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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

Mutations of the conserved positively charged amino acids in NS4B AH1 affect the morphology of double-membrane vesicles.(A) Electron microscopy (EM) analyses of H7-T7-IZ cells transfected with empty vector (mock) or T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring wild-type (wt) or mutant NS4B. Cells were fixed 24 h post-transfection and processed for EM as described in the Materials and Methods section. The scale bar corresponds to 500 nm. Note that double-membrane vesicles (DMV) induced by mutants K18A, K20A and K18A/K20A are larger as compared to wt and E8A/E15A. (B) Graphical representation of DMV diameter induced by wt and NS4B mutants. The analysis is based on at least 621 DMVs from at least 10 different transfected cells. Mean value ± SEM for wt = 178.4±3.1 nm (n = 666); K18A = 298.3±5.1 (n = 832); K20A = 295.1±5.0 (n = 621); K18A/K20A = 374.0±5.0 (n = 1317). Horizontal lines (red) indicate mean values. * P≤0.0001; ns, non significant (P>0.25).
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ppat-1004501-g005: Mutations of the conserved positively charged amino acids in NS4B AH1 affect the morphology of double-membrane vesicles.(A) Electron microscopy (EM) analyses of H7-T7-IZ cells transfected with empty vector (mock) or T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring wild-type (wt) or mutant NS4B. Cells were fixed 24 h post-transfection and processed for EM as described in the Materials and Methods section. The scale bar corresponds to 500 nm. Note that double-membrane vesicles (DMV) induced by mutants K18A, K20A and K18A/K20A are larger as compared to wt and E8A/E15A. (B) Graphical representation of DMV diameter induced by wt and NS4B mutants. The analysis is based on at least 621 DMVs from at least 10 different transfected cells. Mean value ± SEM for wt = 178.4±3.1 nm (n = 666); K18A = 298.3±5.1 (n = 832); K20A = 295.1±5.0 (n = 621); K18A/K20A = 374.0±5.0 (n = 1317). Horizontal lines (red) indicate mean values. * P≤0.0001; ns, non significant (P>0.25).

Mentions: To gain deeper insight into the consequences of removal of the positively charged residues flanking AH1, we investigated the ultrastructure of membrane rearrangements induced by the different NS4B mutants by EM. To this end, the mutants were expressed in Huh-7 cells using a T7 RNA polymerase-based NS3-5B polyprotein expression system, as above. Previous work had shown that DMVs are formed in this system and that these closely resemble the HCV-induced membrane rearrangements observed in the context of subgenomic RNA replication and HCV infection [13], [24]. As shown in Figure 5A, regular round-shaped DMVs were readily observed for mutants K18A/K20A, K18A and K20A but, strikingly, these exhibited a large increase in diameter as compared to the ones formed by the wild-type construct. By contrast, mutant E8A/E15A, which replicated almost as wild-type, formed DMVs that were indistinguishable from wild-type. Quantitation of the DMV diameter for each construct demonstrated that DMVs formed by mutants K18A/K20A, K18A and K20A were significantly larger than the ones formed by the wild-type as well as by mutant E8A/E15A (Fig. 5B; see Figure Legend for details). Taken together, these observations indicate that the loss of one of the conserved positively charged residues flanking AH1, i.e. Lys 18 and/or Lys 20, results in the formation of larger DMVs that do not support HCV RNA replication.


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)

Mutations of the conserved positively charged amino acids in NS4B AH1 affect the morphology of double-membrane vesicles.(A) Electron microscopy (EM) analyses of H7-T7-IZ cells transfected with empty vector (mock) or T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring wild-type (wt) or mutant NS4B. Cells were fixed 24 h post-transfection and processed for EM as described in the Materials and Methods section. The scale bar corresponds to 500 nm. Note that double-membrane vesicles (DMV) induced by mutants K18A, K20A and K18A/K20A are larger as compared to wt and E8A/E15A. (B) Graphical representation of DMV diameter induced by wt and NS4B mutants. The analysis is based on at least 621 DMVs from at least 10 different transfected cells. Mean value ± SEM for wt = 178.4±3.1 nm (n = 666); K18A = 298.3±5.1 (n = 832); K20A = 295.1±5.0 (n = 621); K18A/K20A = 374.0±5.0 (n = 1317). Horizontal lines (red) indicate mean values. * P≤0.0001; ns, non significant (P>0.25).
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ppat-1004501-g005: Mutations of the conserved positively charged amino acids in NS4B AH1 affect the morphology of double-membrane vesicles.(A) Electron microscopy (EM) analyses of H7-T7-IZ cells transfected with empty vector (mock) or T7 RNA polymerase-driven N3-5B polyprotein expression constructs harboring wild-type (wt) or mutant NS4B. Cells were fixed 24 h post-transfection and processed for EM as described in the Materials and Methods section. The scale bar corresponds to 500 nm. Note that double-membrane vesicles (DMV) induced by mutants K18A, K20A and K18A/K20A are larger as compared to wt and E8A/E15A. (B) Graphical representation of DMV diameter induced by wt and NS4B mutants. The analysis is based on at least 621 DMVs from at least 10 different transfected cells. Mean value ± SEM for wt = 178.4±3.1 nm (n = 666); K18A = 298.3±5.1 (n = 832); K20A = 295.1±5.0 (n = 621); K18A/K20A = 374.0±5.0 (n = 1317). Horizontal lines (red) indicate mean values. * P≤0.0001; ns, non significant (P>0.25).
Mentions: To gain deeper insight into the consequences of removal of the positively charged residues flanking AH1, we investigated the ultrastructure of membrane rearrangements induced by the different NS4B mutants by EM. To this end, the mutants were expressed in Huh-7 cells using a T7 RNA polymerase-based NS3-5B polyprotein expression system, as above. Previous work had shown that DMVs are formed in this system and that these closely resemble the HCV-induced membrane rearrangements observed in the context of subgenomic RNA replication and HCV infection [13], [24]. As shown in Figure 5A, regular round-shaped DMVs were readily observed for mutants K18A/K20A, K18A and K20A but, strikingly, these exhibited a large increase in diameter as compared to the ones formed by the wild-type construct. By contrast, mutant E8A/E15A, which replicated almost as wild-type, formed DMVs that were indistinguishable from wild-type. Quantitation of the DMV diameter for each construct demonstrated that DMVs formed by mutants K18A/K20A, K18A and K20A were significantly larger than the ones formed by the wild-type as well as by mutant E8A/E15A (Fig. 5B; see Figure Legend for details). Taken together, these observations indicate that the loss of one of the conserved positively charged residues flanking AH1, i.e. Lys 18 and/or Lys 20, results in the formation of larger DMVs that do not support HCV RNA replication.

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