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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 conserved positively charged amino acids in NS4B AH1 are essential for RNA replication. (A) AH1 mutants introduced into a subgenomic replicon construct harboring a firefly luciferase reporter gene were analyzed in Huh-7.5 cells by luciferase activity measurement at 4, 24, 48 and 72 h post-electroporation of in vitro transcribed RNA. Relative light units (RLU) were normalized to values measured at 4 h. ΔGDD represents a replication-deficient control with an inactivating deletion in the RNA-dependent RNA polymerase active site. Results from a representative experiment performed in triplicate are shown. (B) Alanine substitution of the conserved positively charged amino acids in AH1 does not alter the subcellular localization of NS4B. T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring the different NS4B mutations were transfected into H7-T7-IZ cells which constitutively express the T7 polymerase, followed by immunofluorescence analyses as described in the Materials and Methods section. Polyclonal antibody #86 against NS4B and monoclonal antibody 9E10 against NS5A, were used as primary antibodies.
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ppat-1004501-g004: The conserved positively charged amino acids in NS4B AH1 are essential for RNA replication. (A) AH1 mutants introduced into a subgenomic replicon construct harboring a firefly luciferase reporter gene were analyzed in Huh-7.5 cells by luciferase activity measurement at 4, 24, 48 and 72 h post-electroporation of in vitro transcribed RNA. Relative light units (RLU) were normalized to values measured at 4 h. ΔGDD represents a replication-deficient control with an inactivating deletion in the RNA-dependent RNA polymerase active site. Results from a representative experiment performed in triplicate are shown. (B) Alanine substitution of the conserved positively charged amino acids in AH1 does not alter the subcellular localization of NS4B. T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring the different NS4B mutations were transfected into H7-T7-IZ cells which constitutively express the T7 polymerase, followed by immunofluorescence analyses as described in the Materials and Methods section. Polyclonal antibody #86 against NS4B and monoclonal antibody 9E10 against NS5A, were used as primary antibodies.

Mentions: The role of the structurally conserved features of AH1 in HCV RNA replication was investigated by the use of a subgenomic JFH1 replicon harboring a luciferase reporter gene. As shown in Figure 4A, alanine substitution of Lys 18 and Lys 20, either simultaneously (K18A/K20A) or individually (K18A and K20A), abrogated HCV RNA replication, as inferred by comparison with the non-replicative ΔGDD polymerase mutant. In addition, insertion of one alanine residue between Lys 18 and Ser 19 (KASK), resulting in a 110° twist of the α-helix and thus an altered positioning of the two lysine residues in positions 18 and 20, abrogated HCV RNA replication. Hence, the positively charged residues flanking AH1 on either side are required for HCV RNA replication. By contrast, alanine substitution of the two conserved glutamate residues highlighted above (E8A/E15A) did only slightly affect RNA replication at an early time point (about 5-fold reduction in relative light units [RLU] at 24 h, but no appreciable difference at 48 and 72 h).


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 conserved positively charged amino acids in NS4B AH1 are essential for RNA replication. (A) AH1 mutants introduced into a subgenomic replicon construct harboring a firefly luciferase reporter gene were analyzed in Huh-7.5 cells by luciferase activity measurement at 4, 24, 48 and 72 h post-electroporation of in vitro transcribed RNA. Relative light units (RLU) were normalized to values measured at 4 h. ΔGDD represents a replication-deficient control with an inactivating deletion in the RNA-dependent RNA polymerase active site. Results from a representative experiment performed in triplicate are shown. (B) Alanine substitution of the conserved positively charged amino acids in AH1 does not alter the subcellular localization of NS4B. T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring the different NS4B mutations were transfected into H7-T7-IZ cells which constitutively express the T7 polymerase, followed by immunofluorescence analyses as described in the Materials and Methods section. Polyclonal antibody #86 against NS4B and monoclonal antibody 9E10 against NS5A, were used as primary antibodies.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4231108&req=5

ppat-1004501-g004: The conserved positively charged amino acids in NS4B AH1 are essential for RNA replication. (A) AH1 mutants introduced into a subgenomic replicon construct harboring a firefly luciferase reporter gene were analyzed in Huh-7.5 cells by luciferase activity measurement at 4, 24, 48 and 72 h post-electroporation of in vitro transcribed RNA. Relative light units (RLU) were normalized to values measured at 4 h. ΔGDD represents a replication-deficient control with an inactivating deletion in the RNA-dependent RNA polymerase active site. Results from a representative experiment performed in triplicate are shown. (B) Alanine substitution of the conserved positively charged amino acids in AH1 does not alter the subcellular localization of NS4B. T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring the different NS4B mutations were transfected into H7-T7-IZ cells which constitutively express the T7 polymerase, followed by immunofluorescence analyses as described in the Materials and Methods section. Polyclonal antibody #86 against NS4B and monoclonal antibody 9E10 against NS5A, were used as primary antibodies.
Mentions: The role of the structurally conserved features of AH1 in HCV RNA replication was investigated by the use of a subgenomic JFH1 replicon harboring a luciferase reporter gene. As shown in Figure 4A, alanine substitution of Lys 18 and Lys 20, either simultaneously (K18A/K20A) or individually (K18A and K20A), abrogated HCV RNA replication, as inferred by comparison with the non-replicative ΔGDD polymerase mutant. In addition, insertion of one alanine residue between Lys 18 and Ser 19 (KASK), resulting in a 110° twist of the α-helix and thus an altered positioning of the two lysine residues in positions 18 and 20, abrogated HCV RNA replication. Hence, the positively charged residues flanking AH1 on either side are required for HCV RNA replication. By contrast, alanine substitution of the two conserved glutamate residues highlighted above (E8A/E15A) did only slightly affect RNA replication at an early time point (about 5-fold reduction in relative light units [RLU] at 24 h, but no appreciable difference at 48 and 72 h).

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