV) infection. Different host DNA repair proteins are involved in the conversion of rcDNA to cccDNA. Here, we reported that the DNA repair factor poly(ADP-ribose) polymerase 1 (PARP1) is engaged in HBV cccDNA formation. PARP1 depletion remarkably impaired HBV replication and cccDNA synthesis. Inhibition of PARP1 poly (ADP-ribosylation) activity by olaparib suppressed cccDNA synthesis both in vitro and in vivo. Specifically, the early stage of cccDNA reservoir establishment was more sensitive to olaparib, suggesting that PARP1 participated in de novo cccDNA formation. Furthermore, PARP1 was activated by recognizing the rcDNA-like lesions directly and combined with other DNA repair proteins. The results presented proposed that the DNA damage-sensing protein PARP1 and poly(ADP-ribosylation) modification play a key role in cccDNA formation, which might be the target for developing the anti-HBV drug. IMPORTANCE The biogenesis and eradication of HBV cccDNA have been a research priority in recent years. In this study, we identified the DNA repair factor PARP1 as a host factor required for the HBV de novo cccDNA formation. HBV infection caused PARylation through PARP1 in Huh7-NTCP cells, primary human hepatocytes, and human-liver chimeric mice. We found that PARP1 could directly bind to the rcDNA lesions and was activated, PARylating other DNA repair proteins. We address the importance of PARP1-mediated PARylation in HBV cccDNA formation, which is a potential therapeutic target for chronic hepatitis B.
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ADN Circular , Hepatitis B , Poli(ADP-Ribosa) Polimerasa-1 , Animales , Reparación del ADN , ADN Circular/genética , ADN Circular/metabolismo , ADN Viral/genética , ADN Viral/metabolismo , Hepatitis B/virología , Virus de la Hepatitis B/genética , Humanos , Ratones , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Provirus/genéticaRESUMEN
BACKGROUND: Prodigiosin (PG), a natural red pigment produced by numerous bacterial species, has been a eye-catching research point in recent years for its anticancer activity. However, the role of PG in the cancer biology of cholangiocarcinoma (CCA) remains vague. METHODS: The proliferation of CCA cells was detected by Cell Counting Kit-8(CCK-8), Colony formation assay and 5-ethynyl-2'-deoxyuridine (EdU) assay. Cell apoptosis was evaluated by flow cytometry assay and western blot assay. The effects of PG or SNAREs on cell autophagy were measured by autophagy flux assay and western blot assay. Xenograft mouse models were used to assess the role of PG in CCA cells in vivo. RESULTS: PG could inhibit the proliferation and viability of CCA cells in a concentration- and time-dependent manner via suppressing the late stage of autophagy. Mechanistically, PG inhibits the fusion of autophagosomes and lysosomes by blocking STX17 and SNAP29, components of soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNAREs)complex. When STX17 and SNAP29 were overexpressed, the inhibitory effect of PG on CCA cells autophagy was relieved. In addition, PG showed obvious inhibitory effects on cancer cell viability but no toxic effects on organs in xenotransplantation models. CONCLUSION: Taken together, our results demonstrated that PG inhibits CCA cell proliferation via suppressing SNAREs-dependent autophagy, implying that PG could be a potential chemotherapy drug for advanced CCA.
RESUMEN
The liver is an immune-privileged organ with a tolerogenic environment for maintaining liver homeostasis. This hepatic tolerance limits the intrahepatic CD8+ T-cell response for eliminating infections. The tolerant microenvironment in the liver is orchestrated by liver-specific immunoregulatory cells that can be functionally regulated by pathogen-associated molecular patterns (PAMPs). Here, we report that flagellin, a key PAMP of gut bacteria, modulates the intrahepatic CD8+ T-cell response by activating the TLR5 signalling pathway of hepatocytes. We found that mice treated with Salmonella-derived recombinant flagellin (SF) by hydrodynamic injection had a significantly elevated IFN-γ production by the intrahepatic lymphocytes in 7 days after injection. This was correlated with a reduced immune suppressive effect of primary mouse hepatocytes (PMHs) in comparison with that of PMHs from mock-injected control mice. In vitro co-culture of SF-treated PMHs with splenocytes revealed that hepatocyte-induced immune suppression is alleviated through activation of the TLR5 but not the NLRC4 signalling pathway, leading to improved activation and function of CD8+ T cells during anti-CD3 stimulation or antigen-specific activation. In an acute HBV replication mouse model established by co-administration of SF together with an HBV-replicating plasmid by hydrodynamic injection, SF significantly enhanced the intrahepatic HBV-specific CD8+ T-cell response against HBV surface antigen. Our results clearly showed that flagellin plays a role in modulating the intrahepatic CD8+ T-cell response by activating the TLR5 pathway in PMHs, which suggests a potential role for gut bacteria in regulating liver immunity.
