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Aminoterminal amphipathic α-helix AH1 of hepatitis C virus nonstructural protein 4B possesses a dual role in RNA replication and virus production.

Gouttenoire J, Montserret R, Paul D, Castillo R, Meister S, Bartenschlager R, Penin F, Moradpour D - PLoS Pathog. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.

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.

No MeSH data available.


Related in: MedlinePlus

The N-terminal part of NS4B assumes a dual topology in a replicative context.(A) Huh7-Lunet cells harboring a subgenomic HCV JFH1 replicon with an HA tag inserted between NS4B AH1 and AH2 (see Materials and Methods section) were subjected to total (0.2% digitonin [Dig 0.2%], upper row) or selective membrane permeabilization (0.05% digitonin [Dig 0.05%], lower row), as described in the Materials and Methods section. As a control, replicon cells were transfected with a CMV promotor-driven core-E1-E2-p7 expression construct. Core and NS5A served as controls for cytosolically oriented proteins, E1 as a control for a luminally oriented protein. Monoclonal antibodies C7-50 against HCV core, A4 against E1, 9E10 against NS5A, and HA-7 against the HA tag were used, followed by anti-mouse IgG Alexa Fluor 488 as secondary antibody. Images were acquired on a confocal laser scanning microscope with the same settings for each antibody and condition. Analogous results were obtained when polyclonal antibody Y-11 against the HA tag was used instead of monoclonal antibody HA-7. (B) Histogram of the fluorescence intensity ratios between selective and total membrane permeabilization conditions. Fluorescence intensities in 10–60 images for each condition were determined by using ImageJ software [70]. (C) H7-T7-IZ cells were transfected with a CMV promotor-driven core-E1-E2-p7 expression construct or T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring the different mutations in HA-tagged NS4B, followed by selective membrane permeabilization and immunofluorescence microscopy using the same antibodies as in panel A. Fluorescence intensity ratios between selective and total membrane permeabilization conditions were determined as in panel B. AH2mut is a previously described mutant harboring alanine substitution of 6 aromatic amino acid residues in NS4B AH2 [15]. This mutant is unable to translocate AH2 through the membrane bilayer. * P≤0.0001; ns, non significant (P>0.25).
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ppat-1004501-g008: The N-terminal part of NS4B assumes a dual topology in a replicative context.(A) Huh7-Lunet cells harboring a subgenomic HCV JFH1 replicon with an HA tag inserted between NS4B AH1 and AH2 (see Materials and Methods section) were subjected to total (0.2% digitonin [Dig 0.2%], upper row) or selective membrane permeabilization (0.05% digitonin [Dig 0.05%], lower row), as described in the Materials and Methods section. As a control, replicon cells were transfected with a CMV promotor-driven core-E1-E2-p7 expression construct. Core and NS5A served as controls for cytosolically oriented proteins, E1 as a control for a luminally oriented protein. Monoclonal antibodies C7-50 against HCV core, A4 against E1, 9E10 against NS5A, and HA-7 against the HA tag were used, followed by anti-mouse IgG Alexa Fluor 488 as secondary antibody. Images were acquired on a confocal laser scanning microscope with the same settings for each antibody and condition. Analogous results were obtained when polyclonal antibody Y-11 against the HA tag was used instead of monoclonal antibody HA-7. (B) Histogram of the fluorescence intensity ratios between selective and total membrane permeabilization conditions. Fluorescence intensities in 10–60 images for each condition were determined by using ImageJ software [70]. (C) H7-T7-IZ cells were transfected with a CMV promotor-driven core-E1-E2-p7 expression construct or T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring the different mutations in HA-tagged NS4B, followed by selective membrane permeabilization and immunofluorescence microscopy using the same antibodies as in panel A. Fluorescence intensity ratios between selective and total membrane permeabilization conditions were determined as in panel B. AH2mut is a previously described mutant harboring alanine substitution of 6 aromatic amino acid residues in NS4B AH2 [15]. This mutant is unable to translocate AH2 through the membrane bilayer. * P≤0.0001; ns, non significant (P>0.25).

Mentions: In a series of preliminary experiments, we found that incubation of fixed cells with 0.05% digitonin for 15 min at 4°C allowed for selective permeabilization of the plasma membrane but not the endoplasmic reticulum (ER) membrane of Huh7-Lunet cells which are highly permissive for HCV replication [25]. By contrast, 0.2% digitonin under the same experimental conditions permeabilized both membrane compartments. As shown in Figure 8A, the cytosolically oriented core and NS5A proteins could be detected at the same fluorescence intensity under both selective and total permeabilization conditions while the ER luminally oriented E1 glycoprotein could be detected only after total membrane permeabilization. Interestingly, the HA tag inserted between NS4B AH1 and AH2 was consistently detected at an approximately 50% reduced fluorescence intensity under selective as opposed to total membrane permeabilization conditions (Fig. 8A and 8B). These results, which were independent from the choice of anti-HA antibody, indicate a dual topology of the HA tag and, thereby, of the N-terminal part of NS4B in a functional, replicative context.


