scholarly journals Regulation of Apolipoprotein E Trafficking by Hepatitis C Virus-Induced Autophagy

2018 ◽  
Vol 92 (14) ◽  
Author(s):  
Ja Yeon Kim ◽  
Jing-hsiung James Ou
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Apolipoprotein E ◽  
Protein Degradation ◽  
Envelope Protein ◽  
Subgenomic Rna ◽  
Viral Particles ◽  
Infected Cells ◽  
Rna Replicon

ABSTRACTApolipoprotein E (ApoE) plays an important role in the maturation and infectivity of hepatitis C virus (HCV). By analyzing the subcellular localization of ApoE in Huh7 hepatoma cells that harbored an HCV subgenomic RNA replicon, we found that ApoE colocalized with autophagosomes. This colocalization was marginally detected in HCV-infected cells, apparently due to the depletion of ApoE by HCV, as treatment with bafilomycin A1 (BafA1), a vacuolar ATPase inhibitor that inhibits autophagic protein degradation, partially restored the ApoE level and enhanced its colocalization with autophagosomes in HCV-infected cells. The role of HCV-induced autophagy in the degradation of ApoE was further supported by the observations that nutrient starvation, which induces autophagic protein degradation, led to the loss of ApoE in HCV subgenomic RNA replicon cells and that the knockdown of ATG7, a protein essential for the formation of autophagic vacuoles, increased the ApoE level in cells with productive HCV replication. Interestingly, the inhibition of autophagy by ATG7 knockdown reduced the colocalization of ApoE with the HCV E2 envelope protein and the HCV titers released from cells. In contrast, the treatment of cells with BafA1 enhanced the colocalization of ApoE and HCV E2 and increased both intracellular and extracellular HCV titers. These results indicated that autophagy played an important role in the trafficking of ApoE in HCV-infected cells. While it led to autophagic degradation of ApoE, it also promoted the interaction between ApoE and HCV E2 to enhance the production of infectious progeny viral particles.IMPORTANCEHepatitis C virus (HCV) is one of the most important human pathogens. Its virion is associated with apolipoprotein E (ApoE), which enhances its infectivity. HCV induces autophagy to enhance its replication. In this report, we demonstrate that autophagy plays an important role in the trafficking of ApoE in HCV-infected cells. This leads to the degradation of ApoE by autophagy. However, if the autophagic protein degradation is inhibited, ApoE is stabilized and colocalized with autophagosomes. This leads to its enhanced colocalization with the HCV E2 envelope protein and increased production of infectious progeny viral particles. If autophagy is inhibited by suppressing the expression of ATG7, a gene essential for the formation of autophagosomes, the colocalization of ApoE with E2 is reduced, resulting in the reduction of progeny viral titers. These results indicate an important role of autophagy in the transport of ApoE to promote the production of infectious HCV particles.

scholarly journals Differential Biophysical Properties of Infectious Intracellular and Secreted Hepatitis C Virus Particles

2006 ◽  
Vol 80 (22) ◽  
pp. 11074-11081 ◽  
Author(s):  
Pablo Gastaminza ◽  
Sharookh B. Kapadia ◽  
Francis V. Chisari
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Buoyant Density ◽  
Infection Model ◽  
Virus Particles ◽  
Viral Egress ◽  
Viral Particles ◽  
A Cell ◽  
Infected Cells

ABSTRACT The recent development of a cell culture infection model for hepatitis C virus (HCV) permits the production of infectious particles in vitro. In this report, we demonstrate that infectious particles are present both within the infected cells and in the supernatant. Kinetic analysis indicates that intracellular particles constitute precursors of the secreted infectious virus. Ultracentrifugation analyses indicate that intracellular infectious viral particles are similar in size (∼65 to 70 nm) but different in buoyant density (∼1.15 to 1.20 g/ml) from extracellular particles (∼1.03 to 1.16 g/ml). These results indicate that infectious HCV particles are assembled intracellularly and that their biochemical composition is altered during viral egress.

scholarly journals Neglected but Important Role of Apolipoprotein E Exchange in Hepatitis C Virus Infection