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Linfocitos T CD8-positivos/inmunología , Virus de la Hepatitis B/fisiología , Hepatitis B/inmunología , Hepatocitos/fisiología , Hígado/inmunología , Salmonella/metabolismo , Receptor Toll-Like 5/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Células Cultivadas , Flagelina/metabolismo , Privilegio Inmunológico , Tolerancia Inmunológica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Receptor Toll-Like 5/genéticaRESUMEN
Encapsidation of pregenomic RNA (pgRNA) is a crucial step in hepatitis B virus (HBV) replication. Binding by viral polymerase (Pol) to the epsilon stem-loop (ε) on the 5'-terminal region (TR) of pgRNA is required for pgRNA packaging. However, the detailed mechanism is not well understood. RNA-binding motif protein 24 (RBM24) inhibits core translation by binding to the 5'-TR of pgRNA. Here, we demonstrate that RBM24 is also involved in pgRNA packaging. RBM24 directly binds to the lower bulge of ε via RNA recognition submotifs (RNPs). RBM24 also interacts with Pol in an RNA-independent manner. The alanine-rich domain (ARD) of RBM24 and the reverse transcriptase (RT) domain of Pol are essential for binding between RBM24 and Pol. In addition, overexpression of RBM24 increases Pol-ε interaction, whereas RBM24 knockdown decreases the interaction. RBM24 was able to rescue binding between ε and mutant Pol lacking ε-binding activity, further showing that RBM24 mediates the interaction between Pol and ε by forming a Pol-RBM24-ε complex. Finally, RBM24 significantly promotes the packaging efficiency of pgRNA. In conclusion, RBM24 mediates Pol-ε interaction and formation of a Pol-RBM24-ε complex, which inhibits translation of pgRNA and results in pgRNA packing into capsids/virions for reverse transcription and DNA synthesis.IMPORTANCE Hepatitis B virus (HBV) is a ubiquitous human pathogen, and HBV infection is a major global health burden. Chronic HBV infection is associated with the development of liver diseases, including fulminant hepatitis, hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. A currently approved vaccine can prevent HBV infection, and medications are able to reduce viral loads and prevent liver disease progression. However, current treatments rarely achieve a cure for chronic infection. Thus, it is important to gain insight into the mechanisms of HBV replication. In this study, we found that the host factor RBM24 is involved in pregenomic RNA (pgRNA) packaging and regulates HBV replication. These findings highlight a potential target for antiviral therapeutics of HBV infection.
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Virus de la Hepatitis B/genética , Hepatitis B/genética , Hepatitis B/virología , ARN Viral/genética , Proteínas de Unión al ARN/genética , ARN/genética , Ensamble de Virus/genética , Cápside/virología , Línea Celular Tumoral , Células Hep G2 , Humanos , Unión Proteica/genética , Motivos de Unión al ARN/genética , ADN Polimerasa Dirigida por ARN/genética , Transcripción Reversa/genética , Replicación Viral/genéticaRESUMEN
Hepatitis B virus (HBV) replication and envelopment is dependent on cellular autophagy. Previously, we have provided evidence for the extensive lysosomal degradation of HBV virions and the hepatitis B surface antigen (HBsAg), which is likely controlled by autophagosome-lysosome fusion. Synaptosomal-associated protein 29 (SNAP29) has been identified as a protein specifically mediating autophagosome-lysosome fusion. Thus, in the present study, we addressed the hypothesis that SNAP29 is required for the autophagic degradation of HBV virions and HBsAg. We found that silencing SNAP29 significantly increased the number of autophagosomes and concomitantly promoted HBV replication and HBsAg production. Conversely, SNAP29 overexpression decreased HBV production. Consistent with this, SNAP29 modulated HBV production by interacting with vesicle-associated membrane protein 8 (VAMP8) and synergistically regulated HBV replication with Rab7 complexes. Moreover, the production and release of the small HBsAg is strongly regulated by SNAP29 expression, suggesting that its export occurs partly through the autophagic pathway. Our findings provide new evidence, strongly suggesting that autophagic degradation critically determines the production of HBV virions and HBsAg and that this is controlled by the SNAP29-VAMP8 interaction.-Lin, Y., Wu, C., Wang, X., Liu, S., Kemper, T., Li, F., Squire, A., Zhu, Y., Zhang, J., Chen, X., Lu, M. Synaptosomal-associated protein 29 is required for the autophagic degradation of hepatitis B virus.
