Hepatitis C virus p7 protein is localized in the endoplasmic reticulum when it is encoded by a replication-competent genome

p7 protein is a small protein encoded by Hepatitis C virus (HCV) that functions as an ion channel in planar lipid bilayers. The function of p7 is vital for the virus life cycle. In this study, the p7 protein of genotype 2a (strain JFH1; the only strain that replicates and produces virus progeny in vitro) was tagged with either an enhanced green fluorescent protein (eGFP) or a haemagglutinin (HA) epitope to facilitate tracking of the protein in the intracellular environment. The tagged viral polyprotein was expressed transiently in the cells after transfection with the recombinant RNA transcripts. Confocal microscopy revealed that the tagged p7 protein was localized in the endoplasmic reticulum (ER) but not associated with mitochondria. Immunoelectron microscopy confirmed the p7 localization data and, moreover, showed that intracellular virus-like particles formed in the cells transfected with the wild-type, but not the recombinant, transcripts. Following a few passages of the transfected cells, the recombinant genome with the HA tag reverted to wild-type and the entire tag was deleted. Therefore, in this study, it has been demonstrated that the p7 protein in the context of the full-length polyprotein encoded by a replication competent genome is only localized to the ER and has a possible role in HCV particle formation.

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Infectious Hepatitis C Virus Pseudo-particles Containing Functional E1–E2 Envelope Protein Complexes

Journal of Experimental Medicine ◽

10.1084/jem.20021756 ◽

2003 ◽

Vol 197 (5) ◽

pp. 633-642 ◽

Cited By ~ 815

Author(s):

Birke Bartosch ◽

Jean Dubuisson ◽

François-Loïc Cosset

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Fluorescent Protein ◽

Protein Complexes ◽

Marker Gene ◽

Infectious Hepatitis ◽

Protein Marker ◽

A Cell ◽

Green Fluorescent

The study of hepatitis C virus (HCV), a major cause of chronic liver disease, has been hampered by the lack of a cell culture system supporting its replication. Here, we have successfully generated infectious pseudo-particles that were assembled by displaying unmodified and functional HCV glycoproteins onto retroviral and lentiviral core particles. The presence of a green fluorescent protein marker gene packaged within these HCV pseudo-particles allowed reliable and fast determination of infectivity mediated by the HCV glycoproteins. Primary hepatocytes as well as hepato-carcinoma cells were found to be the major targets of infection in vitro. High infectivity of the pseudo-particles required both E1 and E2 HCV glycoproteins, and was neutralized by sera from HCV-infected patients and by some anti-E2 monoclonal antibodies. In addition, these pseudo-particles allowed investigation of the role of putative HCV receptors. Although our results tend to confirm their involvement, they provide evidence that neither LDLr nor CD81 is sufficient to mediate HCV cell entry. Altogether, these studies indicate that these pseudo-particles may mimic the early infection steps of parental HCV and will be suitable for the development of much needed new antiviral therapies.

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Protein-Protein Interactions between Hepatitis C Virus Nonstructural Proteins

Journal of Virology ◽

10.1128/jvi.77.9.5401-5414.2003 ◽

2003 ◽

Vol 77 (9) ◽

pp. 5401-5414 ◽

Cited By ~ 131

Author(s):

Maria Dimitrova ◽

Isabelle Imbert ◽

Marie Paule Kieny ◽

Catherine Schuster

Keyword(s):

Endoplasmic Reticulum ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Protein Interactions ◽

Laser Scanning ◽

Ex Vivo ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Nonstructural Proteins

ABSTRACT Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.

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Naturally Occurring Hepatitis C Virus Subgenomic Deletion Mutants Replicate Efficiently in Huh-7 Cells and Are trans-Packaged In Vitro To Generate Infectious Defective Particles

Journal of Virology ◽

10.1128/jvi.00308-09 ◽

2009 ◽

Vol 83 (18) ◽

pp. 9079-9093 ◽

Cited By ~ 32

Author(s):

