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

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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Determinants of Hepatitis C Virus p7 Ion Channel Function and Drug Sensitivity Identified In Vitro

Journal of Virology ◽

10.1128/jvi.00521-09 ◽

2009 ◽

Vol 83 (16) ◽

pp. 7970-7981 ◽

Cited By ~ 52

Author(s):

Corine StGelais ◽

Toshana L. Foster ◽

Mark Verow ◽

Elizabeth Atkins ◽

Colin W. G. Fishwick ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Ion Channel ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Channel Activity ◽

Amino Terminal ◽

Channel Function ◽

P7 Channel

ABSTRACT Hepatitis C virus (HCV) chronically infects 170 million individuals, causing severe liver disease. Although antiviral chemotherapy exists, the current regimen is ineffective in 50% of cases due to high levels of innate virus resistance. New, virus-specific therapies are forthcoming although their development has been slow and they are few in number, driving the search for new drug targets. The HCV p7 protein forms an ion channel in vitro and is critical for the secretion of infectious virus. p7 displays sensitivity to several classes of compounds, making it an attractive drug target. We recently demonstrated that p7 compound sensitivity varies according to viral genotype, yet little is known of the residues within p7 responsible for channel activity or drug interactions. Here, we have employed a liposome-based assay for p7 channel function to investigate the genetic basis for compound sensitivity. We demonstrate using chimeric p7 proteins that neither the two trans-membrane helices nor the p7 basic loop individually determines compound sensitivity. Using point mutation analysis, we identify amino acids important for channel function and demonstrate that null mutants exert a dominant negative effect over wild-type protein. We show that, of the three hydrophilic regions within the amino-terminal trans-membrane helix, only the conserved histidine at position 17 is important for genotype 1b p7 channel activity. Mutations predicted to play a structural role affect both channel function and oligomerization kinetics. Lastly, we identify a region at the p7 carboxy terminus which may act as a specific sensitivity determinant for the drug amantadine.

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Hepatitis C Virus-Like Particle Morphogenesis

Journal of Virology ◽

10.1128/jvi.76.8.4073-4079.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 4073-4079 ◽

Cited By ~ 77

Author(s):

Emmanuelle Blanchard ◽

Denys Brand ◽

Sylvie Trassard ◽

Alain Goudeau ◽

Philippe Roingeard

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Mammalian Cells ◽

Semliki Forest Virus ◽

Virus Like Particle ◽

Genes Encoding ◽

Host Cell Interactions ◽

Viral Morphogenesis ◽

First Time

ABSTRACT Although much is known about the hepatitis C virus (HCV) genome, first cloned in 1989, little is known about HCV structure and assembly due to the lack of an efficient in vitro culture system for HCV. Using a recombinant Semliki forest virus replicon expressing genes encoding HCV structural proteins, we observed for the first time the assembly of these proteins into HCV-like particles in mammalian cells. This system opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions.

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

Journal of General Virology ◽

10.1099/vir.0.82049-0 ◽

2007 ◽

Vol 88 (1) ◽

pp. 134-142 ◽

Cited By ~ 31

Author(s):

G. Haqshenas ◽

J. M. Mackenzie ◽

X. Dong ◽

E. J. Gowans

Keyword(s):

Endoplasmic Reticulum ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Lipid Bilayers ◽

Fluorescent Protein ◽

Wild Type ◽

Rna Transcripts ◽

Green Fluorescent ◽

Viral Polyprotein

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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Gene Therapeutic Approach for Inhibiting Hepatitis C Virus Replication Using a Recombinant Protein That Controls Interferon Expression

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00682-10 ◽

2010 ◽

Vol 54 (12) ◽

pp. 5048-5056 ◽

Cited By ~ 3

Author(s):

Chul Hyun Joo ◽

Uk Lee ◽

Young Ran Nam ◽

Jae U. Jung ◽

Heuiran Lee ◽

...

Keyword(s):

Transcription Factor ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Cultured Cells ◽

Viral Vector ◽

Wide Spectrum ◽

Intracellular Localization ◽

Treatment Modalities ◽

In Cells

ABSTRACT The hepatitis C virus (HCV) is a continuing threat to public health. The systemic administration of interferon alpha with ribavirin is the only currently approved treatment. However, this treatment is associated with a wide spectrum of systemic side effects that limits its effectiveness; thus, there is an urgent need for new treatment modalities. In this study, we describe a novel anti-HCV strategy employing a recombinant transcription factor that we have engineered in such a way that NS3/4a viral protease controls its intracellular localization, thereby restoring interferon secretion specifically in cells infected with HCV. Proof-of-concept experiments validated the strategy, showing that the recombinant transcription factor was triggered to stimulate interferon promoter by NS3/4A and remained inactive in cells without NS3/4a. Using an adenovirus-associated viral vector delivery system, we found that the recombinant transcription factor inhibited HCV replication effectively in vitro in cultured cells.