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Autofagia , Antígenos de Superficie de la Hepatitis B/metabolismo , Hepatitis B/metabolismo , Proteínas Qb-SNARE/fisiología , Proteínas Qc-SNARE/fisiología , Proteínas R-SNARE/metabolismo , Sinaptosomas/metabolismo , Animales , Autofagosomas/metabolismo , Bovinos , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica , Silenciador del Gen , Células Hep G2 , Hepatitis B/virología , Virus de la Hepatitis B , Humanos , Lisosomas/metabolismo , Fusión de Membrana , ARN Interferente Pequeño/metabolismo , Albúmina Sérica Bovina/metabolismo , Virión , Replicación ViralRESUMEN
BACKGROUND: Hepatitis E virus (HEV) generally causes self-limiting viral hepatitis. However, in pregnant women, HEV infection can be severe and has been associated with up to 30% mortality in the third trimester. Additionally, HEV infection in pregnancy is also associated with high rates of preterm labor and vertical transmission. MAIN BODY: HEV is now recognized as a global health problem in both developing and industrialized countries. HEV can be transmitted via the fecal-oral route, zoonotic route, and blood transfusion route. An altered immune status, hormonal levels, and viral factors may be related to the severity of the disease. Currently, no established treatment is available for HEV in pregnant women. A Chinese vaccine has been demonstrated to be protective against HEV in the general population and seems to be safe in pregnancy; however, its safety and efficacy in a large population of pregnant women remain to be determined. CONCLUSION: This review summarizes the current knowledge about HEV infection during pregnancy and focuses on the epidemiology, clinical manifestations, mechanisms underlying severe liver injury, and management and prevention of HEV infection during pregnancy. Considering that HEV infection during pregnancy may result in poor outcomes, screening for and monitoring HEV infection early in pregnancy should be taken into account. In addition, a better understanding of the pathogenesis will help to develop potential treatment strategies targeting HEV infection in pregnancy.
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Hepatitis E/epidemiología , Hepatitis E/fisiopatología , Hígado/patología , Complicaciones Infecciosas del Embarazo/virología , Femenino , Humanos , Transmisión Vertical de Enfermedad Infecciosa , Hígado/virología , Trabajo de Parto Prematuro/virología , Embarazo , Complicaciones Infecciosas del Embarazo/fisiopatologíaRESUMEN
While the majority of worldwide hepatitis E viral (HEV) infections that occur in people are from contaminated water or food sources, there has also been a steadily rising number of reported cases of transfusion-transmitted HEV (TT-HEV) in blood donation recipients. For most, HEV infection is acute, self-limiting and asymptomatic. However, patients that are immunocompromised, especially transplant patients, are at much higher risk for developing chronic infections, which can progress to cirrhosis and liver failure, along with overall increased mortality. Because of the rising trend of HEV serological prevalence among the global population, and the fact that TT-HEV infection can cause serious clinical consequences among those patients most at need for blood donation, the need for screening for TT-HEV has been gaining in prominence as an important public health concern for both developing and developed countries. In the review, we summarise evidence for and notable cases of TT-HEV infections, the various aspects of HEV screening protocols and recent trends in the implementation of TT-HEV broad-based blood screening programmes.
