Mobility of the hepatitis C virus NS4B protein on the endoplasmic reticulum membrane and membrane-associated foci

The hepatitis C virus (HCV) non-structural protein NS4B induces morphological changes in the endoplasmic reticulum (ER) membrane that may have a direct role in viral RNA replication. A chimeric GFP–NS4B fusion protein located to the ER membrane and to foci that were attached to the ER. These membrane-associated foci (MAFs) could be related to the membrane alterations observed in cells that replicate HCV RNA. The relationship of MAFs to pre-existing cellular structures is not known. Indirect immunofluorescence analysis demonstrated that they did not contain a cellular marker for vesicles, which have been implicated in the replication of other viruses. From photobleaching studies to examine diffusion of NS4B, the GFP-tagged protein had reduced mobility on MAFs compared with on the ER membrane. This slower mobility suggested that NS4B is likely to form different interactions on MAFs and the ER.

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Generation of T-cell receptors targeting a genetically stable and immunodominant cytotoxic T-lymphocyte epitope within hepatitis C virus non-structural protein 3

Journal of General Virology ◽

10.1099/vir.0.037903-0 ◽

2012 ◽

Vol 93 (2) ◽

pp. 247-258 ◽

Cited By ~ 9

Author(s):

Anna Pasetto ◽

Lars Frelin ◽

Anette Brass ◽

Anila Yasmeen ◽

Sarene Koh ◽

...

Keyword(s):

T Cells ◽

Hepatitis C Virus ◽

Hepatitis C ◽

T Cell ◽

Cytotoxic T Lymphocyte ◽

Hcv Rna ◽

High Avidity ◽

Immune Recognition ◽

Structural Protein ◽

Human Hepatoma Cells

Hepatitis C virus (HCV) is a major cause of severe liver disease, and one major contributing factor is thought to involve a dysfunction of virus-specific T-cells. T-cell receptor (TCR) gene therapy with HCV-specific TCRs would increase the number of effector T-cells to promote virus clearance. We therefore took advantage of HLA-A2 transgenic mice to generate multiple TCR candidates against HCV using DNA vaccination followed by generation of stable T-cell–BW (T-BW) tumour hybrid cells. Using this approach, large numbers of non-structural protein 3 (NS3)-specific functional T-BW hybrids can be generated efficiently. These predominantly target the genetically stable HCV genotype 1 NS31073–1081 CTL epitope, frequently associated with clearance of HCV in humans. These T-BW hybrid clones recognized the NS31073 peptide with a high avidity. The hybridoma effectively recognized virus variants and targeted cells with low HLA-A2 expression, which has not been reported previously. Importantly, high-avidity murine TCRs effectively redirected human non-HCV-specific T-lymphocytes to recognize human hepatoma cells with HCV RNA replication driven by a subgenomic HCV replicon. Taken together, TCR candidates with a range of functional avidities, which can be used to study immune recognition of HCV-positive targets, have been generated. This has implications for TCR-related immunotherapy against HCV.

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Endoplasmic Reticulum Detergent-Resistant Membranes Accommodate Hepatitis C Virus Proteins for Viral Assembly

Cells ◽

10.3390/cells8050487 ◽

2019 ◽

Vol 8 (5) ◽

pp. 487 ◽

Cited By ~ 3

Author(s):

Audrey Boyer ◽

Julie Dreneau ◽

Amélie Dumans ◽

Julien Burlaud-Gaillard ◽

Anne Bull-Maurer ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Protein Interactions ◽

Gradient Centrifugation ◽

Cellular Membrane ◽

Structural Protein ◽

Membrane Structures ◽

Detergent Resistant Membranes ◽

Lysis Buffer

During Hepatitis C virus (HCV) morphogenesis, the non-structural protein 2 (NS2) brings the envelope proteins 1 and 2 (E1, E2), NS3, and NS5A together to form a complex at the endoplasmic reticulum (ER) membrane, initiating HCV assembly. The nature of the interactions in this complex is unclear, but replication complex and structural proteins have been shown to be associated with cellular membrane structures called detergent-resistant membranes (DRMs). We investigated the role of DRMs in NS2 complex formation, using a lysis buffer combining Triton and n-octyl glucoside, which solubilized both cell membranes and DRMs. When this lysis buffer was used on HCV-infected cells and the resulting lysates were subjected to flotation gradient centrifugation, all viral proteins and DRM-resident proteins were found in soluble protein fractions. Immunoprecipitation assays demonstrated direct protein–protein interactions between NS2 and E2 and E1 proteins, and an association of NS2 with NS3 through DRMs. The well-folded E1E2 complex and NS5A were not associated, instead interacting separately with the NS2-E1-E2-NS3 complex through less stable DRMs. Core was also associated with NS2 and the E1E2 complex through these unstable DRMs. We suggest that DRMs carrying this NS2-E1-E2-NS3-4A-NS5A-core complex may play a central role in HCV assembly initiation, potentially as an assembly platform.