Aminoterminal amphipathic α-helix AH1 of hepatitis C virus nonstructural protein 4B possesses a dual role in RNA replication and virus production.

Gouttenoire J, Montserret R, Paul D, Castillo R, Meister S, Bartenschlager R, Penin F, Moradpour D - PLoS Pathog. (2014)

The N-terminal part of NS4B assumes a dual topology in a replicative context.(A) Huh7-Lunet cells harboring a subgenomic HCV JFH1 replicon with an HA tag inserted between NS4B AH1 and AH2 (see Materials and Methods section) were subjected to total (0.2% digitonin [Dig 0.2%], upper row) or selective membrane permeabilization (0.05% digitonin [Dig 0.05%], lower row), as described in the Materials and Methods section. As a control, replicon cells were transfected with a CMV promotor-driven core-E1-E2-p7 expression construct. Core and NS5A served as controls for cytosolically oriented proteins, E1 as a control for a luminally oriented protein. Monoclonal antibodies C7-50 against HCV core, A4 against E1, 9E10 against NS5A, and HA-7 against the HA tag were used, followed by anti-mouse IgG Alexa Fluor 488 as secondary antibody. Images were acquired on a confocal laser scanning microscope with the same settings for each antibody and condition. Analogous results were obtained when polyclonal antibody Y-11 against the HA tag was used instead of monoclonal antibody HA-7. (B) Histogram of the fluorescence intensity ratios between selective and total membrane permeabilization conditions. Fluorescence intensities in 10–60 images for each condition were determined by using ImageJ software [70]. (C) H7-T7-IZ cells were transfected with a CMV promotor-driven core-E1-E2-p7 expression construct or T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring the different mutations in HA-tagged NS4B, followed by selective membrane permeabilization and immunofluorescence microscopy using the same antibodies as in panel A. Fluorescence intensity ratios between selective and total membrane permeabilization conditions were determined as in panel B. AH2mut is a previously described mutant harboring alanine substitution of 6 aromatic amino acid residues in NS4B AH2 [15]. This mutant is unable to translocate AH2 through the membrane bilayer. * P≤0.0001; ns, non significant (P>0.25).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4231108&req=5

ppat-1004501-g008: The N-terminal part of NS4B assumes a dual topology in a replicative context.(A) Huh7-Lunet cells harboring a subgenomic HCV JFH1 replicon with an HA tag inserted between NS4B AH1 and AH2 (see Materials and Methods section) were subjected to total (0.2% digitonin [Dig 0.2%], upper row) or selective membrane permeabilization (0.05% digitonin [Dig 0.05%], lower row), as described in the Materials and Methods section. As a control, replicon cells were transfected with a CMV promotor-driven core-E1-E2-p7 expression construct. Core and NS5A served as controls for cytosolically oriented proteins, E1 as a control for a luminally oriented protein. Monoclonal antibodies C7-50 against HCV core, A4 against E1, 9E10 against NS5A, and HA-7 against the HA tag were used, followed by anti-mouse IgG Alexa Fluor 488 as secondary antibody. Images were acquired on a confocal laser scanning microscope with the same settings for each antibody and condition. Analogous results were obtained when polyclonal antibody Y-11 against the HA tag was used instead of monoclonal antibody HA-7. (B) Histogram of the fluorescence intensity ratios between selective and total membrane permeabilization conditions. Fluorescence intensities in 10–60 images for each condition were determined by using ImageJ software [70]. (C) H7-T7-IZ cells were transfected with a CMV promotor-driven core-E1-E2-p7 expression construct or T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring the different mutations in HA-tagged NS4B, followed by selective membrane permeabilization and immunofluorescence microscopy using the same antibodies as in panel A. Fluorescence intensity ratios between selective and total membrane permeabilization conditions were determined as in panel B. AH2mut is a previously described mutant harboring alanine substitution of 6 aromatic amino acid residues in NS4B AH2 [15]. This mutant is unable to translocate AH2 through the membrane bilayer. * P≤0.0001; ns, non significant (P>0.25).
Mentions: In a series of preliminary experiments, we found that incubation of fixed cells with 0.05% digitonin for 15 min at 4°C allowed for selective permeabilization of the plasma membrane but not the endoplasmic reticulum (ER) membrane of Huh7-Lunet cells which are highly permissive for HCV replication [25]. By contrast, 0.2% digitonin under the same experimental conditions permeabilized both membrane compartments. As shown in Figure 8A, the cytosolically oriented core and NS5A proteins could be detected at the same fluorescence intensity under both selective and total permeabilization conditions while the ER luminally oriented E1 glycoprotein could be detected only after total membrane permeabilization. Interestingly, the HA tag inserted between NS4B AH1 and AH2 was consistently detected at an approximately 50% reduced fluorescence intensity under selective as opposed to total membrane permeabilization conditions (Fig. 8A and 8B). These results, which were independent from the choice of anti-HA antibody, indicate a dual topology of the HA tag and, thereby, of the N-terminal part of NS4B in a functional, replicative context.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.

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.

No MeSH data available.


Related in: MedlinePlus