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Seguridad de la Sangre , Transfusión Sanguínea , Virus de la Hepatitis E , Hepatitis E/sangre , Hepatitis E/transmisión , Donantes de Sangre , HumanosRESUMEN
As a virus-encoded actin nucleation promoting factor (NPF), P78/83 induces actin polymerization to assist in Autographa californica multiple nucleopolyhedrovirus (AcMNPV) propagation. According to our previous study, although P78/83 actively undergoes ubiquitin-independent proteasomal degradation, AcMNPV encodes budded virus/occlusion derived virus (BV/ODV)-C42 (C42), which allows P78/83 to function as a stable NPF by inhibiting its degradation during viral infection. However, whether there are other viral proteins involved in regulating P78/83-induced actin polymerization has yet to be determined. In this study, we found that Ac102, an essential viral gene product previously reported to play a key role in mediating the nuclear accumulation of actin during AcMNPV infection, is a novel regulator of P78/83-induced actin polymerization. By characterizing an ac102 knockout bacmid, we demonstrated that Ac102 participates in regulating nuclear actin polymerization as well as the morphogenesis and distribution of capsid structures in the nucleus. These regulatory effects are heavily dependent on an interaction between Ac102 and C42. Further investigation revealed that Ac102 binds to C42 to suppress K48-linked ubiquitination of C42, which decreases C42 proteasomal degradation and consequently allows P78/83 to function as a stable NPF to induce actin polymerization. Thus, Ac102 and C42 form a regulatory cascade to control viral NPF activity, representing a sophisticated mechanism for AcMNPV to orchestrate actin polymerization in both a ubiquitin-dependent and ubiquitin-independent manner.IMPORTANCE Actin is one of the most functionally important proteins in eukaryotic cells. Morphologically, actin can be found in two forms: a monomeric form called globular actin (G-actin) and a polymeric form called filamentous actin (F-actin). G-actin can polymerize to form F-actin, and nucleation promoting factor (NPF) is the initiator of this process. Many viral pathogens harness the host actin polymerization machinery to assist in virus propagation. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) induces actin polymerization in host cells. P78/83, a viral NPF, is responsible for this process. Previously, we identified that BV/ODV-C42 (C42) binds to P78/83 and protects it from degradation. In this report, we determined that another viral protein, Ac102, is involved in modulating C42 ubiquitination and, consequently, ensures P78/83 activity as an NPF to initiate actin polymerization. This regulatory cascade represents a novel mechanism by which a virus can harness the cellular actin cytoskeleton to assist in viral propagation.
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Actinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Complejo Mediador/metabolismo , Nucleopoliedrovirus/crecimiento & desarrollo , Replicación Viral/fisiología , Animales , Proteínas de la Cápside/metabolismo , Línea Celular , Núcleo Celular/virología , Técnicas de Inactivación de Genes , Péptidos y Proteínas de Señalización Intracelular/genética , Nucleopoliedrovirus/metabolismo , Polimerizacion , Células Sf9 , Spodoptera/virología , UbiquitinaciónRESUMEN
Actin, nucleation-promoting factors (NPFs), and the actin-related protein 2/3 complex (Arp2/3) are key elements of the cellular actin polymerization machinery. With nuclear actin polymerization implicated in ever-expanding biological processes and the discovery of the nuclear import mechanisms of actin and NPFs, determining Arp2/3 nucleo-cytoplasmic shuttling mechanism is important for understanding the function of nuclear actin. A unique feature of alphabaculovirus infection of insect cells is the robust nuclear accumulation of Arp2/3, which induces actin polymerization in the nucleus to assist in virus replication. We found that Ac34, a viral late gene product encoded by the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is involved in Arp2/3 nuclear accumulation during virus infection. Further assays revealed that the subcellular distribution of Arp2/3 under steady-state conditions is controlled by chromosomal maintenance 1 (CRM1)-dependent nuclear export. Upon AcMNPV infection, Ac34 inhibits CRM1 pathway and leads to Arp2/3 retention in the nucleus.
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Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Carioferinas/metabolismo , Nucleopoliedrovirus/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Replicación Viral/fisiología , Animales , Núcleo Celular/metabolismo , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Células Sf9 , Transfección , Proteínas Virales/metabolismo , Proteína Exportina 1RESUMEN
Ceruloplasmin (CP) is mainly synthesized by hepatocytes and plays an essential role in iron metabolism. Previous reports have shown that CP levels correlate negatively with disease progression in patients with chronic hepatitis B. However, the function of CP in the hepatitis B virus (HBV) life cycle and the mechanism underlying the above correlation remain unclear. Here, we report that CP can selectively inhibit the production of extracellular HBV virions without altering intracellular viral replication. HBV expression can also downregulate the expression of CP. Knockdown of CP using small interfering RNA significantly increased the level of extracellular HBV virions in both Huh7 and HepG2.2.15 cells, while overexpression of CP decreased this level. Mechanistically, CP could specifically interact with the HBV middle surface protein (MHB). Using an HBV replication-competent clone unable to express MHBs, we demonstrated that the overexpression of CP did not affect the production of extracellular HBV virions in the absence of MHBs. Furthermore, introduction of an MHB expression construct could rescue the impairment in virion production caused by CP. Taken together, our results suggest that CP may be an important host factor that targets MHBs during the envelopment and/or release of virions.
