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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

Sequence analysis of the N-terminal part of HCV NS4B.Multiple alignment of NS4B amino acid (aa) 1–70 sequences from representative HCV strains of confirmed genotypes [66] are shown (http://euhcvdb.ibcp.fr; [41]). Genotype, GenBank accession number, and strain are indicated for each sequence. Amino acids are numbered with respect to NS4B (top row). The consensus sequence (top row) was deduced from the ClustalW multiple alignment of the indicated NS4B sequences [43]. To highlight the aa variability at each position, aa identical to the consensus sequence are indicated by hyphens. The degree of aa physicochemical conservation at each position can be inferred from the similarity index according to ClustalW convention (asterisk, invariant; colon, highly similar; dot, similar) [43] and the consensus hydropathic pattern: o, hydrophobic position (Pro, Val, Leu, Ile, Met, Phe, Tyr, Trp); n, neutral position (Gly, Ala, Ser, Thr); i, hydrophilic position (Asn, Gln, Asp, Glu, His, Lys, Arg); v, variable position (i.e. when both hydrophobic and hydrophilic residues are observed at a given position). To highlight the variable sequence positions in NS4B, conserved hydrophilic and hydrophobic positions are highlighted in yellow and gray, respectively. Residues are color-coded according to the Wimley and White hydrophobicity scales [67]: hydrophobic residues are black (Pro, Val, Leu, Ile, Met, Phe, Tyr, Trp); polar residues are orange (Gly, Ala, Ser, Thr, Asn and Gln); positively and negatively charged groups of basic (His, Lys, Arg) and acidic residues (Glu, Asp) are blue and red, respectively. Consensus secondary structure predictions of NS4B from representative HCV strains (Second. struct. cons.) are indicated as helical (h, blue) or undetermined (coil [c], orange). Predictions were made by using the web-based algorithms SOPM, HNNC, DSC, GOR IV, PHD, Predator and SIMPA96 available at the NPSA website (http://npsa-pbil.ibcp.fr; [42] and refs. therein). NMR AH2 structure (bottom row) denotes the conformation of residues determined previously by nuclear magnetic resonance (PDB entry 2JXF; [26]). Residue conformations are indicated as helical (H) or undetermined (C).
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ppat-1004501-g001: Sequence analysis of the N-terminal part of HCV NS4B.Multiple alignment of NS4B amino acid (aa) 1–70 sequences from representative HCV strains of confirmed genotypes [66] are shown (http://euhcvdb.ibcp.fr; [41]). Genotype, GenBank accession number, and strain are indicated for each sequence. Amino acids are numbered with respect to NS4B (top row). The consensus sequence (top row) was deduced from the ClustalW multiple alignment of the indicated NS4B sequences [43]. To highlight the aa variability at each position, aa identical to the consensus sequence are indicated by hyphens. The degree of aa physicochemical conservation at each position can be inferred from the similarity index according to ClustalW convention (asterisk, invariant; colon, highly similar; dot, similar) [43] and the consensus hydropathic pattern: o, hydrophobic position (Pro, Val, Leu, Ile, Met, Phe, Tyr, Trp); n, neutral position (Gly, Ala, Ser, Thr); i, hydrophilic position (Asn, Gln, Asp, Glu, His, Lys, Arg); v, variable position (i.e. when both hydrophobic and hydrophilic residues are observed at a given position). To highlight the variable sequence positions in NS4B, conserved hydrophilic and hydrophobic positions are highlighted in yellow and gray, respectively. Residues are color-coded according to the Wimley and White hydrophobicity scales [67]: hydrophobic residues are black (Pro, Val, Leu, Ile, Met, Phe, Tyr, Trp); polar residues are orange (Gly, Ala, Ser, Thr, Asn and Gln); positively and negatively charged groups of basic (His, Lys, Arg) and acidic residues (Glu, Asp) are blue and red, respectively. Consensus secondary structure predictions of NS4B from representative HCV strains (Second. struct. cons.) are indicated as helical (h, blue) or undetermined (coil [c], orange). Predictions were made by using the web-based algorithms SOPM, HNNC, DSC, GOR IV, PHD, Predator and SIMPA96 available at the NPSA website (http://npsa-pbil.ibcp.fr; [42] and refs. therein). NMR AH2 structure (bottom row) denotes the conformation of residues determined previously by nuclear magnetic resonance (PDB entry 2JXF; [26]). Residue conformations are indicated as helical (H) or undetermined (C).

Mentions: Sequence analyses and structure predictions were performed to assess the degree of conservation of the N-terminal part of NS4B and to identify potential structural determinants. The degree of aa conservation among different genotypes was investigated by ClustalW alignment of 27 reference sequences representative of the major HCV genotypes and subtypes. This alignment revealed that the segment comprising aa 50–70, including part of amphipathic α-helix AH2 (aa 42–69), is well conserved, whereas the aa 1–50 segment, including predicted amphipathic α-helix AH1, appears highly variable except for a few well-conserved positions (e.g., basic residues at positions 18 and 20 and a proline at position 38 (Fig. 1). However, the apparent variability is limited at most positions since the observed residues exhibit similar physicochemical properties, as indicated both by the similarity pattern (colons and dots) and the hydropathic pattern, where o, i, and n denote hydrophobic, hydrophilic, and neutral residues, respectively (see Legend to Figure 1 for details). Moreover, all secondary structure prediction methods indicate the presence of an α-helix in the segment comprising aa 5–35 in all genotypes. Hence, despite the apparent aa variability, conservation of the hydropathic pattern suggests that the overall structure of AH1 is conserved.


