scholarly journals Cholesterol sensing by CD81 is important for hepatitis C virus entry

2019 ◽  
Author(s):  
Machaela Palor ◽  
Lenka Stejskal ◽  
Piya Mandal ◽  
Annasara Lenman ◽  
Pia Maria Alberione ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Function ◽  
Binding Pocket ◽  
Receptor Activity ◽  
Conformational Switching ◽  
Dynamic Simulations ◽  
Molecular Scaffold ◽  
Health And Disease ◽  
Cholesterol Binding

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational opening of the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol sensing mechanism; however, the relevance of this for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81-cholesterol association, but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol unbound) or closed (cholesterol bound) conformation. The open mutant of CD81 exhibited reduced receptor activity whereas the closed mutant enhanced activity. These data are consistent with CD81 cholesterol sensing resulting in a switch between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81-partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry and CD81’s function as a molecular scaffold; these insights are relevant to CD81’s varied roles in both health and disease.

scholarly journals Cholesterol sensing by CD81 is important for hepatitis C virus entry

2020 ◽  
Vol 295 (50) ◽  
pp. 16931-16948
Author(s):  
Machaela Palor ◽  
Lenka Stejskal ◽  
Piya Mandal ◽  
Annasara Lenman ◽  
María Pía Alberione ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Function ◽  
Binding Pocket ◽  
Receptor Activity ◽  
Conformational Switching ◽  
Dynamic Simulations ◽  
Molecular Scaffold ◽  
Health And Disease ◽  
Cholesterol Binding

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81–cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81–partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in both health and disease.

scholarly journals Molecular Mechanism of a Thumb Domain Hepatitis C Virus Nonnucleoside RNA-Dependent RNA Polymerase Inhibitor

2006 ◽  
Vol 50 (12) ◽  
pp. 4103-4113 ◽  
Author(s):  
Anita Y. M. Howe ◽  
Huiming Cheng ◽  
Ian Thompson ◽  
Srinivas K. Chunduru ◽  
Steve Herrmann ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
De Novo ◽  
Fold Increase ◽  
Binding Pocket ◽  
Drug Binding ◽  
Single Mutation ◽  
X Ray Crystallography ◽  
Thumb Domain ◽  
Ns5b Polymerase

ABSTRACT A new pyranoindole class of small-molecule inhibitors was studied to understand viral resistance and elucidate the mechanism of inhibition in hepatitis C virus (HCV) replication. HCV replicon variants less susceptible to inhibition by the pyranoindoles were selected in Huh-7 hepatoma cells. Variant replicons contained clusters of mutations in the NS5B polymerase gene corresponding to the drug-binding pocket on the surface of the thumb domain identified by X-ray crystallography. An additional cluster of mutations present in part of a unique β-hairpin loop was also identified. The mutations were characterized by using recombinant replicon variants engineered with the corresponding amino acid substitutions. A single mutation (L419M or M423V), located at the pyranoindole-binding site, resulted in an 8- to 10-fold more resistant replicon, while a combination mutant (T19P, M71V, A338V, M423V, A442T) showed a 17-fold increase in drug resistance. The results of a competition experiment with purified NS5B enzyme with GTP showed that the inhibitory activity of the pyranoindole inhibitor was not affected by GTP at concentrations up to 250 μM. Following de novo initiation, the presence of a pyranoindole inhibitor resulted in the accumulation of a five-nucleotide oligomer, with a concomitant decrease in higher-molecular-weight products. The results of these studies have confirmed that pyranoindoles target the NS5B polymerase through interactions at the thumb domain. This inhibition is independent of GTP concentrations and is likely mediated by an allosteric blockade introduced by the inhibitor during the transition to RNA elongation after the formation of an initiation complex.

scholarly journals Hepatitis C Virus Envelope Glycoprotein E2 Glycans Modulate Entry, CD81 Binding, and Neutralization

2007 ◽  
Vol 81 (15) ◽  
pp. 8072-8079 ◽  
Author(s):  
Emilia Falkowska ◽  
Francis Kajumo ◽  
Edie Garcia ◽  
John Reinus ◽  
Tatjana Dragic
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Entry ◽  
Protein Function ◽  
Neutralizing Antibodies ◽  
Target Cells ◽  
Virus Envelope ◽  
New Strategy ◽  
Viral Envelopes ◽  
Cd81 Binding