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Differences in the expression of the hepatitis C virus core+1 open reading frame between a nuclear and a cytoplasmic expression system

Journal of General Virology ◽

10.1099/vir.0.83260-0 ◽

2008 ◽

Vol 89 (1) ◽

pp. 222-231 ◽

Cited By ~ 10

Author(s):

Niki Vassilaki ◽

Katerina I. Kalliampakou ◽

Penelope Mavromara

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Mammalian Cells ◽

Expression System ◽

Open Reading Frame ◽

Reading Frame ◽

Transcription System ◽

The Core ◽

Internal Core

The hepatitis C virus (HCV) genome possesses an open reading frame (ORF) overlapping the core gene at +1 nucleotide (core+1 ORF). Initial in vitro studies suggested that the core+1 ORF is translated by a ribosomal −2/+1 frameshift mechanism during elongation of the viral polyprotein. Recent studies, however, based on transfection of mammalian cells with reporter constructs have shown that translation of the core+1 ORF is mediated from internal core+1 codons. To resolve the apparent discrepancies associated with the mechanism of core+1 translation, we examined the expression of the HCV-1 and HCV-1a (H) core+1 ORF in a cytoplasmic transcription system based on Huh-7/T7 cells that constitutively synthesize the T7 RNA polymerase in comparison to that in Huh-7 cells. We showed that the efficiency of both the −2/+1 and −1/+2 frameshift events operating at the HCV-1 core codons 8–11 is significantly enhanced in the Huh-7/T7 cytoplasmic transcription system and is dependent on the presence of the consecutive adenine (A) residues within core codons 8–11. In contrast, internal translation initiation at core+1 codons 85/87 occurs in both the nuclear and cytoplasmic transcription systems and is not repressed by the ribosomal frameshifting event. Finally, although core+1 codons 85/87 is the most efficient site for internal initiation of core+1 translation, it may not be unique, as additional internal core+1 codon(s) appear to drive translation at low levels.

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Intracellular localization of full-length and truncated hepatitis C virus core protein expressed in mammalian cells

Journal of Hepatology ◽

10.1016/s0168-8278(05)80157-6 ◽

1994 ◽

Vol 20 (6) ◽

pp. 833-836 ◽

Cited By ~ 42

Author(s):

Antonella Ravaggi ◽

Gioacchino Natoli ◽

Daniele Primi ◽

Alberto Albertini ◽

Massimo Levrero ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Mammalian Cells ◽

Core Protein ◽

Intracellular Localization ◽

Full Length ◽

Virus Core

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Functional analysis of hepatitis C virus E2 glycoproteins and virus-like particles reveals structural dissimilarities between different forms of E2

Journal of General Virology ◽

10.1099/0022-1317-82-8-1877 ◽

2001 ◽

Vol 82 (8) ◽

pp. 1877-1883 ◽

Cited By ~ 140

Author(s):

Ania Owsianka ◽

Reginald F. Clayton ◽

Lawrence D. Loomis-Price ◽

Jane A. McKeating ◽

Arvind H. Patel

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Structure Function ◽

Mammalian Cells ◽

Function Analysis ◽

Virus Like Particles ◽

Binding Characteristics ◽

Virus Interaction ◽

Cd81 Binding

Structure–function analysis of the hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, has been difficult due to the unavailability of HCV virions. Truncated soluble forms of E2 have been used as models to study virus interaction with the putative HCV receptor CD81, but they may not fully mimic E2 structures on the virion. Here, we compared the CD81-binding characteristics of truncated E2 (E2660) and full-length (FL) E1E2 complex expressed in mammalian cells, and of HCV virus-like particles (VLPs) generated in insect cells. All three glycoprotein forms interacted with human CD81 in an in vitro binding assay, allowing us to test a panel of well-characterized anti-E2 monoclonal antibodies (MAbs) for their ability to inhibit the glycoprotein–CD81 interaction. MAbs specific for E2 amino acid (aa) regions 396–407, 412–423 and 528–535 blocked binding to CD81 of all antigens tested. However, MAbs specific for regions 432–443, 436–443 and 436–447 inhibited the interaction of VLPs, but not of E2660 or the FL E1E2 complex with CD81, indicating the existence of structural differences amongst the E2 forms. These findings underscore the need to carefully select an appropriate ligand for structure–function analysis.

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