Laura Pacini ◽

Rita Graziani ◽

Linda Bartholomew ◽

Raffaele De Francesco ◽

Giacomo Paonessa

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Line ◽

Genome Structure ◽

Structural Proteins ◽

Wild Type Virus ◽

Deletion Mutants ◽

Wild Type ◽

Naturally Occurring

ABSTRACT Naturally occurring hepatitis C virus (HCV) subgenomic RNAs have been found in several HCV patients. These subgenomic deletion mutants, mostly lacking the genes encoding envelope glycoproteins, were found in both liver and serum, where their relatively high abundance suggests that they are capable of autonomous replication and can be packaged and secreted in viral particles, presumably harboring the envelope proteins from wild type virus coinfecting the same cell. We recapitulated some of these natural subgenomic deletions in the context of the isolate JFH-1 and confirmed these hypotheses in vitro. In Huh-7.5 cells, these deletion-containing genomes show robust replication and can be efficiently trans-packaged and infect naïve Huh-7.5 cells when cotransfected with the full-length wild-type J6/JFH genome. The genome structure of these natural subgenomic deletion mutants was dissected, and the maintenance of both core and NS2 regions was proven to be significant for efficient replication and trans-packaging. To further explore the requirements needed to achieve trans-complementation, we provided different combinations of structural proteins in trans. Optimal trans-complementation was obtained when fragments of the polyprotein encompassing core to p7 or E1 to NS2 were expressed. Finally, we generated a stable helper cell line, constitutively expressing the structural proteins from core to p7, which efficiently supports trans-complementation of a subgenomic deletion encompassing amino acids 284 to 732. This cell line can produce and be infected by defective particles, thus representing a powerful tool to investigate the life cycle and relevance of natural HCV subgenomic deletion mutants in vivo.

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Hepatitis C Virus F Protein Is a Short-Lived Protein Associated with the Endoplasmic Reticulum

Journal of Virology ◽

10.1128/jvi.77.2.1578-1583.2003 ◽

2003 ◽

Vol 77 (2) ◽

pp. 1578-1583 ◽

Cited By ~ 54

Author(s):

Zhenming Xu ◽

Jinah Choi ◽

Wen Lu ◽

Jing-hsiung Ou

Keyword(s):

Endoplasmic Reticulum ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Half Life ◽

Proteasome Inhibitors ◽

Subcellular Fractionation ◽

Biological Properties ◽

F Protein ◽

Proteasome Pathway

ABSTRACT Hepatitis C virus (HCV) F protein is a newly discovered HCV gene product that is expressed by translational ribosomal frameshift. Little is known about the biological properties of this protein. By performing pulse-chase labeling experiments, we demonstrate here that the F protein is a labile protein with a half-life of <10 min in Huh7 hepatoma cells and in vitro. The half-life of the F protein could be substantially increased by proteasome inhibitors, suggesting that the rapid degradation of the F protein is mediated by the proteasome pathway. Further immunofluorescence staining and subcellular fractionation experiments indicate that the F protein is primarily associated with the endoplasmic reticulum. This subcellular localization is similar to those of HCV core and NS5A proteins, raising the possibility that the F protein may participate in HCV morphogenesis or replication.

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Modulation of Virulence by Two Acidified Nitrite-Responsive Loci of Salmonella enterica Serovar Typhimurium

Infection and Immunity ◽

10.1128/iai.71.6.3196-3205.2003 ◽

2003 ◽

Vol 71 (6) ◽

pp. 3196-3205 ◽

Cited By ~ 39

Author(s):

Charles C. Kim ◽

Denise Monack ◽

Stanley Falkow

Keyword(s):

Salmonella Enterica ◽

Fluorescent Protein ◽

Dependent Manner ◽

Wild Type ◽

Independent Manner ◽

Inducible Promoters ◽

Serovar Typhimurium ◽

Bacterial Genes ◽

Green Fluorescent

ABSTRACT Two acidified nitrite-inducible genes of Salmonella enterica serovar Typhimurium were identified with a green fluorescent protein-based promoter-trap screen. The nitrite-inducible promoters were located upstream of loci that we designated nipAB and nipC, which correspond to hcp-hcr (hybrid cluster protein) of Escherichia coli and norA of Alcaligenes eutrophus, respectively. Maximal induction of the promoters by nitrite was dependent on pH. The nipAB promoter was regulated by oxygen in an Fnr-dependent manner. The nipC promoter was also regulated by oxygen but in an Fnr-independent manner. The promoters were upregulated in activated RAW264.7 macrophage-like cells, which produce NO via the inducible nitric oxide synthase (iNOS), and the induction was inhibited by aminoguanidine, an inhibitor of iNOS. Although the nipAB and nipC mutants displayed no defects under a variety of in vitro conditions or in tissue culture infections, they exhibited lower oral 50% lethal doses (LD50s) than did the wild type in C57BL/6J mouse infections. The lower LD50s reflected an unexpected increased ability of small inoculating doses of the mutant bacteria to cause lethal infection 2 to 3 weeks after challenge, compared to a similar challenge dose of wild-type bacteria. We conclude that these genes are regulated by physiological nitrogen oxides and that the absence of these bacterial genes in some way diminishes the ability of mice to clear a low dose infection.

