Limits...
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

Analysis of pseudorevertants selected with NS4B AH1 mutant K18A.(A) Selected amino acid changes identified following transfection of a selectable subgenomic replicon harboring the NS4B AH1 K18A mutation (see Table 1) were reengineered into a subgenomic HCV replicon harboring a luciferase reporter gene. In vitro transcribed RNAs were electroporated into Huh-7.5 cells, followed by luciferase activity measurement at 4, 24, 48 and 72 h post-electroporation. Relative light units (RLU) were normalized to values measured at 4 h. Results from two independent experiments were pooled (n = 8 for each construct and time point). For statistical analyses, RLU measured at 48 h were compared to the 48-h values obtained with mutant K18A. *, P<0.001. (B) Structure models of the different AH1 sequences were established by using Swiss-PdbViewer 4.01 software [69] and drawn by using VMD 1.9 software [68].
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231108&req=5

ppat-1004501-g006: Analysis of pseudorevertants selected with NS4B AH1 mutant K18A.(A) Selected amino acid changes identified following transfection of a selectable subgenomic replicon harboring the NS4B AH1 K18A mutation (see Table 1) were reengineered into a subgenomic HCV replicon harboring a luciferase reporter gene. In vitro transcribed RNAs were electroporated into Huh-7.5 cells, followed by luciferase activity measurement at 4, 24, 48 and 72 h post-electroporation. Relative light units (RLU) were normalized to values measured at 4 h. Results from two independent experiments were pooled (n = 8 for each construct and time point). For statistical analyses, RLU measured at 48 h were compared to the 48-h values obtained with mutant K18A. *, P<0.001. (B) Structure models of the different AH1 sequences were established by using Swiss-PdbViewer 4.01 software [69] and drawn by using VMD 1.9 software [68].

Mentions: In order to further assess the importance of the positively charged residues flanking AH1, mutations K18A, K20A and K18A/K20A were introduced into a selectable subgenomic JFH1 replicon harboring a neomycin resistance cassette in the first cistron. The corresponding in vitro transcribed RNAs were electroporated into Huh-7.5 cells, followed by selection with 500 µg/ml G418 for 3 weeks. About 70 G418-resistant colonies per µg of electroporated RNA were obtained for mutant K18A whereas mutants K18A/K20A and K20A did not yield any viable colonies. Total RNA was extracted from pooled colonies and the NS3-5B region amplified by RT-PCR, followed by cloning of amplicons and sequencing of 10 DNA subclones. Sequence analyses revealed that the K18A mutation in AH1 was retained in all clones. Amino acid changes identified elsewhere in the NS3-5B region are listed in Table 1. Strikingly, mutation P189L in domain I of NS5A was found in 9 out of 10 clones. The only clone which did not harbor this aa change in NS5A showed a substitution of Gln 26 by arginine (Q26R) in NS4B. Interestingly, this mutation results in a new positively charged residue flanking the hydrophobic side of AH1 (Fig. 3). Selection of a compensatory positively charged residue flanking AH1 was also observed in clones harboring pseudoreversions Q22R and Q27R, which coexisted with the NS5A P189L change. The relevance of these compensatory mutations was tested by reintroducing them, alone or in combination with K18A, into a JFH1 subgenomic replicon harboring a luciferase reporter gene. As shown in Figure 6A, the NS5A P189L change alone did not confer any replication advantage to the wild-type replicon and did only slightly (about 2-fold) improve replication of the NS4B K18A mutant. However, the Q22R and Q26R changes in NS4B AH1 strongly enhanced RNA replication capacity of mutant K18A (about 1.5 log for Q22R and 1.0 log for Q26R). Remarkably, the addition of NS5A change P189L to the NS4B K18A/Q22R mutant completely rescued RNA replication, thereby supporting the notion of a functional interaction between NS4B AH1 and NS5A. Finally, the NS4B Q27R change, identified in one clone harboring a number of additional aa changes (Table 1), did not rescue RNA replication of mutant K18A.