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)

Sequence analysis of the N-terminal part of HCV NS4B.Multiple alignment of NS4B amino acid (aa) 1–70 sequences from representative HCV strains of confirmed genotypes [66] are shown (http://euhcvdb.ibcp.fr; [41]). Genotype, GenBank accession number, and strain are indicated for each sequence. Amino acids are numbered with respect to NS4B (top row). The consensus sequence (top row) was deduced from the ClustalW multiple alignment of the indicated NS4B sequences [43]. To highlight the aa variability at each position, aa identical to the consensus sequence are indicated by hyphens. The degree of aa physicochemical conservation at each position can be inferred from the similarity index according to ClustalW convention (asterisk, invariant; colon, highly similar; dot, similar) [43] and the consensus hydropathic pattern: o, hydrophobic position (Pro, Val, Leu, Ile, Met, Phe, Tyr, Trp); n, neutral position (Gly, Ala, Ser, Thr); i, hydrophilic position (Asn, Gln, Asp, Glu, His, Lys, Arg); v, variable position (i.e. when both hydrophobic and hydrophilic residues are observed at a given position). To highlight the variable sequence positions in NS4B, conserved hydrophilic and hydrophobic positions are highlighted in yellow and gray, respectively. Residues are color-coded according to the Wimley and White hydrophobicity scales [67]: hydrophobic residues are black (Pro, Val, Leu, Ile, Met, Phe, Tyr, Trp); polar residues are orange (Gly, Ala, Ser, Thr, Asn and Gln); positively and negatively charged groups of basic (His, Lys, Arg) and acidic residues (Glu, Asp) are blue and red, respectively. Consensus secondary structure predictions of NS4B from representative HCV strains (Second. struct. cons.) are indicated as helical (h, blue) or undetermined (coil [c], orange). Predictions were made by using the web-based algorithms SOPM, HNNC, DSC, GOR IV, PHD, Predator and SIMPA96 available at the NPSA website (http://npsa-pbil.ibcp.fr; [42] and refs. therein). NMR AH2 structure (bottom row) denotes the conformation of residues determined previously by nuclear magnetic resonance (PDB entry 2JXF; [26]). Residue conformations are indicated as helical (H) or undetermined (C).
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Related In: Results  -  Collection

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ppat-1004501-g001: Sequence analysis of the N-terminal part of HCV NS4B.Multiple alignment of NS4B amino acid (aa) 1–70 sequences from representative HCV strains of confirmed genotypes [66] are shown (http://euhcvdb.ibcp.fr; [41]). Genotype, GenBank accession number, and strain are indicated for each sequence. Amino acids are numbered with respect to NS4B (top row). The consensus sequence (top row) was deduced from the ClustalW multiple alignment of the indicated NS4B sequences [43]. To highlight the aa variability at each position, aa identical to the consensus sequence are indicated by hyphens. The degree of aa physicochemical conservation at each position can be inferred from the similarity index according to ClustalW convention (asterisk, invariant; colon, highly similar; dot, similar) [43] and the consensus hydropathic pattern: o, hydrophobic position (Pro, Val, Leu, Ile, Met, Phe, Tyr, Trp); n, neutral position (Gly, Ala, Ser, Thr); i, hydrophilic position (Asn, Gln, Asp, Glu, His, Lys, Arg); v, variable position (i.e. when both hydrophobic and hydrophilic residues are observed at a given position). To highlight the variable sequence positions in NS4B, conserved hydrophilic and hydrophobic positions are highlighted in yellow and gray, respectively. Residues are color-coded according to the Wimley and White hydrophobicity scales [67]: hydrophobic residues are black (Pro, Val, Leu, Ile, Met, Phe, Tyr, Trp); polar residues are orange (Gly, Ala, Ser, Thr, Asn and Gln); positively and negatively charged groups of basic (His, Lys, Arg) and acidic residues (Glu, Asp) are blue and red, respectively. Consensus secondary structure predictions of NS4B from representative HCV strains (Second. struct. cons.) are indicated as helical (h, blue) or undetermined (coil [c], orange). Predictions were made by using the web-based algorithms SOPM, HNNC, DSC, GOR IV, PHD, Predator and SIMPA96 available at the NPSA website (http://npsa-pbil.ibcp.fr; [42] and refs. therein). NMR AH2 structure (bottom row) denotes the conformation of residues determined previously by nuclear magnetic resonance (PDB entry 2JXF; [26]). Residue conformations are indicated as helical (H) or undetermined (C).
Mentions: Sequence analyses and structure predictions were performed to assess the degree of conservation of the N-terminal part of NS4B and to identify potential structural determinants. The degree of aa conservation among different genotypes was investigated by ClustalW alignment of 27 reference sequences representative of the major HCV genotypes and subtypes. This alignment revealed that the segment comprising aa 50–70, including part of amphipathic α-helix AH2 (aa 42–69), is well conserved, whereas the aa 1–50 segment, including predicted amphipathic α-helix AH1, appears highly variable except for a few well-conserved positions (e.g., basic residues at positions 18 and 20 and a proline at position 38 (Fig. 1). However, the apparent variability is limited at most positions since the observed residues exhibit similar physicochemical properties, as indicated both by the similarity pattern (colons and dots) and the hydropathic pattern, where o, i, and n denote hydrophobic, hydrophilic, and neutral residues, respectively (see Legend to Figure 1 for details). Moreover, all secondary structure prediction methods indicate the presence of an α-helix in the segment comprising aa 5–35 in all genotypes. Hence, despite the apparent aa variability, conservation of the hydropathic pattern suggests that the overall structure of AH1 is conserved.

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