2LVG

NMR structure of HCV Non-structural protein AB, NS4B(1-40)

Summary for 2LVG

• Entry DOI: 10.2210/pdb2lvg/pdb
• Related: 2jxf
• NMR Information: BMRB: 18568
• Descriptor: Non-structural protein 4B (1 entity in total)
• Functional Keywords: membrane protein
• Biological source: Hepatitis C virus JFH-1 (HCV)
• Cellular location: Core protein p21: Host endoplasmic reticulum membrane ; Single-pass membrane protein . Core protein p19: Virion . Envelope glycoprotein E1: Virion membrane ; Single-pass type I membrane protein . Envelope glycoprotein E2: Virion membrane ; Single-pass type I membrane protein . p7: Host endoplasmic reticulum membrane ; Multi-pass membrane protein . Protease NS2-3: Host endoplasmic reticulum membrane ; Multi-pass membrane protein . Serine protease NS3: Host endoplasmic reticulum membrane ; Peripheral membrane protein . Non-structural protein 4A: Host endoplasmic reticulum membrane ; Single-pass type I membrane protein . Non-structural protein 4B: Host endoplasmic reticulum membrane ; Multi-pass membrane protein . Non-structural protein 5A: Host endoplasmic reticulum membrane ; Peripheral membrane protein . RNA-directed RNA polymerase: Host endoplasmic reticulum membrane ; Single-pass type I membrane protein : Q99IB8
• Total number of polymer chains: 1
• Total formula weight: 4385.05

Authors

Montserret, R.,Penin, F. (deposition date: 2012-07-05, release date: 2013-06-19, Last modification date: 2024-05-15)

Primary citation

Gouttenoire, J.,Montserret, R.,Paul, D.,Castillo, R.,Meister, S.,Bartenschlager, R.,Penin, F.,Moradpour, D.
Aminoterminal Amphipathic alpha-Helix AH1 of Hepatitis C Virus Nonstructural Protein 4B Possesses a Dual Role in RNA Replication and Virus Production.
Plos Pathog., 10:e1004501-e1004501, 2014

PubMed 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.

PubMed: 25392992
DOI: 10.1371/journal.ppat.1004501

Experimental method

SOLUTION NMR