scholarly journals Cochaperone Activity of Human Butyrate-Induced Transcript 1 Facilitates Hepatitis C Virus Replication through an Hsp90-Dependent Pathway

2009 ◽  
Vol 83 (20) ◽  
pp. 10427-10436 ◽  
Author(s):  
Shuhei Taguwa ◽  
Hiroto Kambara ◽  
Hiroko Omori ◽  
Hideki Tani ◽  
Takayuki Abe ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Heat Shock Protein 90 ◽  
Dependent Manner ◽  
Replication Complex ◽  
Fk506 Binding Protein ◽  
Unfolded Protein ◽  
Hsp90 Chaperone ◽  
Protein Response

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a component of the replication complex consisting of several host and viral proteins. We have previously reported that human butyrate-induced transcript 1 (hB-ind1) recruits heat shock protein 90 (Hsp90) and FK506-binding protein 8 (FKBP8) to the replication complex through interaction with NS5A. To gain more insights into the biological functions of hB-ind1 in HCV replication, we assessed the potential cochaperone-like activity of hB-ind1, because it has significant homology with cochaperone p23, which regulates Hsp90 chaperone activity. The chimeric p23 in which the cochaperone domain was replaced with the p23-like domain of hB-ind1 exhibited cochaperone activity comparable to that of the authentic p23, inhibiting the glucocorticoid receptor signaling in an Hsp90-dependent manner. Conversely, the chimeric hB-ind1 in which the p23-like domain was replaced with the cochaperone domain of p23 resulted in the same level of recovery of HCV propagation as seen in the authentic hB-ind1 in cells with knockdown of the endogenous hB-ind1. Immunofluorescence analyses revealed that hB-ind1 was colocalized with NS5A, FKBP8, and double-stranded RNA in the HCV replicon cells. HCV replicon cells exhibited a more potent unfolded-protein response (UPR) than the parental and the cured cells upon treatment with an inhibitor for Hsp90. These results suggest that an Hsp90-dependent chaperone pathway incorporating hB-ind1 is involved in protein folding in the membranous web for the circumvention of the UPR and that it facilitates HCV replication.

scholarly journals Characterization of Soluble Hepatitis C Virus RNA-Dependent RNA Polymerase Expressed in Escherichia coli

1999 ◽  
Vol 73 (2) ◽  
pp. 1649-1654 ◽  
Author(s):  
Eric Ferrari ◽  
Jacquelyn Wright-Minogue ◽  
Jane W. S. Fang ◽  
Bahige M. Baroudy ◽  
Johnson Y. N. Lau ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Nonstructural Protein ◽  
Full Length ◽  
Dependent Manner ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Hydrophobic Domain ◽  
Hcv Ns5b

ABSTRACT Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BΔCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.

scholarly journals Identification of a Novel Determinant for Membrane Association in Hepatitis C Virus Nonstructural Protein 4B

2009 ◽  
Vol 83 (12) ◽  
pp. 6257-6268 ◽  
Author(s):  
Jérôme Gouttenoire ◽  
Valérie Castet ◽  
Roland Montserret ◽  
Naveen Arora ◽  
Vincent Raussens ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Attenuated Total Reflection ◽  
Membrane Extraction ◽  
Phospholipid Bilayer ◽  
Membrane Association ◽  
Acceptor Site ◽  
Replication Complex ◽  
Α Helix

ABSTRACT Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic α-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic α-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B.

scholarly journals Efficient Rescue of Hepatitis C Virus RNA Replication by trans-Complementation with Nonstructural Protein 5A

2005 ◽  
Vol 79 (2) ◽  
pp. 896-909 ◽  
Author(s):  
Nicole Appel ◽  
Ulrike Herian ◽  
Ralf Bartenschlager
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Functional Organization ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Hcv Rna ◽  
Replication Complex ◽  
Diarrhea Virus ◽  
Amino Terminal ◽  
Essential Components

ABSTRACT Studies of Hepatitis C virus (HCV) RNA replication have become possible with the development of subgenomic replicons. This system allows the functional analysis of the essential components of the viral replication complex, which so far are poorly defined. In the present study we wanted to investigate whether lethal mutations in HCV nonstructural genes can be rescued by trans-complementation. Therefore, a series of replicon RNAs carrying mutations in NS3, NS4B, NS5A, and NS5B that abolish replication were transfected into Huh-7 hepatoma cells harboring autonomously replicating helper RNAs. Similar to data described for the Bovine viral diarrhea virus (C. W. Grassmann, O. Isken, N. Tautz, and S. E. Behrens, J. Virol. 75:7791-7802, 2001), we found that only NS5A mutants could be efficiently rescued. There was no evidence for RNA recombination between helper and mutant RNAs, and we did not observe reversions in the transfected mutants. Furthermore, we established a transient complementation assay based on the cotransfection of helper and mutant RNAs. Using this assay, we extended our results and demonstrated that (i) inactivating NS5A mutations affecting the amino-terminal amphipathic helix cannot be complemented in trans; (ii) replication of the helper RNA is not necessary to allow efficient trans-complementation; and (iii) the minimal sequence required for trans-complementation of lethal NS5A mutations is NS3 to -5A, whereas NS5A expressed alone does not restore RNA replication. In summary, our results provide the first insight into the functional organization of the HCV replication complex.