2016 ◽  
Vol 90 (21) ◽  
pp. 9632-9643 ◽  
Author(s):  
Zaili Yang ◽  
Xiaoning Wang ◽  
Xiumei Chi ◽  
Fanfan Zhao ◽  
Jinxu Guo ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Surface ◽  
Apolipoprotein E ◽  
Virus Assembly ◽  
Hcv Infection ◽  
Apoe Level ◽  
New Perspective ◽  
Apoe Expression

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic liver disease, infecting approximately 170 million people worldwide. HCV assembly is tightly associated with the lipoprotein pathway. Exchangeable apolipoprotein E (apoE) is incorporated on infectious HCV virions and is important for infectious HCV virion morphogenesis and entry. Moreover, the virion apoE level is positively correlated with its ability to escape E2 antibody neutralization. However, the role of apoE exchange in the HCV life cycle is unclear. In this study, the relationship between apoE expression and cell permissiveness to HCV infection was assessed by infecting apoE knockdown and derived apoE rescue cell lines with HCV. Exchange of apoE between lipoproteins and HCV lipoviral particles (LVPs) was evaluated by immunoprecipitation, infectivity testing, and viral genome quantification. Cell and heparin column binding assays were applied to determine the attachment efficiency of LVPs with different levels of incorporated apoE. The results showed that cell permissiveness for HCV infection was determined by exogenous apoE-associated lipoproteins. Furthermore, apoE exchange did occur between HCV LVPs and lipoproteins, which was important to maintain a high apoE level on LVPs. Lipid-free apoE was capable of enhancing HCV infectivity for apoE knockdown cells but not apoE rescue cells. A higher apoE level on LVPs conferred more efficient LVP attachment to both the cell surface and heparin beads. This study revealed that exogenous apoE-incorporating lipoproteins from uninfected hepatocytes safeguarded the apoE level of LVPs for more efficient attachment during HCV infection. IMPORTANCE In this study, a neglected but important role of apoE exchange in HCV LVP infectivity after virus assembly and release was identified. The data indicated that apoE expression level in uninfected cells is important for high permissiveness to HCV infection. Secreted apoE-associated lipoprotein specifically enhances infection of HCV LVPs. apoE exchange between HCV LVP and lipoproteins is important to maintain an adequate apoE level on LVPs for their efficient attachment to cell surface. These data defined for the first time an extracellular role of exchangeable apoE in HCV infection and suggested that exchangeable apolipoproteins reach a natural equilibrium between HCV LVPs and lipoprotein particles, which provides a new perspective to the understanding of the heterogeneity of HCV LVPs in composition.

scholarly journals The Autophagosomal SNARE Protein Syntaxin 17 Is an Essential Factor for the Hepatitis C Virus Life Cycle

2016 ◽  
Vol 90 (13) ◽  
pp. 5989-6000 ◽  
Author(s):  
Huimei Ren ◽  
Fabian Elgner ◽  
Bingfu Jiang ◽  
Kiyoshi Himmelsbach ◽  
Regina Medvedev ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
De Novo ◽  
Receptor Protein ◽  
Snare Protein ◽  
Small Interfering Rnas ◽  
Lysosomal Degradation ◽  
Viral Particles ◽  
Infected Cells