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Ceruloplasmina/metabolismo , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/crecimiento & desarrollo , Virus de la Hepatitis B/inmunología , Adulto , Línea Celular , Ceruloplasmina/análisis , Femenino , Hepatitis B Crónica/virología , Hepatocitos/virología , Humanos , Masculino , Persona de Mediana Edad , Mapeo de Interacción de ProteínasRESUMEN
Lipid droplet (LD) accumulation in hepatocytes is a typical character of steatosis. Hepatitis C virus (HCV) infection, one of the risk factors related to steatosis, induced LD accumulation in cultured cells. However, the mechanisms of which HCV induce LD formation are not fully revealed. Previously we identified cytosolic phospholipase A2 gamma (PLA2G4C) as a host factor upregulated by HCV infection and involved in HCV replication. Here we further revealed that PLA2G4C plays an important role in LD biogenesis and refined the functional analysis of PLA2G4C in LD biogenesis and HCV assembly. LD formation upon fatty acid and HCV stimulation in PLA2G4C knockdown cells was impaired and could not be restored by complementation with PLA2G4A. PLA2G4C was tightly associated in the membrane with the domain around the amino acid residues 260-292, normally in ER but relocated into LDs upon oleate stimulation. Mutant PLA2G4C without enzymatic activity was not able to restore LD formation in PLA2G4C knockdown cells. Thus, both the membrane attachment and the enzymatic activity of PLA2G4C were required for its function in LD formation. The participation of PLA2G4C in LD formation is correlated with its involvement in HCV assembly. Finally, PLA2G4C overexpression itself led to LD formation in hepatic cells and enhanced LD accumulation in the liver of high-fat diet (HFD)-fed mice, suggesting its potential role in fatty liver disease.
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Citosol/metabolismo , Fosfolipasas A2 Grupo IV/metabolismo , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos/fisiología , Aminoácidos/metabolismo , Animales , Línea Celular , Citosol/virología , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Ácidos Grasos/metabolismo , Células HEK293 , Hepacivirus/patogenicidad , Hepatitis C/metabolismo , Hepatocitos/metabolismo , Hepatocitos/virología , Humanos , Gotas Lipídicas/virología , Hígado/metabolismo , Hígado/virología , Masculino , Membranas/metabolismo , Membranas/virología , Ratones , Ratones Endogámicos C57BLRESUMEN
Actin polymerization induced by nucleation promoting factors (NPFs) is one of the most fundamental biological processes in eukaryotic cells. NPFs contain a conserved output domain (VCA domain) near the C terminus, which interacts with and activates the cellular actin-related protein 2/3 complex (Arp2/3) to induce actin polymerization and a diverse regulatory domain near the N terminus. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) nucleocapsid protein P78/83 is a virus-encoded NPF that contains a C-terminal VCA domain and induces actin polymerization in virus-infected cells. However, there is no similarity between the N terminus of P78/83 and that of other identified NPFs, suggesting that P78/83 may possess a unique regulatory mechanism. In this study, we identified a multifunctional regulatory sequence (MRS) located near the N terminus of P78/83 and determined that one of its functions is to serve as a degron to mediate P78/83 degradation in a proteasome-dependent manner. In AcMNPV-infected cells, the MRS also binds to another nucleocapsid protein, BV/ODV-C42, which stabilizes P78/83 and modulates the P78/83-Arp2/3 interaction to orchestrate actin polymerization. In addition, the MRS is also essential for the incorporation of P78/83 into the nucleocapsid, ensuring virion mobility powered by P78/83-induced actin polymerization. The triple functions of the MRS enable P78/83 to serve as an essential viral protein in the AcMNPV replication cycle, and the possible roles of the MRS in orchestrating the virus-induced actin polymerization and viral genome decapsidation are discussed.
