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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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Membrane association of NS4B carboxy terminal domain. Huh7 cells transfected with NS4B-CTD-Myc, NS4B-CTD tripleE-Myc or NS4B-CTD Helix-swop-Myc were subjected to sucrose density gradient centrifugation. Cell lysates were loaded under a sucrose gradient from 10–80% w/v and part of the lysate was used as a loading control (L). Fractions were taken from top (fraction 1) to bottom (fraction 23) and separated by SDS-PAGE. Followed by immunoblot analysis for Calnexin, Transferrin Receptor (TfR), Cytochrome C oxidase subunit IV (COX-IV) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). NS4B-CTD and NS4B-CTD tripleE were assayed using an antibody against Myc-epitope. M indicates where molecular weight marker was loaded.
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Figure 2: Membrane association of NS4B carboxy terminal domain. Huh7 cells transfected with NS4B-CTD-Myc, NS4B-CTD tripleE-Myc or NS4B-CTD Helix-swop-Myc were subjected to sucrose density gradient centrifugation. Cell lysates were loaded under a sucrose gradient from 10–80% w/v and part of the lysate was used as a loading control (L). Fractions were taken from top (fraction 1) to bottom (fraction 23) and separated by SDS-PAGE. Followed by immunoblot analysis for Calnexin, Transferrin Receptor (TfR), Cytochrome C oxidase subunit IV (COX-IV) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). NS4B-CTD and NS4B-CTD tripleE were assayed using an antibody against Myc-epitope. M indicates where molecular weight marker was loaded.

Mentions: Membrane association of proteins can be investigated in a membrane floatation assay. In such an assay, a continuous-density gradient is loaded on top of a cell extract and subjected to centrifugation. Membranes and associated proteins float into the gradient, while cytosolic proteins stay in the loaded bottom fraction. To examine the suggested membrane association characteristics of the NS4B-CTD a membrane floatation assay was performed. Figure 2 shows the results of that assay, in which a cell lysate of Huh7 cells transfected with NS4B-CTD was used. Fractions were collected from the top (10%) to the bottom (80%) of the gradient and the odd fractions were analyzed by western blotting. As a control for cytosolic proteins, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used. As expected, GAPDH was retained in the bottom fractions 21 and 23 of the density gradient, where the cell extract was loaded (Fig. 2). Calnexin, Transferrin receptor (TfR) and Cytochrome C oxidase subunit IV (COX-IV) are transmembrane proteins and float into the gradient, they are mainly observed in fractions 9 and 11 (Fig. 2). Since calnexin, TfR and COX-IV reside on different membranes in the cell (ER, the endocytic pathway and mitochondria), their distribution differs slightly (Fig. 2). The NS4B-CTD is detected in fractions 7 to 13 and 21 and 23 with its highest signal in fraction 11 (Fig. 2). In conclusion, similar to membrane proteins the NS4B-CTD floats into the gradient, implying membrane association of the NS4B-CTD. Together, the punctate structures in immunofluorescence and the floatation into the membrane floatation gradient, suggest association of the CTD of NS4B to membranes.


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)

Membrane association of NS4B carboxy terminal domain. Huh7 cells transfected with NS4B-CTD-Myc, NS4B-CTD tripleE-Myc or NS4B-CTD Helix-swop-Myc were subjected to sucrose density gradient centrifugation. Cell lysates were loaded under a sucrose gradient from 10–80% w/v and part of the lysate was used as a loading control (L). Fractions were taken from top (fraction 1) to bottom (fraction 23) and separated by SDS-PAGE. Followed by immunoblot analysis for Calnexin, Transferrin Receptor (TfR), Cytochrome C oxidase subunit IV (COX-IV) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). NS4B-CTD and NS4B-CTD tripleE were assayed using an antibody against Myc-epitope. M indicates where molecular weight marker was loaded.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Membrane association of NS4B carboxy terminal domain. Huh7 cells transfected with NS4B-CTD-Myc, NS4B-CTD tripleE-Myc or NS4B-CTD Helix-swop-Myc were subjected to sucrose density gradient centrifugation. Cell lysates were loaded under a sucrose gradient from 10–80% w/v and part of the lysate was used as a loading control (L). Fractions were taken from top (fraction 1) to bottom (fraction 23) and separated by SDS-PAGE. Followed by immunoblot analysis for Calnexin, Transferrin Receptor (TfR), Cytochrome C oxidase subunit IV (COX-IV) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). NS4B-CTD and NS4B-CTD tripleE were assayed using an antibody against Myc-epitope. M indicates where molecular weight marker was loaded.
Mentions: Membrane association of proteins can be investigated in a membrane floatation assay. In such an assay, a continuous-density gradient is loaded on top of a cell extract and subjected to centrifugation. Membranes and associated proteins float into the gradient, while cytosolic proteins stay in the loaded bottom fraction. To examine the suggested membrane association characteristics of the NS4B-CTD a membrane floatation assay was performed. Figure 2 shows the results of that assay, in which a cell lysate of Huh7 cells transfected with NS4B-CTD was used. Fractions were collected from the top (10%) to the bottom (80%) of the gradient and the odd fractions were analyzed by western blotting. As a control for cytosolic proteins, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used. As expected, GAPDH was retained in the bottom fractions 21 and 23 of the density gradient, where the cell extract was loaded (Fig. 2). Calnexin, Transferrin receptor (TfR) and Cytochrome C oxidase subunit IV (COX-IV) are transmembrane proteins and float into the gradient, they are mainly observed in fractions 9 and 11 (Fig. 2). Since calnexin, TfR and COX-IV reside on different membranes in the cell (ER, the endocytic pathway and mitochondria), their distribution differs slightly (Fig. 2). The NS4B-CTD is detected in fractions 7 to 13 and 21 and 23 with its highest signal in fraction 11 (Fig. 2). In conclusion, similar to membrane proteins the NS4B-CTD floats into the gradient, implying membrane association of the NS4B-CTD. Together, the punctate structures in immunofluorescence and the floatation into the membrane floatation gradient, suggest association of the CTD of NS4B to membranes.

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