Aminoterminal amphipathic α-helix AH1 of hepatitis C virus nonstructural protein 4B possesses a dual role in RNA replication and virus production.

Détails

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Etat: Public
Version: de l'auteur
ID Serval
serval:BIB_E6C14F253970
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Aminoterminal amphipathic α-helix AH1 of hepatitis C virus nonstructural protein 4B possesses a dual role in RNA replication and virus production.
Périodique
Plos Pathogens
Auteur(s)
Gouttenoire J., Montserret R., Paul D., Castillo R., Meister S., Bartenschlager R., Penin F., Moradpour D.
ISSN
1553-7374 (Electronic)
ISSN-L
1553-7366
Statut éditorial
Publié
Date de publication
2014
Peer-reviewed
Oui
Volume
10
Numéro
11
Pages
e1004501
Langue
anglais
Résumé
Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. In concert with other nonstructural proteins, it induces a specific membrane rearrangement, designated as membranous web, which serves as a scaffold for the HCV replicase. The N-terminal part of NS4B comprises a predicted and a structurally resolved amphipathic α-helix, designated as AH1 and AH2, respectively. Here, we report a detailed structure-function analysis of NS4B AH1. Circular dichroism and nuclear magnetic resonance structural analyses revealed that AH1 folds into an amphipathic α-helix extending from NS4B amino acid 4 to 32, with positively charged residues flanking the helix. These residues are conserved among hepaciviruses. Mutagenesis and selection of pseudorevertants revealed an important role of these residues in RNA replication by affecting the biogenesis of double-membrane vesicles making up the membranous web. Moreover, alanine substitution of conserved acidic residues on the hydrophilic side of the helix reduced infectivity without significantly affecting RNA replication, indicating that AH1 is also involved in virus production. Selective membrane permeabilization and immunofluorescence microscopy analyses of a functional replicon harboring an epitope tag between NS4B AH1 and AH2 revealed a dual membrane topology of the N-terminal part of NS4B during HCV RNA replication. Luminal translocation was unaffected by the mutations introduced into AH1, but was abrogated by mutations introduced into AH2. In conclusion, our study reports the three-dimensional structure of AH1 from HCV NS4B, and highlights the importance of positively charged amino acid residues flanking this amphipathic α-helix in membranous web formation and RNA replication. In addition, we demonstrate that AH1 possesses a dual role in RNA replication and virus production, potentially governed by different topologies of the N-terminal part of NS4B.
Mots-clé
Amino Acid Sequence, Cell Line, Tumor, Cell Membrane/metabolism, Hepacivirus/chemistry, Hepacivirus/genetics, Hepatitis C/virology, Humans, Models, Molecular, Models, Structural, Molecular Sequence Data, Mutation, Protein Structure, Secondary, Protein Structure, Tertiary, RNA, Viral/genetics, Replicon, Sequence Alignment, Viral Nonstructural Proteins/chemistry, Viral Nonstructural Proteins/genetics, Virus Replication
Pubmed
Web of science
Open Access
Oui
Création de la notice
27/12/2014 10:30
Dernière modification de la notice
20/08/2019 17:09
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