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Actinas/metabolismo , Proteínas de la Nucleocápside/genética , Nucleopoliedrovirus/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Interacciones Huésped-Patógeno , Proteínas de Insectos/metabolismo , Microscopía Fluorescente , Modelos Genéticos , Datos de Secuencia Molecular , Mariposas Nocturnas/virología , Proteínas de la Nucleocápside/metabolismo , Nucleopoliedrovirus/metabolismo , Nucleopoliedrovirus/fisiología , Polimerizacion , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Homología de Secuencia de Aminoácido , Células Sf9 , Spodoptera , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
UNLABELLED: Several members of the phospholipase family have been reported to be involved in hepatitis C virus (HCV) replication. Here, we identified another phospholipase, phosphatidylserine-specific phospholipase A1 (PLA1A), as a host factor involved in HCV assembly. PLA1A was upregulated by HCV infection, and PLA1A knockdown significantly reduced J399EM (genotype 2a) HCV propagation at the assembly step but not the entry, RNA replication, and protein translation steps of the life cycle. Protein localization and interaction analysis further revealed a role of PLA1A in the interaction of NS2-E2 and NS2-NS5A, as the formation of the NS2-E2 and NS2-NS5A complexes was weakened in the absence of PLA1A. In addition, PLA1A stabilized the NS2/NS5A dotted structure during infection. These data suggest that PLA1A plays an important role in bridging the membrane-associated NS2-E2 complex and the NS5A-associated replication complex via its interaction with E2, NS2, and NS5A, which leads to a coordinating interaction between the structural and nonstructural proteins and facilitates viral assembly. IMPORTANCE: Hepatitis C virus (HCV) genomic replication is driven by the replication complex and occurs at the membranous web, while the lipid droplet is the organelle in which virion assembly is initiated. In this study, we identified phosphatidylserine-specific phospholipase A1 (PLA1A), a member of phospholipase A 1 family, as a novel host factor involved in the assembly process of HCV. PLA1A, which is induced by HCV infection at a late infection stage, interacts with HCV E2, NS2, and NS5A proteins and enhances and stabilizes the NS2-E2 and NS2-NS5A complex formation, which is essential for viral assembly. Thus, PLA1A is an important host factor which is involved in the initiation of the viral assembly in close proximity to Core-decorated lipid droplets through bringing together the HCV replication complex and envelope complex.
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Hepacivirus/fisiología , Interacciones Huésped-Patógeno , Fosfolipasas A1/metabolismo , Multimerización de Proteína , Proteínas no Estructurales Virales/metabolismo , Ensamble de Virus , Línea Celular , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , HumanosRESUMEN
Ultra-deep pyrosequencing (UDPS) was used to analyse the dynamics of quasispecies and resistant mutations during telbivudine (LDT) treatment of hepatitis B patients. Twenty-six HBeAg-positive chronic hepatitis B patients were treated with LDT for a period of 104âweeks and were characterized as 16 responders, six partial responders and four viral breakthrough patients based on hepatitis B virus (HBV) DNA levels. The plasma samples were subjected to UDPS of the reverse transcriptase (RT) region of HBV. Mutations rtM204I, rtL80I and rtL80V were detected in at least three of the four viral breakthrough patients, indicating the significant roles of the mutations in resistance to LDT. The degree of complexity of viral quasispecies remained in a steady state in the absence of selection pressure, but increased after the LDT treatment. The complexity in the responder group at week 12 was significantly higher than that in the group comprising partial responders and viral breakthrough patients. In vitro replication efficiency analyses showed that the RT mutations had different impacts on HBV replication, with a tendency of rtM204I>rtL80V>rtL80I. Furthermore, double mutations rtL80I/M204I and rtL80V/M204V had replication efficiency similar to that of rtL80I and rtL80V, respectively. Consistent with previous studies, mutation rtM204I was found to be highly resistant to LDT. However, in contrast with their sensitivity to lamivudine, rtL80I and rtL80V were moderately resistant to LDT. Our results indicated that rtL80I and rtL80V may not only serve as replication complementary mutations to rtM204I, but also directly contribute to the LDT resistance.