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A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria

Journal of General Virology ◽

10.1099/vir.0.19634-0 ◽

2004 ◽

Vol 85 (2) ◽

pp. 451-461 ◽

Cited By ~ 125

Author(s):

Stephen D. C. Griffin ◽

Ruth Harvey ◽

Dean S. Clarke ◽

Wendy S. Barclay ◽

Mark Harris ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Ion Channel ◽

Lipid Bilayers ◽

Mammalian Cells ◽

Intracellular Localization ◽

Channel Activity ◽

Diarrhoea Virus ◽

Basic Loop

We previously identified the function of the hepatitis C virus (HCV) p7 protein as an ion channel in artificial lipid bilayers and demonstrated that this in vitro activity is inhibited by amantadine. Here we show that the ion channel activity of HCV p7 expressed in mammalian cells can substitute for that of influenza virus M2 in a cell-based assay. This was also the case for the p7 from the related virus, bovine viral diarrhoea virus (BVDV). Moreover, amantadine was shown to abrogate HCV p7 function in this assay at a concentration that specifically inhibits M2. Mutation of a conserved basic loop located between the two predicted trans-membrane alpha helices rendered HCV p7 non-functional as an ion channel. The intracellular localization of p7 was unaffected by this mutation and was found to overlap significantly with membranes associated with mitochondria. Demonstration of p7 ion channel activity in cellular membranes and its inhibition by amantadine affirm the protein as a target for future anti-viral chemotherapy.

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Conserved C-Terminal Threonine of Hepatitis C Virus NS3 Regulates Autoproteolysis and Prevents Product Inhibition

Journal of Virology ◽

10.1128/jvi.78.2.700-709.2004 ◽

2004 ◽

Vol 78 (2) ◽

pp. 700-709 ◽

Cited By ~ 15

Author(s):

Wenyan Wang ◽

Frederick C. Lahser ◽

MinKyung Yi ◽

Jacquelyn Wright-Minogue ◽

Ellen Xia ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

In Vitro Transcription ◽

Product Inhibition ◽

Cleavage Sites ◽

Wild Type ◽

Genome Sequences ◽

Mutant Proteins ◽

C Terminus

ABSTRACT Inspection of over 250 hepatitis C virus (HCV) genome sequences shows that a threonine is strictly conserved at the P1 position in the NS3-NS4A (NS3-4A) autoproteolysis junction, while a cysteine is maintained as the P1 residue in all of the putative trans cleavage sites (NS4A-4B, NS4B-5A, and NS5A-5B). To understand why T631 is conserved at the NS3-4A junction of HCV, a series of in vitro transcription-translation studies were carried out using wild-type and mutant (T631C) NS3-4A constructs bearing native, truncated, and mutant NS4A segments. The autocleavage of the wild-type junction was found to be dependent on the presence of the central cofactor domain of NS4A (residues 21 to 34). In contrast, all NS3-4A T631C mutant proteins underwent self-cleavage even in the absence of the cofactor. Subgenomic replicons derived from the Con1 strain of HCV and bearing the T631C mutation showed reduced levels of colony formation in transfection studies. Similarly, replicons derived from a second genotype 1b virus, HCV-N, demonstrated a comparable reduction in replication efficiency in transient-transfection assays. These data suggest that the threonine is conserved at position 631 because it serves two functions: (i) to slow processing at the NS3-4A cleavage site, ensuring proper intercalation of the NS4A cofactor with NS3 prior to polyprotein scission, and (ii) to prevent subsequent product inhibition by the NS3 C terminus.