ABSTRACT Hepatitis C virus (HCV) is a major human pathogen that causes serious liver disease, including cirrhosis and hepatocellular carcinoma. The primary target cells of HCV are hepatocytes, and entry is restricted by interactions of the envelope glycoproteins, E1 and E2, with cellular receptors. E1 and E2 form noncovalently linked heterodimers and are heavily glycosylated. Glycans contribute to protein folding and transport as well as protein function. In addition, glycans associated with viral envelopes mask important functional domains from the immune system and attenuate viral immunogenicity. Here, we explored the role of N- and O-linked glycans on E2, which is the receptor binding subunit of the HCV envelope. We identified a number of glycans that are critical for viral entry. Importantly, we showed that the removal of several glycans significantly increased the inhibition of entry by sera from HCV-positive individuals. Only some of the glycans that affected entry and neutralization were also important for CD81 binding. Our results show that HCV envelope-associated glycans play a crucial role in masking functionally important regions of E2 and suggest a new strategy for eliciting highly neutralizing antibodies against this virus.

scholarly journals Early IFNβ secretion determines variable downstream IL-12p70 responses upon TLR4 activation in health and disease

2020 ◽  
Author(s):  
Celine Posseme ◽  
Alba Llibre ◽  
Bruno Charbit ◽  
Vincent Bondet ◽  
Vincent Rouilly ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Immune Responses ◽  
Clinical Relevance ◽  
Epigenetic Variation ◽  
Healthy Donors ◽  
Systems Immunology ◽  
Health And Disease ◽  
Pre Treatment ◽  
Tlr4 Activation

SummaryThe IL-12 family of cytokines comprises the only heterodimeric cytokines mediating diverse functional effects. We previously observed a bi-modal IL-12p70 response to LPS in healthy donors of the Milieu Interieur cohort. Herein, we demonstrate that IFNβ expression serves as an upstream determinant of variable IL-12p70 production. Integrative modelling of proteomic, genetic, epigenomic and cellular data confirmed IFNβ as key for regulation of LPS induced IL12A and IL-12p70 variability. The clinical relevance was supported by reduced and variable IL-12p70 responses in individuals infected with the hepatitis C virus (HCV), and findings that IFN-based therapy for HCV is more likely to fail in those patients with dysregulated pre-treatment IL-12p70 responses. In sum, our systems immunology approach has defined a better understanding of IL-12p70 and IFNβ in healthy and infected persons, providing insights into how common genetic and epigenetic variation may impact immune responses to bacterial infection in health and disease.

scholarly journals Identification of a Novel Sequence at the 3′ End of the GB Virus B Genome

1999 ◽  
Vol 73 (12) ◽  
pp. 10546-10550 ◽  
Author(s):  
Andrea Sbardellati ◽  
Elisa Scarselli ◽  
Licia Tomei ◽  
Alexander S. Kekulé ◽  
Cinzia Traboni
Keyword(s):  
Animal Model ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Function ◽  
Genome Organization ◽  
Tissue Tropism ◽  
Genomic Rna ◽  
B Genome ◽  
The Family ◽  
Chimeric Viruses

ABSTRACT GB virus B (GBV-B) is a virus of the familyFlaviviridae that infects small primates (Saguinus sp. [tamarins]) and shows similarities to hepatitis C virus (HCV) in genome organization, protein function, tissue tropism, and pathogenicity. This suggests the possibility of using tamarins infected by GBV-B or GBV-B/HCV chimeric viruses as a surrogate animal model of HCV infection. To achieve the construction of such chimeric viruses, it is essential to produce a complete and infectious GBV-B genomic RNA. We have identified a novel sequence at the 3′ end of the GBV-B genome and show that it can be arranged in a secondary structure resembling that of the 3′ end of the HCV genome, which is known to be essential for infectivity.

scholarly journals De Novo Initiation Pocket Mutations Have Multiple Effects on Hepatitis C Virus RNA-Dependent RNA Polymerase Activities

2004 ◽  
Vol 78 (22) ◽  
pp. 12207-12217 ◽  
Author(s):  
C. T. Ranjith-Kumar ◽  
R. T. Sarisky ◽  
L. Gutshall ◽  
M. Thomson ◽  
C. C. Kao
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
De Novo ◽  
Specific Interaction ◽  
Binding Pocket ◽  
Hcv Rna ◽  
Elongation Complex ◽  
Rna Dependent Rna Polymerase ◽  
Template Switch