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Antivirales/uso terapéutico , Farmacorresistencia Viral , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B Crónica/tratamiento farmacológico , Mutación/efectos de los fármacos , Timidina/análogos & derivados , Adolescente , Adulto , Anciano , Femenino , Virus de la Hepatitis B/clasificación , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B Crónica/virología , Humanos , Masculino , Persona de Mediana Edad , Telbivudina , Timidina/uso terapéutico , Adulto JovenRESUMEN
UNLABELLED: Hepatitis B virus (HBV) quasispecies contain a large number of variants that serve as a reservoir for viral selection under antiviral treatment and the immune response, leading to the acute exacerbation and subsequent development of liver failure. However, there is no clear experimental evidence for a significant role of HBV quasispecies in viral pathogenesis. In the present study, HBV sequences were amplified from a patient with severe liver disease and used for construction of HBV replication-competent plasmids. Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining were performed to analyze the expression, secretion, and subcellular localization of viral proteins in vitro. Viral replication intermediates were detected by Southern blotting. HBV gene expression and replication and the induction of specific immune responses in an HBV hydrodynamic injection (HI) mouse model were investigated. The results demonstrated that two naturally occurring HBV variants, SH and SH-DPS, were identified. The variant SH-DPS expressed only a nonexportable hepatitis B virus surface antigen (HBsAg) with abnormal intracellular accumulation. The coexistence of the HBV variants at a ratio of 1 to 4 (SH to SH-DPS) increased HBV replication. Significantly stronger intrahepatic cytotoxic T lymphocyte (CTL) responses and antibody responses specific to HBsAg were induced in mice by the HBV variants when coapplied by HI. These findings uncovered an unexpected aspect of HBV quasispecies: the coexistence of different variants can significantly modulate specific host immune responses, representing a novel mechanism for the immunopathogenesis of HBV infection. IMPORTANCE: Hepatitis B virus (HBV) is an important human pathogen. HBV quasispecies with genetically heterogenous variants are thought to play a role in the progression of HBV-associated liver diseases. So far, direct evidence is available in only a few cases to confirm the proposed role of HBV variants in the pathogenesis. We report here that the coexistence of two naturally occurring HBV variants at a ratio of 1 to 4 increased HBV replication and induced significantly stronger intrahepatic cytotoxic T lymphocyte responses and antibody responses specific to HBV surface antigen (HBsAg) in mice. Our discovery uncovered an unexpected aspect of HBV quasispecies: the coexistence of different variants can significantly modulate specific host immune responses and may enhance immune-mediated liver damage under some circumstances, representing a novel mechanism for the immunopathogenesis of HBV infection.
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Variación Genética , Anticuerpos contra la Hepatitis B/sangre , Virus de la Hepatitis B/inmunología , Virus de la Hepatitis B/fisiología , Hepatitis B/virología , Inmunidad Celular , Replicación Viral , Adulto , Animales , ADN Viral/química , ADN Viral/genética , Modelos Animales de Enfermedad , Hepatitis B/inmunología , Virus de la Hepatitis B/clasificación , Virus de la Hepatitis B/genética , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
Orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) is a transcription factor stimulated by many factors and plays pivotal roles in metabolism, proliferation and apoptosis. In this study, the expression of NR4A1 in Huh7.5.1 cells was significantly upregulated by hepatitis C virus (HCV) infection. The silencing of NR4A1 inhibited the entry of HCV and reduced the specific infectivity of secreted HCV particles but had only minor or no effect on the genome replication and translation, virion assembly and virus release steps of the virus life cycle. Further experiments demonstrated that the silencing of NR4A1 affected virus entry through pan-downregulation of the expression of HCV receptors scavenger receptor BI, occludin, claudin-1 and epidermal growth factor receptor but not CD81. The reduced specific infectivity of HCV in the knockdown cells was due to decreased apolipoprotein E (ApoE) expression. These results explain the delayed spread of HCV in NR4A1 knockdown Huh7.5.1 cells. Thus, NR4A1 plays a role in HCV replication through regulating the expression of HCV receptors and ApoE, and facilitates HCV entry and spread.
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Apolipoproteínas E/biosíntesis , Hepacivirus/fisiología , Hepatocitos/virología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Receptores Virales/biosíntesis , Internalización del Virus , Línea Celular Tumoral , Regulación de la Expresión Génica , Silenciador del Gen , Humanos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/antagonistas & inhibidoresRESUMEN
Prostate cancer presents as a challenging disease, as it is often characterized as an immunologically "cold" tumor, leading to suboptimal outcomes with current immunotherapeutic approaches in clinical settings. Photodynamic therapy (PDT) harnesses reactive oxygen species generated by photosensitizers (PSs) to disrupt the intracellular redox equilibrium. This process induces DNA damage in both the mitochondria and nucleus, activating the process of immunogenic cell death (ICD) and the cGAS-STING pathway. Ultimately, this cascade of events leads to the initiation of antitumor immune responses. Nevertheless, existing PSs face challenges, including suboptimal tumor targeting, aggregation-induced quenching, and insufficient oxygen levels in the tumor regions. To this end, a versatile bionic nanoplatform has been designed for the simultaneous delivery of the aggregation-induced emission PS TPAQ-Py-PF6 and paclitaxel (PTX). The cell membrane camouflage of the nanoplatform leads to its remarkable abilities in tumor targeting and cellular internalization. Upon laser irradiation, the utilization of TPAQ-Py-PF6 in conjunction with PTX showcases a notable and enhanced synergistic antitumor impact. Additionally, the nanoplatform has the capability of initiating the cGAS-STING pathway, leading to the generation of cytokines. The presence of damage-associated molecular patterns induced by ICD collaborates with these aforementioned cytokines lead to the recruitment and facilitation of dendritic cell maturation. Consequently, this elicits a systemic immune response against tumors. In summary, this promising strategy highlights the use of a multifunctional biomimetic nanoplatform, combining chemotherapy, PDT, and immunotherapy to enhance the effectiveness of antitumor treatment.