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)

Analysis of pseudorevertants selected with NS4B AH1 mutant K18A.(A) Selected amino acid changes identified following transfection of a selectable subgenomic replicon harboring the NS4B AH1 K18A mutation (see Table 1) were reengineered into a subgenomic HCV replicon harboring a luciferase reporter gene. In vitro transcribed RNAs were electroporated into Huh-7.5 cells, followed by luciferase activity measurement at 4, 24, 48 and 72 h post-electroporation. Relative light units (RLU) were normalized to values measured at 4 h. Results from two independent experiments were pooled (n = 8 for each construct and time point). For statistical analyses, RLU measured at 48 h were compared to the 48-h values obtained with mutant K18A. *, P<0.001. (B) Structure models of the different AH1 sequences were established by using Swiss-PdbViewer 4.01 software [69] and drawn by using VMD 1.9 software [68].
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004501-g006: Analysis of pseudorevertants selected with NS4B AH1 mutant K18A.(A) Selected amino acid changes identified following transfection of a selectable subgenomic replicon harboring the NS4B AH1 K18A mutation (see Table 1) were reengineered into a subgenomic HCV replicon harboring a luciferase reporter gene. In vitro transcribed RNAs were electroporated into Huh-7.5 cells, followed by luciferase activity measurement at 4, 24, 48 and 72 h post-electroporation. Relative light units (RLU) were normalized to values measured at 4 h. Results from two independent experiments were pooled (n = 8 for each construct and time point). For statistical analyses, RLU measured at 48 h were compared to the 48-h values obtained with mutant K18A. *, P<0.001. (B) Structure models of the different AH1 sequences were established by using Swiss-PdbViewer 4.01 software [69] and drawn by using VMD 1.9 software [68].
Mentions: In order to further assess the importance of the positively charged residues flanking AH1, mutations K18A, K20A and K18A/K20A were introduced into a selectable subgenomic JFH1 replicon harboring a neomycin resistance cassette in the first cistron. The corresponding in vitro transcribed RNAs were electroporated into Huh-7.5 cells, followed by selection with 500 µg/ml G418 for 3 weeks. About 70 G418-resistant colonies per µg of electroporated RNA were obtained for mutant K18A whereas mutants K18A/K20A and K20A did not yield any viable colonies. Total RNA was extracted from pooled colonies and the NS3-5B region amplified by RT-PCR, followed by cloning of amplicons and sequencing of 10 DNA subclones. Sequence analyses revealed that the K18A mutation in AH1 was retained in all clones. Amino acid changes identified elsewhere in the NS3-5B region are listed in Table 1. Strikingly, mutation P189L in domain I of NS5A was found in 9 out of 10 clones. The only clone which did not harbor this aa change in NS5A showed a substitution of Gln 26 by arginine (Q26R) in NS4B. Interestingly, this mutation results in a new positively charged residue flanking the hydrophobic side of AH1 (Fig. 3). Selection of a compensatory positively charged residue flanking AH1 was also observed in clones harboring pseudoreversions Q22R and Q27R, which coexisted with the NS5A P189L change. The relevance of these compensatory mutations was tested by reintroducing them, alone or in combination with K18A, into a JFH1 subgenomic replicon harboring a luciferase reporter gene. As shown in Figure 6A, the NS5A P189L change alone did not confer any replication advantage to the wild-type replicon and did only slightly (about 2-fold) improve replication of the NS4B K18A mutant. However, the Q22R and Q26R changes in NS4B AH1 strongly enhanced RNA replication capacity of mutant K18A (about 1.5 log for Q22R and 1.0 log for Q26R). Remarkably, the addition of NS5A change P189L to the NS4B K18A/Q22R mutant completely rescued RNA replication, thereby supporting the notion of a functional interaction between NS4B AH1 and NS5A. Finally, the NS4B Q27R change, identified in one clone harboring a number of additional aa changes (Table 1), did not rescue RNA replication of mutant K18A.

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