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Inhibitors of Endoplasmic Reticulum α-Glucosidases Potently Suppress Hepatitis C Virus Virion Assembly and Release

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01319-10 ◽

2010 ◽

Vol 55 (3) ◽

pp. 1036-1044 ◽

Cited By ~ 47

Author(s):

Xiaowang Qu ◽

Xiaoben Pan ◽

Jessica Weidner ◽

Wenquan Yu ◽

Dominic Alonzi ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Nonstructural Protein ◽

Hcv Rna ◽

Virion Assembly ◽

Subsequent Degradation ◽

Important Host ◽

Antiviral Targets ◽

Sugar Derivatives

ABSTRACTα-Glucosidases I and II are endoplasmic reticulum-resident enzymes that are essential for N-linked glycan processing and subsequent proper folding of glycoproteins. In this report, we first demonstrate that downregulation of the expression of α-glucosidase I, II, or both in Huh7.5 cells by small hairpin RNA technology inhibited the production of hepatitis C virus (HCV). In agreement with the essential role of α-glucosidases in HCV envelope glycoprotein processing and folding, treatment of HCV-infected cells with a panel of imino sugar derivatives, which are competitive inhibitors of α-glucosidases, did not affect intracellular HCV RNA replication and nonstructural protein expression but resulted in the inhibition of glycan processing and subsequent degradation of HCV E2 glycoprotein. As a consequence, HCV virion assembly and secretion were inhibited. In searching for imino sugars with better antiviral activity, we found that a novel imino sugar, PBDNJ0804, had a superior ability to inhibit HCV virion assembly and secretion. In summary, we demonstrated that glucosidases are important host factor-based antiviral targets for HCV infection. The low likelihood of drug-resistant virus emergence and potent antiviral efficacy of the novel glucosidase inhibitor hold promise for its development as a therapeutic agent for the treatment of chronic hepatitis C.

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Hepatitis C virus NS5A protein interacts with 2′,5′-oligoadenylate synthetase and inhibits antiviral activity of IFN in an IFN sensitivity-determining region-independent manner

Journal of General Virology ◽

10.1099/vir.0.19513-0 ◽

2004 ◽

Vol 85 (4) ◽

pp. 959-969 ◽

Cited By ~ 81

Author(s):

Takashi Taguchi ◽

Motoko Nagano-Fujii ◽

Masato Akutsu ◽

Hiroyasu Kadoya ◽

Shinji Ohgimoto ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Activity ◽

Cultured Cells ◽

Single Point ◽

Point Mutations ◽

Hcv Rna ◽

Structural Protein ◽

Oligoadenylate Synthetase ◽

Independent Manner

The non-structural protein 5A (NS5A) of hepatitis C virus (HCV) has been implicated in inhibition of antiviral activity of IFN. While previous studies have suggested an interaction between NS5A and the double-stranded RNA-dependent protein kinase (PKR), the possibility still remains that interaction with another molecule(s) is involved in the NS5A-mediated inhibition of IFN. In the present study, we investigated a possible interaction between NS5A and 2′,5′-oligoadenylate synthetase (2-5AS), another key molecule in antiviral activity. We observed that NS5A physically interacted with 2-5AS in cultured cells, with an N-terminal portion of NS5A [aa 1–148; NS5A(1–148)] and two separate portions of 2-5AS (aa 52–104 and 184–275) being involved in the interaction. Single point mutations at residue 37 of NS5A affected the degree of the interaction with 2-5AS, with a Phe-to-Leu mutation (F37L) augmenting and a Phe-to-Asn mutation (F37N) diminishing it. Virus rescue assay revealed that the full-length NS5A (NS5A-F) and NS5A(1–148), the latter of which contains neither the IFN sensitivity-determining region (ISDR) nor the PKR-binding domain, significantly counteracted the antiviral activity of IFN. Introduction of a F37N mutation into NS5A(1–148) impaired the otherwise more significant IFN-inhibitory activity of NS5A(1–148). It was also found that the F37N mutation was highly disadvantageous for the replication of an HCV RNA replicon. Taken together, our results suggest the possibility that NS5A interacts with 2-5AS and inhibits the antiviral activity of IFN in an ISDR-independent manner.