ABSTRACT The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has several distinct biochemical activities, including initiation of RNA synthesis by a de novo mechanism, extension from a primed template, nontemplated nucleotide addition, and synthesis of a recombinant RNA product from two or more noncovalently linked templates (template switch). All of these activities require specific interaction with nucleoside triphosphates (NTPs). Based on the structure of the HCV RdRp bound to NTP (S. Bressanelli, L. Tomei, F. A. Rey, and R. DeFrancesco, J. Virol. 76:3482-3492, 2002), we mutated the amino acid residues that contact the putative initiation GTP and examined the effects on the various activities. Although all mutations retained the ability for primer extension, alanine substitution at R48, R158, R386, R394, or D225 decreased de novo initiation, and two or more mutations abolished de novo initiation. While the prototype enzyme had a Km for GTP of 3.5 μM, all of the mutations except one had Km s that were three- to sevenfold higher. These results demonstrate that the affected residues are functionally required to interact with the initiation nucleotide. Unexpectedly, many of the mutations also affected the addition of nontemplated nucleotide, indicating that residues in the initiating NTP (NTPi)-binding pocket are required for nontemplated nucleotide additions. Interestingly, mutations in D225 are dramatically affected in template switch, indicating that this residue of the NTPi pocket also interacts with components in the elongation complex. We also examined the interaction of ribavirin triphosphate with the NTPi-binding site.

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

2001 ◽  
Vol 82 (9) ◽  
pp. 2243-2250 ◽  
Author(s):  
A. R. Ciccaglione ◽  
A. Costantino ◽  
C. Marcantonio ◽  
M. Equestre ◽  
A. Geraci ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Membrane Permeability ◽  
Protein Function ◽  
Critical Role ◽  
Cell Lysis ◽  
Hydrophobic Region ◽  
E Coli ◽  
Conserved Amino Acids

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.

scholarly journals Contribution of Redox Status to Hepatitis C Virus E2 Envelope Protein Function and Antigenicity

2008 ◽  
Vol 283 (39) ◽  
pp. 26340-26348 ◽  
Author(s):  
Emmanuel Fenouillet ◽  
Dimitri Lavillette ◽  
Silvia Loureiro ◽  
George Krashias ◽  
Guillemette Maurin ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Envelope Protein ◽  
Protein Function ◽  
Redox Status

scholarly journals Regulatory Role of Phospholipids in Hepatitis C Virus Replication and Protein Function

Pathogens ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 102
Author(s):  
Anna V. Bulankina ◽  
Rebecca M. Richter ◽  
Christoph Welsch
Keyword(s):  
Hepatitis C Virus ◽  
Lipid Metabolism ◽  
Hepatitis C ◽  
Structure Function ◽  
Protein Function ◽  
Intracellular Membranes ◽  
Infected Cells ◽  
Function Relationship ◽  
Phosphatidylinositol 4 ◽  
Relationship Of

Positive-strand RNA viruses such as hepatitis C virus (HCV) hijack key factors of lipid metabolism of infected cells and extensively modify intracellular membranes to support the viral lifecycle. While lipid metabolism plays key roles in viral particle assembly and maturation, viral RNA synthesis is closely linked to the remodeling of intracellular membranes. The formation of viral replication factories requires a number of interactions between virus proteins and host factors including lipids. The structure–function relationship of those proteins is influenced by their lipid environments and lipids that selectively modulate protein function. Here, we review our current understanding on the roles of phospholipids in HCV replication and of lipid–protein interactions in the structure–function relationship of the NS5A protein. NS5A is a key factor in membrane remodeling in HCV-infected cells and is known to recruit phosphatidylinositol 4-kinase III alpha to generate phosphatidylinositol 4-phosphate at the sites of replication. The dynamic interplay between lipids and viral proteins within intracellular membranes is likely key towards understanding basic mechanisms in the pathobiology of virus diseases, the mode of action of specific antiviral agents and related drug resistance mechanisms.

scholarly journals Polymorphisms in melanoma differentiation-associated gene 5 link protein function to clearance of hepatitis C virus

Hepatology ◽  
2015 ◽  
Vol 61 (2) ◽  
pp. 460-470 ◽  
Author(s):  
Franziska S. Hoffmann ◽  
Andreas Schmidt ◽  
Meike Dittmann Chevillotte ◽  
Christian Wisskirchen ◽  
Johannes Hellmuth ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Function ◽  
Link Protein
Sign in / Sign up

Export Citation Format

Share Document