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Hepatitis C virus NS4B carboxy terminal domain is a membrane binding domain.

Liefhebber JM, Brandt BW, Broer R, Spaan WJ, van Leeuwen HC - Virol. J. (2009)

Bottom Line: All HCV proteins are associated to membranes, pointing out the importance of membranes for HCV.Membrane association was confirmed by swopping the membrane contacting helix of d-LDH with the corresponding domain of the 4B-CTD.Together these data show that NS4B-CTD is associated to membranes, similar to the prokaryotic d-LDH MBD, and is important for replication.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, 2300 RC Leiden, The Netherlands. J.M.P.Liefhebber@lumc.nl

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.

Conclusion: Together these data show that NS4B-CTD is associated to membranes, similar to the prokaryotic d-LDH MBD, and is important for replication.

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Effect of NS4B carboxy terminal mutations on colony formation using selectable replicons. Colony formation assay in which Huh7 cells are transfected with in vitro transcribed replicon RNA that contain NS4B-CTD mutations. Colonies were stained using Coomassie blue. Wild-type is pFK5.1. Mock transfected cells as the control. The NS4B-CTD mutations TripleE, E247, E248 and helix-swop in pFK5.1 are explained in figure 4.
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Figure 5: Effect of NS4B carboxy terminal mutations on colony formation using selectable replicons. Colony formation assay in which Huh7 cells are transfected with in vitro transcribed replicon RNA that contain NS4B-CTD mutations. Colonies were stained using Coomassie blue. Wild-type is pFK5.1. Mock transfected cells as the control. The NS4B-CTD mutations TripleE, E247, E248 and helix-swop in pFK5.1 are explained in figure 4.

Mentions: To examine the importance of the NS4B-CTD positively charged residues for RNA replication, we exchanged these amino acids involved in membrane association for negatively charged glutamic acids in selectable subgenomic replicons [9]. Huh7 cells transfected with replicon RNA that carry the three negatively charged residues (NS4B-CTD tripleE) did not yield any viable colonies (Fig. 5). Moreover the single mutations K247E and R248E were replication defective and gave no colonies (Fig. 5). Thus the positive residues are clearly indispensible for viral RNA replication in cell culture, suggesting that loss in membrane association leads to a replication defect. These results, together with the possible functional parallel between d-LDH MBD and the NS4B-CTD, prompted us to swop the membrane binding helix from d-LDH MBD (PPRMKNWRDK) into replicons (helix-swop, Fig. 3a) and determine colony formation. These replicons in which eight amino acids are exchanged indeed formed several viable colonies (40 colony forming units per ug (CFU) of transfected replicon RNA) (Fig. 5). Clearly, far less colonies were formed relative to wild type (~10.000 CFU), but the replication defect from the helix swop is less than the negative charged mutations, where no colonies were formed. Two separate replicon colonies derived from the NS4B-CTD helix-swop were expanded, RNA isolated and sequenced to analyze whether they still contained the original mutations. Interestingly, the complete introduced helix was retained, confirming the importance of this membrane contacting helix.


Hepatitis C virus NS4B carboxy terminal domain is a membrane binding domain.

Liefhebber JM, Brandt BW, Broer R, Spaan WJ, van Leeuwen HC - Virol. J. (2009)

Effect of NS4B carboxy terminal mutations on colony formation using selectable replicons. Colony formation assay in which Huh7 cells are transfected with in vitro transcribed replicon RNA that contain NS4B-CTD mutations. Colonies were stained using Coomassie blue. Wild-type is pFK5.1. Mock transfected cells as the control. The NS4B-CTD mutations TripleE, E247, E248 and helix-swop in pFK5.1 are explained in figure 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698844&req=5

Figure 5: Effect of NS4B carboxy terminal mutations on colony formation using selectable replicons. Colony formation assay in which Huh7 cells are transfected with in vitro transcribed replicon RNA that contain NS4B-CTD mutations. Colonies were stained using Coomassie blue. Wild-type is pFK5.1. Mock transfected cells as the control. The NS4B-CTD mutations TripleE, E247, E248 and helix-swop in pFK5.1 are explained in figure 4.
Mentions: To examine the importance of the NS4B-CTD positively charged residues for RNA replication, we exchanged these amino acids involved in membrane association for negatively charged glutamic acids in selectable subgenomic replicons [9]. Huh7 cells transfected with replicon RNA that carry the three negatively charged residues (NS4B-CTD tripleE) did not yield any viable colonies (Fig. 5). Moreover the single mutations K247E and R248E were replication defective and gave no colonies (Fig. 5). Thus the positive residues are clearly indispensible for viral RNA replication in cell culture, suggesting that loss in membrane association leads to a replication defect. These results, together with the possible functional parallel between d-LDH MBD and the NS4B-CTD, prompted us to swop the membrane binding helix from d-LDH MBD (PPRMKNWRDK) into replicons (helix-swop, Fig. 3a) and determine colony formation. These replicons in which eight amino acids are exchanged indeed formed several viable colonies (40 colony forming units per ug (CFU) of transfected replicon RNA) (Fig. 5). Clearly, far less colonies were formed relative to wild type (~10.000 CFU), but the replication defect from the helix swop is less than the negative charged mutations, where no colonies were formed. Two separate replicon colonies derived from the NS4B-CTD helix-swop were expanded, RNA isolated and sequenced to analyze whether they still contained the original mutations. Interestingly, the complete introduced helix was retained, confirming the importance of this membrane contacting helix.

Bottom Line: All HCV proteins are associated to membranes, pointing out the importance of membranes for HCV.Membrane association was confirmed by swopping the membrane contacting helix of d-LDH with the corresponding domain of the 4B-CTD.Together these data show that NS4B-CTD is associated to membranes, similar to the prokaryotic d-LDH MBD, and is important for replication.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, 2300 RC Leiden, The Netherlands. J.M.P.Liefhebber@lumc.nl

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.

Conclusion: Together these data show that NS4B-CTD is associated to membranes, similar to the prokaryotic d-LDH MBD, and is important for replication.

Show MeSH
Related in: MedlinePlus