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Evidence for immune activation in patients with residual hepatitis C virus RNA long after successful treatment with IFN and ribavirin

Journal of General Virology ◽

10.1099/vir.0.064709-0 ◽

2014 ◽

Vol 95 (9) ◽

pp. 2004-2009 ◽

Cited By ~ 8

Author(s):

Marek Radkowski ◽

Jolanta Opoka-Kegler ◽

Kamila Caraballo Cortes ◽

Iwona Bukowska-Ośko ◽

Karol Perlejewski ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Successful Treatment ◽

Immune Activation ◽

Hcv Rna ◽

Structural Protein ◽

Inflammatory Protein ◽

Virus Rna ◽

Tnf Α ◽

Macrophage Inflammatory Protein

Low-level hepatitis C virus (HCV) RNA may persist in PBMCs after successful treatment of chronic hepatitis C, but the consequences of this phenomenon are unclear. Forty-nine patients who achieved a sustained virological response (SVR) after pegylated IFN and ribavirin therapy were analysed 52–66 months after the SVR. HCV RNA was detected in PBMCs from 18 patients (47.4 %), and PBMCs in two patients stained positive for non-structural protein 3 (NS3). Quantification of various cytokine and chemokine transcripts in PBMCs revealed that levels of IL-6, IL-8, IL-12, TNF-α and macrophage inflammatory protein 1β were significantly higher in HCV-positive patients than in HCV-negative individuals. In conclusion, persistence of HCV RNA in PBMCs of patients with a SVR appears to be associated with immune activation.

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

Journal of General Virology ◽

10.1099/vir.0.81701-0 ◽

2006 ◽

Vol 87 (7) ◽

pp. 1935-1945 ◽

Cited By ~ 77

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.

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Hepatitis C Virus Lipoviroparticles Assemble in the Endoplasmic Reticulum (ER) and Bud off from the ER to the Golgi Compartment in COPII Vesicles

Journal of Virology ◽

10.1128/jvi.00499-17 ◽

2017 ◽

Vol 91 (15) ◽

Cited By ~ 17

Author(s):

Gulam H. Syed ◽

Mohsin Khan ◽

Song Yang ◽

Aleem Siddiqui

Keyword(s):

Electron Microscopy ◽

Endoplasmic Reticulum ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Immunogold Electron Microscopy ◽

Hcv Rna ◽

Core Proteins ◽

Golgi Compartment ◽

Copii Vesicles

ABSTRACT Hepatitis C virus (HCV) exists as a lipoprotein-virus hybrid lipoviroparticle (LVP). In vitro studies have demonstrated the importance of apolipoproteins in HCV secretion and infectivity, leading to the notion that HCV coopts the secretion of very-low-density lipoprotein (VLDL) for its egress. However, the mechanisms involved in virus particle assembly and egress are still elusive. The biogenesis of VLDL particles occurs in the endoplasmic reticulum (ER), followed by subsequent lipidation in the ER and Golgi compartment. The secretion of mature VLDL particles occurs through the Golgi secretory pathway. HCV virions are believed to latch onto or fuse with the nascent VLDL particle in either the ER or the Golgi compartment, resulting in the generation of LVPs. In our attempt to unravel the collaboration between HCV and VLDL secretion, we studied HCV particles budding from the ER en route to the Golgi compartment in COPII vesicles. Biophysical characterization of COPII vesicles fractionated on an iodixanol gradient revealed that HCV RNA is enriched in the highly buoyant COPII vesicle fractions and cofractionates with apolipoprotein B (ApoB), ApoE, and the HCV core and envelope proteins. Electron microscopy of immunogold-labeled microsections revealed that the HCV envelope and core proteins colocalize with apolipoproteins and HCV RNA in Sec31-coated COPII vesicles. Ultrastructural analysis also revealed the presence of HCV structural proteins, RNA, and apolipoproteins in the Golgi stacks. These findings support the hypothesis that HCV LVPs assemble in the ER and are transported to the Golgi compartment in COPII vesicles to embark on the Golgi secretory route. IMPORTANCE HCV assembly and release accompany the formation of LVPs that circulate in the sera of HCV patients and are also produced in an in vitro culture system. The pathway of HCV morphogenesis and secretion has not been fully understood. This study investigates the exact site where the association of HCV virions with host lipoproteins occurs. Using immunoprecipitation of COPII vesicles and immunogold electron microscopy (EM), we characterize the existence of LVPs that cofractionate with lipoproteins, viral proteins, RNA, and vesicular components. Our results show that this assembly occurs in the ER, and LVPs thus formed are carried through the Golgi network by vesicular transport. This work provides a unique insight into the HCV LVP assembly process within infected cells and offers opportunities for designing antiviral therapeutic cellular targets.

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