Abstract
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.
Highlights
Hepatitis C virus (HCV) infection is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide, with a peak of the disease burden expected in around 10 years [1]
Nonstructural protein 4B (NS4B) AH1 contributes to proper double membrane vesicles (DMVs) formation To gain deeper insight into the consequences of removal of the positively charged residues flanking AH1, we investigated the ultrastructure of membrane rearrangements induced by the different NS4B mutants by electron microscopy (EM)
In a series of preliminary experiments, we found that incubation of fixed cells with 0.05% digitonin for 15 min at 4uC allowed for selective permeabilization of the plasma membrane but not the endoplasmic reticulum (ER) membrane of Huh7-Lunet cells which are highly permissive for hepatitis C virus (HCV) replication [25]
Summary
Hepatitis C virus (HCV) infection is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide, with a peak of the disease burden expected in around 10 years [1]. HCV and GB virus B have been classified in the Hepacivirus genus within the Flaviviridae family, which includes the genera Flavivirus, Pestivirus and Pegivirus [2]. Additional closely related viruses have been identified recently in horses as well as other animal species, and have been classified in the Hepacivirus and Pegivirus genera, including nonprimate hepaciviruses (NPHV) [3,4]. HCV contains a 9.6-kb positive-strand RNA genome encoding a polyprotein precursor that is co- and posttranslationally processed into ten structural and nonstructural proteins [2,5]. As all positive-strand RNA viruses, HCV replicates its genome in a membrane-associated replication complex composed of viral proteins, replicating RNA, rearranged intracellular membranes and additional host factors [6,7,8,9]. Nonstructural proteins 3 through 5B are essential for HCV RNA replication, and their functional complex is referred to as replicase
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