ABSTRACTSyntaxin 17 is an autophagosomal SNARE (solubleN-ethylmaleimide-sensitive factor attachment protein receptor) protein required for the fusion of autophagosomes with lysosomes to form autolysosomes and thereby to deliver the enclosed contents for degradation. Hepatitis C virus (HCV) induces autophagy. In light of the observation that the number of viral particles formed by HCV-infected cells is much greater than the number of infectious viral particles finally released by HCV-infected cells, the regulation of fusion between autophagosomes and lysosomes might fulfill a key function controlling the number of released virions. HCV-replicating cells possess a decreased amount of syntaxin 17 due to impaired expression and increased turnover of syntaxin 17. Overexpression of syntaxin 17 in HCV-replicating cells diminishes the number of released infectious viral particles and decreases the amount of intracellular retained viral particles by favoring the formation of autolysosomes, in which HCV particles are degraded. Inhibition of lysosomal acidification by bafilomycin rescues the decreased release of virions from syntaxin 17-overexpressing cells, while induction of autophagy by rapamycin enforces the impairment of release under these conditions. Vice versa, inhibition of syntaxin 17 expression by specific small interfering RNAs results in an elevated amount of intracellular retained viral particles and facilitates the release of HCV virions by impairment of autophagosome-lysosome fusion. HCV genome replication, however, is not affected by modulation of syntaxin 17 expression. These data identify syntaxin 17 to be a novel factor controlling the release of HCV. This is achieved by regulation of autophagosome-lysosome fusion, which affects the equilibrium between the release of infectious viral particles and lysosomal degradation of intracellular retained viral particles.IMPORTANCEHepatitis C virus (HCV) induces autophagy. Syntaxin 17 is an autophagosomal SNARE protein required for the fusion of autophagosomes with lysosomes. In HCV-infected cells, a major fraction of thede novo-synthesized viral particles is not released but is intracellularly degraded. In this context, the effect of HCV on the amount and distribution of syntaxin 17 and the relevance of syntaxin 17 for the viral life cycle were investigated. This study demonstrates that the amount of syntaxin 17 decreased in HCV-replicating cells. In addition, syntaxin 17 is identified to be a novel factor controlling the release of HCV, and the relevance of autophagosome-lysosome fusion as a regulator of the amount of released viral particles is revealed. Taken together, these findings indicate that syntaxin 17 is involved in the regulation of autophagosome-lysosome fusion and thereby affects the equilibrium between the release of infectious viral particles and the lysosomal degradation of intracellularly retained viral particles.

scholarly journals The inflammasome components NLRP3 and ASC act in concert with IRGM to rearrange the Golgi during HCV infection.

2020 ◽  
Author(s):  
Coralie F. Daussy ◽  
Sarah C. Monard ◽  
Coralie Guy ◽  
Sara Muñoz-González ◽  
Maxime Chazal ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hcv Infection ◽  
Pyrin Domain ◽  
Inflammasome Component ◽  
Infected Cells ◽  
Golgi Fragmentation ◽  
Golgi Protein ◽  
Golgi Structure

Hepatitis C virus (HCV) infection triggers Golgi fragmentation through the Golgi-resident protein immunity-related GTPase M (IRGM). Here, we report the role of NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) and ASC (Apoptosis-associated speck-like protein containing a CARD), two inflammasome components, in the initial events leading to this fragmentation. We show that ASC resides at the Golgi with IRGM at homeostasis. Upon infection, ASC dissociates from both IRGM and Golgi and associates with HCV-induced NLRP3. NLRP3 silencing inhibits Golgi fragmentation. ASC silencing disrupts the Golgi structure in both control and infected cells and reduces the localization of IRGM at the Golgi. IRGM-depletion in the ASC silenced cells cannot totally restore the Golgi structure. These data highlight a role for ASC, upstream of the formation of the inflammasome, in regulating IRGM through its control on the Golgi. A similar mechanism occurs in response to Nigericin treatment, but not in cells infected with another member of the Flaviviridae family, Zika virus (ZIKV). We propose a model for a newly ascribed function of the inflammasome components in Golgi structural remodeling during certain stimuli. IMPORTANCE Numerous pathogens can affect cellular homeostasis and organelle dynamics. Hepatitis C virus (HCV) triggers Golgi fragmentation through the immunity-related GTPase M (IRGM), a resident Golgi protein to enhance its lipid supply for replication. Here, we reveal the role of the inflammasome components NLRP3 and ASC in this process, thus uncovering a new interplay between effectors of inflammation and viral infection or stress. We show that the inflammasome component ASC resides at the Golgi under homeostasis and associates with IRGM. Upon HCV infection, ASC is recruited to NLRP3 and dissociates from IRGM causing Golgi fragmentation. Our results uncover that aside from their known function in the inflammation response, this host defense regulators also ensure the maintenance of intact intracellular structure in homeostasis status, while their activation relieves factors leading to Golgi remodeling.

scholarly journals Non-structural protein 4A of Hepatitis C virus accumulates on mitochondria and renders the cells prone to undergoing mitochondria-mediated apoptosis

