Mutagenesis of hepatitis C virus E1 protein affects its membrane-permeabilizing activity

The E1 glycoprotein of hepatitis C virus is a transmembrane glycoprotein with a C-terminal anchor domain. When expressed in Escherichia coli, E1 induces a change in membrane permeability that is toxic to the bacterial cell. The C-terminal hydrophobic region (aa 331–383) of E1 is mainly responsible for membrane association and for inducing changes in membrane permeability. These observed changes are similar to those produced in E. coli by influenza virus M2, human immunodeficiency virus gp41 and poliovirus 3AB proteins, whose hydrophobic domains are thought to cause pore formation in biological membranes. To further characterize the activity of E1 at a molecular level, the membrane-permeabilizing ability of a second internal hydrophobic region (aa 262–291) was examined by expressing different deletion mutants of E1 in an E. coli system that is widely used for analysing membrane-active proteins from other animal viruses. Moreover, highly conserved amino acids in the C-terminal hydrophobic region were mutated to identify residues that are critical for inducing changes in membrane permeability. Analysis of cell growth curves of recombinant cultures and membrane-permeability assays revealed that synthesis of this fragment increased the flux of small compounds through the membrane and caused progressive cell lysis, suggesting that this domain has membrane-active properties. Furthermore, analysis of C-terminal mutants indicated that the conserved amino acids Arg339, Trp368 and Lys370 play a critical role in protein function, as both cell lysis and changes in membrane permeability induced by the wild-type clone could be blocked by substitutions in these positions.

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Faculty Opinions recommendation of Critical role of cyclophilin A and its prolyl-peptidyl isomerase activity in the structure and function of the hepatitis C virus replication complex.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1162955.623591 ◽

2009 ◽

Author(s):

Teresa Compton

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Replication ◽

Critical Role ◽

Cyclophilin A ◽

Structure And Function ◽

Replication Complex ◽

Isomerase Activity ◽

And Function

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Branched‐chain amino acids reduce hepatic iron accumulation and oxidative stress in hepatitis C virus polyprotein‐expressing mice

Liver International ◽

10.1111/liv.12675 ◽

2014 ◽

Vol 35 (4) ◽

pp. 1303-1314 ◽

Cited By ~ 16

Author(s):

Masaaki Korenaga ◽

Sohji Nishina ◽

Keiko Korenaga ◽

Yasuyuki Tomiyama ◽

Naoko Yoshioka ◽

...

Keyword(s):

Oxidative Stress ◽

Amino Acids ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Branched Chain Amino Acids ◽

Iron Accumulation ◽

Hepatic Iron ◽

Branched Chain ◽

Virus Polyprotein ◽

And Oxidative Stress

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Binding Dynamics of Hepatitis C Virus' NS5A Amphipathic Peptide to Cell and Model Membranes

Journal of Virology ◽

10.1128/jvi.02783-06 ◽

2007 ◽

Vol 81 (12) ◽

pp. 6682-6689 ◽

Cited By ~ 26

Author(s):

Nam-Joon Cho ◽

Kwang Ho Cheong ◽

ChoongHo Lee ◽

Curtis W. Frank ◽

Jeffrey S. Glenn

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Critical Role ◽

Membrane Binding ◽

Lipid Binding ◽

Model Membranes ◽

Cellular Membranes ◽

Kinetic Measurements ◽

Amphipathic Peptide ◽

Target Membrane

ABSTRACT Membrane association of the hepatitis C virus NS5A protein is required for viral replication. This association is dependent on an N-terminal amphipathic helix (AH) within NS5A and is restricted to a subset of host cell intracellular membranes. The mechanism underlying this specificity is not known, but it may suggest a novel strategy for developing specific antiviral therapy. Here we have probed the mechanistic details of NS5A AH-mediated binding to both cell-derived and model membranes by use of biochemical membrane flotation and quartz crystal microbalance (QCM) with dissipation. With both assays, we observed AH-mediated binding to model lipid bilayers. When cell-derived membranes were coated on the quartz nanosensor, however, significantly more binding was detected, and the QCM-derived kinetic measurements suggested the existence of an interacting receptor in the target membranes. Biochemical flotation assays performed with trypsin-treated cell-derived membranes exhibited reduced AH-mediated membrane binding, while membrane binding of control cytochrome b5 remained unaffected. Similarly, trypsin treatment of the nanosensor coated with cellular membranes abolished AH peptide binding to the cellular membranes but did not affect the binding of a control lipid-binding peptide. These results therefore suggest that a protein plays a critical role in mediating and stabilizing the binding of NS5A's AH to its target membrane. These results also demonstrate the successful development of a new nanosensor technology ideal both for studying the interaction between a protein and its target membrane and for developing inhibitors of that interaction.

