PMID- 25392992
OWN - NLM
STAT- MEDLINE
DCOM- 20160426
LR  - 20181113
IS  - 1553-7374 (Electronic)
IS  - 1553-7366 (Print)
IS  - 1553-7366 (Linking)
VI  - 10
IP  - 10
DP  - 2014 Oct
TI  - Aminoterminal amphipathic alpha-helix AH1 of hepatitis C virus nonstructural protein 
      4B possesses a dual role in RNA replication and virus production.
PG  - e1004501
LID - 10.1371/journal.ppat.1004501 [doi]
LID - e1004501
AB  - 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 alpha-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 alpha-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 
      alpha-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.
FAU - Gouttenoire, Jerome
AU  - Gouttenoire J
AD  - Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire 
      Vaudois, University of Lausanne, Lausanne, Switzerland.
FAU - Montserret, Roland
AU  - Montserret R
AD  - Institut de Biologie et Chimie des Proteines, Bases Moleculaires et Structurales 
      des Systemes Infectieux, UMR 5086, CNRS, Labex Ecofect, University of Lyon, Lyon, 
      France.
FAU - Paul, David
AU  - Paul D
AD  - Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 
      Heidelberg, Germany.
FAU - Castillo, Rosa
AU  - Castillo R
AD  - Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire 
      Vaudois, University of Lausanne, Lausanne, Switzerland.
FAU - Meister, Simon
AU  - Meister S
AD  - Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire 
      Vaudois, University of Lausanne, Lausanne, Switzerland.
FAU - Bartenschlager, Ralf
AU  - Bartenschlager R
AD  - Department of Infectious Diseases, Molecular Virology, University of Heidelberg, 
      Heidelberg, Germany.
FAU - Penin, Francois
AU  - Penin F
AD  - Institut de Biologie et Chimie des Proteines, Bases Moleculaires et Structurales 
      des Systemes Infectieux, UMR 5086, CNRS, Labex Ecofect, University of Lyon, Lyon, 
      France.
FAU - Moradpour, Darius
AU  - Moradpour D
AD  - Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire 
      Vaudois, University of Lausanne, Lausanne, Switzerland.
LA  - eng
SI  - PDB/2LVG
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20141113
PL  - United States
TA  - PLoS Pathog
JT  - PLoS pathogens
JID - 101238921
RN  - 0 (NS4B protein, flavivirus)
RN  - 0 (RNA, Viral)
RN  - 0 (Viral Nonstructural Proteins)
SB  - IM
MH  - Amino Acid Sequence
MH  - Cell Line, Tumor
MH  - Cell Membrane/metabolism
MH  - Hepacivirus/chemistry/genetics/*metabolism/ultrastructure
MH  - Hepatitis C/*virology
MH  - Humans
MH  - Models, Molecular
MH  - Models, Structural
MH  - Molecular Sequence Data
MH  - Mutation
MH  - Protein Structure, Secondary
MH  - Protein Structure, Tertiary
MH  - RNA, Viral/genetics
MH  - Replicon
MH  - Sequence Alignment
MH  - Viral Nonstructural Proteins/chemistry/genetics/*metabolism
MH  - Virus Replication
PMC - PMC4231108
COIS- The authors have declared that no competing interests exist.
EDAT- 2014/11/14 06:00
MHDA- 2016/04/27 06:00
PMCR- 2014/11/13
CRDT- 2014/11/14 06:00
PHST- 2014/08/14 00:00 [received]
PHST- 2014/10/02 00:00 [accepted]
PHST- 2014/11/14 06:00 [entrez]
PHST- 2014/11/14 06:00 [pubmed]
PHST- 2016/04/27 06:00 [medline]
PHST- 2014/11/13 00:00 [pmc-release]
AID - PPATHOGENS-D-14-01967 [pii]
AID - 10.1371/journal.ppat.1004501 [doi]
PST - epublish
SO  - PLoS Pathog. 2014 Nov 13;10(10):e1004501. doi: 10.1371/journal.ppat.1004501. 
      eCollection 2014 Oct.