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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

Alanine substitution of the conserved acidic residues on the hydrophilic side of NS4B AH1 affects virus production.(A) Extra- and intracellular infectivity after electroporation of a replication-deficient (ΔGDD), wild-type (wt), E8A/E15A mutant and envelope glycoprotein-deficient (ΔE1E2) full-length Jc1 genomes was determined by 50% tissue culture infective dose (TCID50) measurement at 48 h post-electroporation. (B) Intra- and extracellular HCV RNA levels were determined by RT-PCR 48 h post-electroporation and normalized to GAPDH mRNA level. Results measured for the wt were set to 100%. Note the use of a linear scale as opposed to the logarithmic scale used in panel A.
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ppat-1004501-g007: Alanine substitution of the conserved acidic residues on the hydrophilic side of NS4B AH1 affects virus production.(A) Extra- and intracellular infectivity after electroporation of a replication-deficient (ΔGDD), wild-type (wt), E8A/E15A mutant and envelope glycoprotein-deficient (ΔE1E2) full-length Jc1 genomes was determined by 50% tissue culture infective dose (TCID50) measurement at 48 h post-electroporation. (B) Intra- and extracellular HCV RNA levels were determined by RT-PCR 48 h post-electroporation and normalized to GAPDH mRNA level. Results measured for the wt were set to 100%. Note the use of a linear scale as opposed to the logarithmic scale used in panel A.

Mentions: As mutant E8A/E15A did not show any significant replication defect, we introduced these aa substitutions into a full-length Jc1 (J6/JFH1 chimeric) HCV genome and assessed virus production by 50% tissue culture infective dose (TCID50) determination. As shown in Figure 7A, infectious virus production by this mutant was strongly reduced, with more than 100-fold lower extra- and intracellular TCID50 yields as compared to the wild-type virus. These results clearly indicate that NS4B AH1 possesses a role in HCV particle production. Quantitation of intra- vs. extracellular TCID50 suggests that the defect is primarily at the level of particle assembly, but an effect on release cannot be excluded (Fig. 7A). Quantitation of intra- vs. extracellular HCV RNA levels demonstrates that mutant E8A/E15A replicates in a full-length viral genome context and confirms the selective defect in virus production of this mutant (Fig. 7B).


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)

Alanine substitution of the conserved acidic residues on the hydrophilic side of NS4B AH1 affects virus production.(A) Extra- and intracellular infectivity after electroporation of a replication-deficient (ΔGDD), wild-type (wt), E8A/E15A mutant and envelope glycoprotein-deficient (ΔE1E2) full-length Jc1 genomes was determined by 50% tissue culture infective dose (TCID50) measurement at 48 h post-electroporation. (B) Intra- and extracellular HCV RNA levels were determined by RT-PCR 48 h post-electroporation and normalized to GAPDH mRNA level. Results measured for the wt were set to 100%. Note the use of a linear scale as opposed to the logarithmic scale used in panel A.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004501-g007: Alanine substitution of the conserved acidic residues on the hydrophilic side of NS4B AH1 affects virus production.(A) Extra- and intracellular infectivity after electroporation of a replication-deficient (ΔGDD), wild-type (wt), E8A/E15A mutant and envelope glycoprotein-deficient (ΔE1E2) full-length Jc1 genomes was determined by 50% tissue culture infective dose (TCID50) measurement at 48 h post-electroporation. (B) Intra- and extracellular HCV RNA levels were determined by RT-PCR 48 h post-electroporation and normalized to GAPDH mRNA level. Results measured for the wt were set to 100%. Note the use of a linear scale as opposed to the logarithmic scale used in panel A.
Mentions: As mutant E8A/E15A did not show any significant replication defect, we introduced these aa substitutions into a full-length Jc1 (J6/JFH1 chimeric) HCV genome and assessed virus production by 50% tissue culture infective dose (TCID50) determination. As shown in Figure 7A, infectious virus production by this mutant was strongly reduced, with more than 100-fold lower extra- and intracellular TCID50 yields as compared to the wild-type virus. These results clearly indicate that NS4B AH1 possesses a role in HCV particle production. Quantitation of intra- vs. extracellular TCID50 suggests that the defect is primarily at the level of particle assembly, but an effect on release cannot be excluded (Fig. 7A). Quantitation of intra- vs. extracellular HCV RNA levels demonstrates that mutant E8A/E15A replicates in a full-length viral genome context and confirms the selective defect in virus production of this mutant (Fig. 7B).

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