2006 ◽  
Vol 87 (7) ◽  
pp. 1935-1945 ◽  
Author(s):  
Yuki Nomura-Takigawa ◽  
Motoko Nagano-Fujii ◽  
Lin Deng ◽  
Sohei Kitazawa ◽  
Satoshi Ishido ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Replication ◽  
Hcv Rna ◽  
Structural Protein ◽  
Cellular Functions ◽  
Replication Complex ◽  
Huh7 Cells ◽  
Infected Cells ◽  
Rna Replicon

Non-structural protein 4A (NS4A) of Hepatitis C virus (HCV) functions as a cofactor for NS3 by forming a complex with it to augment its enzymic activities. NS4A also forms a complex with other HCV proteins, such as NS4B/NS5A, to facilitate the formation of the viral RNA replication complex on the endoplasmic reticulum (ER) membrane. In addition to its essential role in HCV replication, NS4A is thought to be involved in viral pathogenesis by affecting cellular functions. In this study, it was demonstrated that NS4A was localized not only on the ER, but also on mitochondria when expressed either alone or together with NS3 in the form of the NS3/4A polyprotein and in the context of HCV RNA replication in Huh7 cells harbouring an HCV RNA replicon. Moreover, NS4A expression altered the intracellular distribution of mitochondria significantly and caused mitochondrial damage, as evidenced by the collapsed mitochondrial transmembrane potential and release of cytochrome c into the cytoplasm, which led ultimately to induction of apoptosis through activation of caspase-3, but not caspase-8. Consistently, Huh7 cells expressing NS3/4A and those harbouring an HCV RNA replicon were shown to be more prone to undergoing actinomycin D-induced, mitochondria-mediated apoptosis, compared with the control Huh7 cells. Taken together, these results suggest the possibility that HCV exerts cytopathic effect (CPE) on the infected cells under certain conditions and that NS4A is responsible, at least in part, for the conditional CPE in HCV-infected cells.

scholarly journals Modeling hepatitis C virus kinetics during liver transplantation reveals the role of the liver in virus clearance

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Louis Shekhtman ◽  
Miquel Navasa ◽  
Natasha Sansone ◽  
Gonzalo Crespo ◽  
Gitanjali Subramanya ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Kinetic Data ◽  
Culture Medium ◽  
Rapid Decline ◽  
Transplant Patients ◽  
Virus Clearance ◽  
Anhepatic Phase ◽  
Infected Cells

While the liver, specifically hepatocytes, are widely accepted as the main source of hepatitis C virus (HCV) production, the role of the liver/hepatocytes in clearance of circulating HCV remains unknown. Frequent HCV kinetic data were recorded and mathematically modeled from 5 liver-transplant patients throughout the anhepatic (absence of liver) phase and for 4 hours post-reperfusion. During the anhepatic phase, HCV remained at pre-anhepatic levels (n=3) or declined (n=2) with t1/2~1h. Immediately post-reperfusion, virus declined in a biphasic manner in 4 patients consisting of a rapid decline (t1/2=5min) followed by a slower decline (t1/2=67min). Consistent with the majority of patients in the anhepatic phase, when we monitored HCV clearance at 37°C from culture medium in the absence/presence of chronically infected hepatoma cells that were inhibited from secreting HCV, the HCV t1/2 in cell culture was longer in the absence of chronically HCV-infected cells. The results suggest that the liver plays a major role in the clearance of circulating HCV and that hepatocytes may be involved.

scholarly journals Osteopontin Regulates Hepatitis C Virus (HCV) Replication and Assembly by Interacting with HCV Proteins and Lipid Droplets and by Binding to Receptors αVβ3 and CD44

2018 ◽  
Vol 92 (13) ◽  
pp. e02116-17 ◽  
Author(s):  
Jawed Iqbal ◽  
Mehuli Sarkar-Dutta ◽  
Steven McRae ◽  
Akshaya Ramachandran ◽  
Binod Kumar ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Droplets ◽  
Critical Role ◽  
Migration And Invasion ◽  
Core Proteins ◽  
Important Relationship ◽  
Infected Cells

