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

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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In vitro hepatitis C virus infection and hepatic choline metabolism

10.1101/746776 ◽

2019 ◽

Author(s):

Kaelan Gobeil Odai ◽

Conor O’Dwyer ◽

Rineke Steenbergen ◽

Tyler A. Shaw ◽

Tyler M. Renner ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cultured Cells ◽

Hcv Infection ◽

Choline Uptake ◽

Choline Transport ◽

Choline Metabolism ◽

Positive Strand Rna ◽

Uptake And Metabolism

AbstractCholine is an essential nutrient required for normal neuronal and muscular development, as well as homeostatic regulation of hepatic metabolism. In the liver, choline is incorporated into the main eukaryotic phospholipid, phosphatidylcholine (PC) and can enter one carbon metabolism via mitochondrial oxidation. Hepatitis C virus (HCV) is a hepatotropic positive-strand RNA virus that similar to other positive-strand RNA viruses can impact phospholipid metabolism. In the current study we sought to interrogate the link between choline transport and early HCV infection. Namely, we aimed to investigate how HCV modulates markers of choline metabolism following in vitro infection, while subsequently assessing how the inhibition of choline uptake and metabolism upon concurrent HCV infection may alter early viral replication. Finally, we assessed whether these parameters were consistent between cells cultured in fetal bovine serum (FBS) or human serum (HS), conditions known to differentially affect in vitro HCV infection. We observed that choline transport in FBS-cultured Huh7.5 cells is facilitated by the intermediate affinity transporter choline transporter-like family (CTL), and that CTL1 expression and the incorporation of choline into PC is diminished in 24 h infected FBS-cultured cells. Reciprocally, limiting the availability of choline for PC synthesis resulted in increased HCV replication at this early stage. In chronically HS-cultured Huh7.5 cells, there were no differences in the expression of choline transporters upon HCV infection or alterations to viral replication when choline transport was inhibited compared to control treatments. However, inhibiting choline uptake and metabolism in this system significantly impaired the production of infectious virions in HS-cultured cells. These results suggest that in addition to a known role of choline kinase, the transport of choline, potentially via CTL1, might also represent an important and regulated process during HCV infection.Abstract Figure

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In Vitro Hepatitis C Virus Infection and Hepatic Choline Metabolism

Viruses ◽

10.3390/v12010108 ◽

2020 ◽

Vol 12 (1) ◽

pp. 108 ◽

Cited By ~ 1

Author(s):

Kaelan Gobeil Odai ◽

Conor O’Dwyer ◽

Rineke Steenbergen ◽

Tyler A. Shaw ◽

Tyler M. Renner ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Post Infection ◽

Cultured Cells ◽

Hcv Infection ◽

Choline Uptake ◽

Choline Metabolism ◽

Positive Strand Rna ◽

Uptake And Metabolism

Choline is an essential nutrient required for normal neuronal and muscular development, as well as homeostatic regulation of hepatic metabolism. In the liver, choline is incorporated into the main eukaryotic phospholipid, phosphatidylcholine (PC), and can enter one-carbon metabolism via mitochondrial oxidation. Hepatitis C virus (HCV) is a hepatotropic positive-strand RNA virus that similar to other positive-strand RNA viruses and can impact phospholipid metabolism. In the current study we sought to interrogate if HCV modulates markers of choline metabolism following in vitro infection, while subsequently assessing if the inhibition of choline uptake and metabolism upon concurrent HCV infection alters viral replication and infectivity. Additionally, we assessed whether these parameters were consistent between cells cultured in fetal bovine serum (FBS) or human serum (HS), conditions known to differentially affect in vitro HCV infection. We observed that choline transport in FBS- and HS-cultured Huh7.5 cells is facilitated by the intermediate affinity transporter, choline transporter-like family (CTL). HCV infection in FBS, but not HS-cultured cells diminished CTL1 transcript and protein expression at 24 h post-infection, which was associated with lower choline uptake and lower incorporation of choline into PC. No changes in other transporters were observed and at 96 h post-infection, all differences were normalized. Reciprocally, limiting the availability of choline for PC synthesis by use of a choline uptake inhibitor resulted in increased HCV replication at this early stage (24 h post-infection) in both FBS- and HS-cultured cells. Finally, in chronic infection (96 h post-infection), inhibiting choline uptake and metabolism significantly impaired the production of infectious virions. These results suggest that in addition to a known role of choline kinase, the transport of choline, potentially via CTL1, might also represent an important and regulated process during HCV 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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Selection of Functional Variants of the NS3-NS4A Protease of Hepatitis C Virus by Using Chimeric Sindbis Viruses

Journal of Virology ◽

10.1128/jvi.73.1.561-575.1999 ◽

1999 ◽

Vol 73 (1) ◽

pp. 561-575 ◽

Cited By ~ 10

Author(s):

Gessica Filocamo ◽

Laura Pacini ◽

Chiara Nardi ◽

Linda Bartholomew ◽

Maria Scaturro ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Sindbis Virus ◽

Cultured Cells ◽

Catalytic Efficiency ◽

Temperature Sensitive ◽

Functional Variants ◽

Efficient Replication ◽

Chimeric Viruses

ABSTRACT The NS3-NS4A serine protease of hepatitis C virus (HCV) mediates four specific cleavages of the viral polyprotein and its activity is considered essential for the biogenesis of the HCV replication machinery. Despite extensive biochemical and structural characterization, the analysis of natural variants of this enzyme has been limited by the lack of an efficient replication system for HCV in cultured cells. We have recently described the generation of chimeric HCV-Sindbis viruses whose propagation depends on the NS3-NS4A catalytic activity. NS3-NS4A gene sequences were fused to the gene coding for the Sindbis virus structural polyprotein in such a way that processing of the chimeric polyprotein, nucleocapsid assembly, and production of infectious viruses required NS3-NS4A-mediated proteolysis (G. Filocamo, L. Pacini, and G. Migliaccio, J. Virol. 71:1417–1427, 1997). Here we report the use of these chimeric viruses to select and characterize active variants of the NS3-NS4A protease. Our original chimeric viruses displayed a temperature-sensitive phenotype and formed lysis plaques much smaller than those formed by wild-type (wt) Sindbis virus. By serially passaging these chimeric viruses on BHK cells, we have selected virus variants which formed lysis plaques larger than those produced by their progenitors and produced NS3-NS4A proteins different in size and/or sequence from those of the original viruses. Characterization of the selected protease variants revealed that all of the mutated proteases still efficiently processed the chimeric polyprotein in infected cells and also cleaved an HCV substrate in vitro. One of the selected proteases was expressed in a bacterial system and showed a catalytic efficiency comparable to that of the wt recombinant protease.

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