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The N-terminal amino acids are essential for the antigenic structure of the hepatitis C virus core protein

Hepatology ◽

10.1016/0270-9139(93)91843-h ◽

1993 ◽

Vol 18 (4) ◽

pp. A79

Author(s):

T GOESER

Keyword(s):

Amino Acids ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Core Protein ◽

Antigenic Structure ◽

Virus Core ◽

Terminal Amino

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Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

Journal of Virology ◽

10.1128/jvi.75.13.6095-6106.2001 ◽

2001 ◽

Vol 75 (13) ◽

pp. 6095-6106 ◽

Cited By ~ 212

Author(s):

Stephen J. Polyak ◽

Khalid S. A. Khabar ◽

Denise M. Paschal ◽

Heather J. Ezelle ◽

Gilles Duverlie ◽

...

Keyword(s):

Amino Acids ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Antiviral Response ◽

Full Length ◽

Interleukin 8 ◽

Antiviral Resistance ◽

Ns5a Protein ◽

Terminal Amino

ABSTRACT Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-ΔN110), 222 (NS5A-ΔN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-ΔN110 and NS5A-ΔN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-κB and AP-1 were important in NS5A-ΔN222 transactivation in the presence of tumor necrosis factor alpha and that NF–IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis.

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Mutational analysis of conserved amino acids in the T cell receptor α-chain transmembrane region: a critical role of leucine 112 and phenylalanine 127 for assembly and surface expression

Molecular Immunology ◽

10.1016/s0161-5890(03)00027-0 ◽

2003 ◽

Vol 39 (15) ◽

pp. 953-963 ◽

Cited By ~ 5

Author(s):

Aparna Bhatnagar ◽

Sven Gülland ◽

Micaela Bascand ◽

Ed Palmer ◽

Terrence G. Gardner ◽

...

Keyword(s):

Amino Acids ◽

T Cell ◽

T Cell Receptor ◽

Mutational Analysis ◽

Critical Role ◽

Cell Receptor ◽

Surface Expression ◽

Transmembrane Region ◽

Conserved Amino Acids

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CRITICAL ROLE OF VAL/GLY86 HLA-DRB DIMORPHISM IN THE NEONATAL RESISTANCE OR SUSCEPTIBILITY TO MATERNAL HEPATITIS C VIRUS INFECTION

The Pediatric Infectious Disease Journal ◽

10.1097/00006454-199710000-00019 ◽

1997 ◽

Vol 16 (10) ◽

pp. 1001-1002 ◽

Cited By ~ 3

Author(s):

Miryam Martinetti ◽

Ilaria Pacati ◽

Cristina Daielli ◽

Laura Salvaneschi ◽

Anna Maccabruni

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

Critical Role ◽

Hepatitis C Virus Infection

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Interactions between Viral Nonstructural Proteins and Host Protein hVAP-33 Mediate the Formation of Hepatitis C Virus RNA Replication Complex on Lipid Raft

Journal of Virology ◽

10.1128/jvi.78.7.3480-3488.2004 ◽

2004 ◽

Vol 78 (7) ◽

pp. 3480-3488 ◽

Cited By ~ 247

Author(s):

Lu Gao ◽

Hideki Aizaki ◽

Jian-Wen He ◽

Michael M. C. Lai

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Lipid Raft ◽

Critical Role ◽

Rna Replication ◽

Dominant Negative ◽

Host Protein ◽

Hcv Rna ◽

Replication Complex ◽

Detergent Resistant Membranes

ABSTRACT The lipid raft membrane has been shown to be the site of hepatitis C virus (HCV) RNA replication. The mechanism of formation of the replication complex is not clear. We show here that the formation of the HCV RNA replication complex on lipid raft (detergent-resistant membranes) requires interactions among the HCV nonstructural (NS) proteins and may be initiated by the precursor of NS4B, which has the intrinsic property of anchoring to lipid raft membrane. In hepatocyte cell lines containing an HCV RNA replicon, most of the other NS proteins, including NS5A, NS5B, and NS3, were also localized to the detergent-resistant membranes. However, when individually expressed, only NS4B was associated exclusively with lipid raft. In contrast, NS5B and NS3 were localized to detergent-sensitive membrane and cytosolic fractions, respectively. NS5A was localized to both detergent-sensitive and -resistant membrane fractions. Furthermore, we show that a cellular vesicle membrane transport protein named hVAP-33 (the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein), which binds to both NS5A and NS5B, plays a critical role in the formation of HCV replication complex. The hVAP-33 protein is partially associated with the detergent-resistant membrane fraction. The expression of dominant-negative mutants and small interfering RNA of hVAP-33 in HCV replicon cells resulted in the relocation of NS5B from detergent-resistant to detergent-sensitive membranes. Correspondingly, the amounts of both HCV RNA and proteins in the cells were reduced, indicating that hVAP-33 is critical for the formation of HCV replication complex and RNA replication. These results indicate that protein-protein interactions among the various HCV NS proteins and hVAP-33 are important for the formation of HCV replication complex.

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