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Phosphoprotein Enriched in Astrocytes-15 kDa Expression Inhibits Astrocyte Migration by a Protein Kinase Cδ-dependent Mechanism

Molecular Biology of the Cell ◽

10.1091/mbc.e05-11-1072 ◽

2006 ◽

Vol 17 (12) ◽

pp. 5141-5152 ◽

Cited By ~ 38

Author(s):

François Renault-Mihara ◽

Frédéric Beuvon ◽

Xavier Iturrioz ◽

Brigitte Canton ◽

Sophie De Bouard ◽

...

Keyword(s):

Protein Kinase ◽

Fluorescent Protein ◽

Constitutive Expression ◽

Mitogen Activated Protein Kinase ◽

Wild Type ◽

Bryostatin 1 ◽

Calcium Calmodulin Dependent ◽

Mitogen Activated Protein ◽

Green Fluorescent

Phosphoprotein enriched in astrocytes-15 kDa (PEA-15), a phosphoprotein enriched in astrocytes, inhibits both apoptosis and proliferation in normal and cancerous cells. Here, analysis of PEA-15 expression in glioblastoma organotypic cultures revealed low levels of PEA-15 in tumor cells migrating away from the explants, regardless of the expression levels in the originating explants. Because glioblastomas are highly invasive primary brain tumors that can originate from astrocytes, we explored the involvement of PEA-15 in the control of astrocyte migration. PEA-15−/− astrocytes presented an enhanced motility in vitro compared with their wild-type counterparts. Accordingly, NIH-3T3 cells transfected by green fluorescent protein-PEA-15 displayed a reduced migration. Reexpression of PEA-15 restored PEA-15−/− astrocyte motility to wild-type levels. Pharmacological manipulations excluded a participation of extracellular signal-regulated kinase/mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and calcium/calmodulin-dependent protein kinase II in this effect of PEA-15. In contrast, treatment by bisindolylmaleimide, Gö6976, and rottlerin, and chronic application of phorbol 12-myristate 13-acetate and/or bryostatin-1 indicated that PKCδ mediated PEA-15 inhibition of astrocyte migration. PEA-15−/− astrocytes constitutively expressed a 40-kDa form of PKCδ that was down-regulated upon PEA-15 reexpression. Together, these data reveal a new function for PEA-15 in the inhibitory control of astrocyte motility through a PKCδ-dependent pathway involving the constitutive expression of a catalytic fragment of PKCδ.

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Insertion of Green Fluorescent Protein into Nonstructural Protein 5A Allows Direct Visualization of Functional Hepatitis C Virus Replication Complexes

Journal of Virology ◽

10.1128/jvi.78.14.7400-7409.2004 ◽

2004 ◽

Vol 78 (14) ◽

pp. 7400-7409 ◽

Cited By ~ 194

Author(s):

Darius Moradpour ◽

Matthew J. Evans ◽

Rainer Gosert ◽

Zhenghong Yuan ◽

Hubert E. Blum ◽

...

Keyword(s):

Hepatitis C Virus ◽

Green Fluorescent Protein ◽

Hepatitis C ◽

Fusion Protein ◽

Fluorescent Protein ◽

Nonstructural Protein ◽

Rna Replication ◽

Direct Visualization ◽

Green Fluorescent ◽

Nonstructural Protein 5A

ABSTRACT Hepatitis C virus (HCV) replicates its genome in a membrane-associated replication complex, composed of viral proteins, replicating RNA and altered cellular membranes. We describe here HCV replicons that allow the direct visualization of functional HCV replication complexes. Viable replicons selected from a library of Tn7-mediated random insertions in the coding sequence of nonstructural protein 5A (NS5A) allowed the identification of two sites near the NS5A C terminus that tolerated insertion of heterologous sequences. Replicons encoding green fluorescent protein (GFP) at these locations were only moderately impaired for HCV RNA replication. Expression of the NS5A-GFP fusion protein could be demonstrated by immunoblot, indicating that the GFP was retained during RNA replication and did not interfere with HCV polyprotein processing. More importantly, expression levels were robust enough to allow direct visualization of the fusion protein by fluorescence microscopy. NS5A-GFP appeared as brightly fluorescing dot-like structures in the cytoplasm. By confocal laser scanning microscopy, NS5A-GFP colocalized with other HCV nonstructural proteins and nascent viral RNA, indicating that the dot-like structures, identified as membranous webs by electron microscopy, represent functional HCV replication complexes. These findings reveal an unexpected flexibility of the C-terminal domain of NS5A and provide tools for studying the formation and turnover of HCV replication complexes in living cells.

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