title: Hepatitis C virus NS4B carboxy terminal domain is a membrane binding domain.
abstract: BACKGROUND
  Hepatitis C virus (HCV) induces membrane rearrangements during replication. All HCV proteins are associated to membranes, pointing out the importance of membranes for HCV. Non structural protein 4B (NS4B) has been reported to induce cellular membrane alterations like the membranous web. Four transmembrane segments in the middle of the protein anchor NS4B to membranes. An amphipatic helix at the amino-terminus attaches to membranes as well. The carboxy-terminal domain (CTD) of NS4B is highly conserved in Hepaciviruses, though its function remains unknown.
  
  
  RESULTS
  A cytosolic localization is predicted for the NS4B-CTD. However, using membrane floatation assays and immunofluorescence, we now show targeting of the NS4B-CTD to membranes. Furthermore, a profile-profile search, with an HCV NS4B-CTD multiple sequence alignment, indicates sequence similarity to the membrane binding domain of prokaryotic D-lactate dehydrogenase (d-LDH). The crystal structure of E. coli d-LDH suggests that the region similar to NS4B-CTD is located in the membrane binding domain (MBD) of d-LDH, implying analogy in membrane association. Targeting of d-LDH to membranes occurs via electrostatic interactions of positive residues on the outside of the protein with negative head groups of lipids. To verify that anchorage of d-LDH MBD and NS4B-CTD is analogous, NS4B-CTD mutants were designed to disrupt these electrostatic interactions. Membrane association was confirmed by swopping the membrane contacting helix of d-LDH with the corresponding domain of the 4B-CTD. Furthermore, the functionality of these residues was tested in the HCV replicon system.
  
  
  CONCLUSIONS
  Together these data show that NS4B-CTD is associated to membranes, similar to the prokaryotic d-LDH MBD, and is important for replication.
authors.affiliation: Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, 2300 RC Leiden, The Netherlands. J.M.P.Liefhebber@lumc.nl
authors.forename: Jolanda M P
authors.initials: JM
authors.lastname: Liefhebber
authors.forename: Bernd W
authors.initials: BW
authors.lastname: Brandt
authors.forename: Rene
authors.initials: R
authors.lastname: Broer
authors.forename: Willy J M
authors.initials: WJ
authors.lastname: Spaan
authors.forename: Hans C
authors.initials: HC
authors.lastname: van Leeuwen
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000821:virology*; D029968:Escherichia coli Proteins / Q000737:chemistry; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C080107:NS4B protein, flavivirus; D017361:Viral Nonstructural Proteins