ABSTRACT Hepatitis C virus (HCV) replication and assembly occur at the specialized site of endoplasmic reticulum (ER) membranes and lipid droplets (LDs), respectively. Recently, several host proteins have been shown to be involved in HCV replication and assembly. In the present study, we demonstrated the important relationship among osteopontin (OPN), the ER, and LDs. OPN is a secreted phosphoprotein, and overexpression of OPN in hepatocellular carcinoma (HCC) tissue can lead to invasion and metastasis. OPN expression is also enhanced in HCV-associated HCC. Our recent studies have demonstrated the induction, proteolytic cleavage, and secretion of OPN in response to HCV infection. We also defined the critical role of secreted OPN in human hepatoma cell migration and invasion through binding to receptors integrin αVβ3 and CD44. However, the role of HCV-induced OPN in the HCV life cycle has not been elucidated. In this study, we showed a significant reduction in HCV replication, assembly, and infectivity in HCV-infected cells transfected with small interfering RNA (siRNA) against OPN, αVβ3, and CD44. We also observed the association of endogenous OPN with HCV proteins (NS3, NS5A, NS4A/B, NS5B, and core). Confocal microscopy revealed the colocalization of OPN with HCV NS5A and core in the ER and LDs, indicating a possible role for OPN in HCV replication and assembly. Interestingly, the secreted OPN activated HCV replication, infectivity, and assembly through binding to αVβ3 and CD44. Collectively, these observations provide evidence that HCV-induced OPN is critical for HCV replication and assembly. IMPORTANCE Recently, our studies uncovered the critical role of HCV-induced endogenous and secreted OPN in migration and invasion of hepatocytes. However, the role of OPN in the HCV life cycle has not been elucidated. In this study, we investigated the importance of OPN in HCV replication and assembly. We demonstrated that endogenous OPN associates with HCV NS3, NS5A, NS5B, and core proteins, which are in close proximity to the ER and LDs. Moreover, we showed that the interactions of secreted OPN with cell surface receptors αVβ3 and CD44 are critical for HCV replication and assembly. These observations provide evidence that HCV-induced endogenous and secreted OPN play pivotal roles in HCV replication and assembly in HCV-infected cells. Taken together, our findings clearly demonstrate that targeting OPN may provide opportunities for therapeutic intervention of HCV pathogenesis.

scholarly journals Peptide inhibitors of hepatitis C virus core oligomerization and virus production

2009 ◽  
Vol 90 (6) ◽  
pp. 1319-1328 ◽  
Author(s):  
S. Kota ◽  
C. Coito ◽  
G. Mousseau ◽  
J.-P. Lavergne ◽  
A. D. Strosberg
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Virus Production ◽  
Peptide Inhibitors ◽  
Rt Pcr ◽  
Protein Protein Interaction ◽  
Virus Core ◽  
Infected Cells

Hepatitis C virus (HCV) nucleocapsid assembly requires dimerization of the core protein, an essential step in the formation of the virus particle. We developed a novel quantitative assay for monitoring this protein–protein interaction, with the goal of identifying inhibitors of core dimerization that might block HCV production in infected Huh-7.5 hepatoma cells. Two core-derived, 18-residue peptides were found that inhibited the dimerization of a fragment of core comprising residues 1–106 (core106) by 68 and 63 %, respectively. A third, related 15-residue peptide displayed 50 % inhibition, with an IC50 of 21.9 μM. This peptide was shown, by fluorescence polarization, to bind directly to core106 with a K d of 1.9 μM and was displaced by the unlabelled peptide with an IC50 of 18.7 μM. When measured by surface plasmon resonance, the same peptide bound core169 with a K d of 7.2 μM. When added to HCV-infected cells, each of the three peptides blocked release, but not replication, of infectious virus. When measured by real-time RT-PCR, the RNA levels were reduced by 7-fold. The 15-residue peptide had no effect on HIV propagation. Such inhibitors may constitute useful tools to investigate the role of core dimerization in the virus cycle.

scholarly journals Correction for Yang et al., “Neglected but Important Role of Apolipoprotein E Exchange in Hepatitis C Virus Infection”

2017 ◽  
Vol 91 (24) ◽  
Author(s):  
Zaili Yang ◽  
Xiaoning Wang ◽  
Xiumei Chi ◽  
Fanfan Zhao ◽  
Jinxu Guo ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Infection ◽  
Apolipoprotein E ◽  
Hepatitis C Virus Infection
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