RESUMEN
Similar to other positive-sense, single-stranded RNA viruses, hepatitis C virus (HCV) replicates its genome in a remodeled intracellular membranous structure known as the membranous web (MW). To date, the process of MW formation remains unclear. It is generally acknowledged that HCV nonstructural protein 4B (NS4B) can induce MW formation through interaction with the cytosolic endoplasmic reticulum (ER) membrane. Many host proteins, such as phosphatidylinositol 4-kinase IIIα (PI4KIIIα), have been identified as critical factors required for this process. We now report a new factor, the cytosolic phospholipase A2 gamma (PLA2G4C), which contributes to MW formation, HCV replication, and assembly. The PLA2G4C gene was identified as a host gene with upregulated expression upon HCV infection. Knockdown of PLA2G4C in HCV-infected cells or HCV replicon-containing cells by small interfering RNA (siRNA) significantly suppressed HCV replication and assembly. In addition, the chemical inhibitor methyl arachidonyl fluorophosphonate (MAFP), which specifically inhibits PLA2, reduced HCV replication and assembly. Electron microscopy demonstrated that MW structure formation was defective after PLA2G4C knockdown in HCV replicon-containing cells. Further analysis by immunostaining and immunoprecipitation assays indicated that PLA2G4C colocalized with the HCV proteins NS4B and NS5A in cells infected with JFH-1 and interacted with NS4B. In addition, PLA2G4C was able to transport the HCV nonstructural proteins from replication sites to lipid droplets, the site for HCV assembly. These data suggest that PLA2G4C plays an important role in the HCV life cycle and might represent a potential target for anti-HCV therapy.
Asunto(s)
Citosol/metabolismo , Fosfolipasas A2 Grupo IV/metabolismo , Hepacivirus/fisiología , Ensamble de Virus/fisiología , Replicación Viral/fisiología , Ácido Araquidónico/farmacología , Ácidos Araquidónicos/farmacología , Western Blotting , Línea Celular , Cartilla de ADN/genética , Retículo Endoplásmico/metabolismo , Técnicas de Silenciamiento del Gen , Fosfolipasas A2 Grupo IV/antagonistas & inhibidores , Humanos , Inmunoprecipitación , Microscopía Fluorescente , Organofosfonatos/farmacología , Plásmidos/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas no Estructurales Virales/metabolismo , Ensamble de Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
A variety of amino acid substitutions, such as K122I and G145R, have been identified around or within the a determinant of hepatitis B surface antigen (HBsAg), impair HBsAg secretion and antibody binding, and may be responsible for immune escape in patients. In this study, we examined how different substitutions at amino acid positions 122 and 145 of HBsAg influence HBsAg expression, secretion, and recognition by anti-HBs antibodies. The results showed that the hydrophobicity, the presence of the phenyl group, and the charges in the side chain of the amino acid residues at position 145 reduced HBsAg secretion and impaired reactivity with anti-HBs antibodies. Only the substitution K122I at position 122 affected HBsAg secretion and recognition by anti-HBs antibodies. Genetic immunization in mice demonstrated that the priming of anti-HBs antibody response was strongly impaired by the substitutions K122I, G145R, and others, like G145I, G145W, and G145E. Mice preimmunized with wild-type HBsAg (wtHBsAg) or variant HBsAg (vtHBsAg) were challenged by hydrodynamic injection (HI) with a replication-competent hepatitis B virus (HBV) clone. HBsAg persisted in peripheral blood for at least 3 days after HI in mice preimmunized with vtHBsAg but was undetectable in mice preimmunized with wtHBsAg, indicating that vtHBsAgs fail to induce proper immune responses for efficient HBsAg clearance. In conclusion, the biochemical properties of amino acid residues at positions 122 and 145 of HBsAg have a major effect on antigenicity and immunogenicity. In addition, the presence of proper anti-HBs antibodies is indispensable for the neutralization and clearance of HBsAg during HBV infection.