scholarly journals GOLT1B Activation in Hepatitis C Virus-Infected Hepatocytes Links ER Trafficking and Viral Replication

Pathogens ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 46
Author(s):  
Jacqueline Butterworth ◽  
Damien Gregoire ◽  
Marion Peter ◽  
Armando Andres Roca Suarez ◽  
Guillaume Desandré ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Single Molecule ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Unfolded Protein ◽  
Expression Of Genes ◽  
The Unfolded Protein Response ◽  
Protein Response

Chronic hepatitis C carries a high risk of development of hepatocellular carcinoma (HCC), triggered by both direct and indirect effects of the virus. We examined cell-autonomous alterations in gene expression profiles associated with hepatitis C viral presence. Highly sensitive single molecule fluorescent in situ hybridization applied to frozen tissue sections of a hepatitis C patient allowed the delineation of clusters of infected hepatocytes. Laser microdissection followed by RNAseq analysis of hepatitis C virus (HCV)-positive and -negative regions from the tumoral and non-tumoral tissues from the same patient revealed HCV-related deregulation of expression of genes in the tumor and in the non-tumoral tissue. However, there was little overlap between both gene sets. Our interest in alterations that increase the probability of tumorigenesis prompted the examination of genes whose expression was increased by the virus in the non-transformed cells and whose level remained high in the tumor. This strategy led to the identification of a novel HCV target gene: GOLT1B, which encodes a protein involved in ER-Golgi trafficking. We further show that GOLT1B expression is induced during the unfolded protein response, that its presence is essential for efficient viral replication, and that its expression is correlated with poor outcome in HCC.

scholarly journals Impact of a baseline polymorphism on the emergence of resistance to the hepatitis C virus nonstructural protein 5a replication complex inhibitor, BMS-790052

Hepatology ◽  
2012 ◽  
Vol 55 (6) ◽  
pp. 1692-1699 ◽  
Author(s):  
Jin-Hua Sun ◽  
Donald R. O'Boyle II ◽  
Yan Zhang ◽  
Chunfu Wang ◽  
Peter Nower ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Replication Complex ◽  
Emergence Of Resistance ◽  
Nonstructural Protein 5A

scholarly journals Hepatitis C Virus RNA Elimination and Development of Resistance in Replicon Cells Treated with BMS-790052

2012 ◽  
Vol 56 (3) ◽  
pp. 1350-1358 ◽  
Author(s):  
Chunfu Wang ◽  
Haichang Huang ◽  
Lourdes Valera ◽  
Jin-Hua Sun ◽  
Donald R. O'Boyle ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Nonstructural Protein ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Dependent Manner ◽  
Resistance Mutations ◽  
Genotype 1A ◽  
Clinical Resistance

ABSTRACTBMS-790052, a first-in-class hepatitis C virus (HCV) replication complex inhibitor, targeting nonstructural protein 5A (NS5A), displays picomolar to nanomolar potency against genotypes 1 to 5. This exceptional potency translated into robust anti-HCV activity in clinical studies with HCV genotype 1-infected subjects. To date, all BMS-790052-associated resistance mutations have mapped to the N-terminal region of NS5A. To further characterize the antiviral activity of BMS-790052, HCV replicon elimination and colony formation assays were performed. Replicon was cleared from genotype 1a and 1b replicon cells in a time- and dose-dependent manner. Elimination of the genotype 1a replicon required longer treatment durations and higher concentrations of BMS-790052 than those for the genotype1b replicon. Single amino acid substitutions that conferred relatively low levels of resistance were observed at early time points and at low doses. Higher doses and longer treatment durations yielded mutations that conferred greater levels of resistance, including linked amino acid substitutions. Replicon cells that survived inhibitor treatment remained fully sensitivity to pegylated alpha interferon (pegIFN-α) and other HCV inhibitors. Moreover, genotype 1a replicon elimination was markedly enhanced when pegIFN-α and BMS-790052 were combined. Resistant variants observed in this study were very similar to those observed in a multiple ascending dose (MAD) monotherapy trial of BMS-790052, validating replicon elimination studies as a model to predict clinical resistance. Insights gained from thein vitroanti-HCV activity and resistance profiles of BMS-790052 will be used to help guide the clinical development of this novel HCV inhibitor.

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