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NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly.

Popescu CI, Callens N, Trinel D, Roingeard P, Moradpour D, Descamps V, Duverlie G, Penin F, Héliot L, Rouillé Y, Dubuisson J - PLoS Pathog. (2011)

Bottom Line: Our data demonstrate molecular interactions between NS2 and p7 and E2.We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization.Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein.

View Article: PubMed Central - PubMed

Affiliation: Inserm U1019, CNRS UMR8204, Center for Infection & Immunity of Lille (CIIL), Institut Pasteur de Lille, Université Lille Nord de France, Lille, France.

ABSTRACT
Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.

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Rationale for mutagenesis in NS2 transmembrane region.(A) Schematic representation of the topology of E2, p7 and NS2 proteins. (B) Position of the inserted alanine residues in the putative N-terminal membrane domain of NS2. Alignments of NS2 membrane domain sequences from HCV strains JFH1 (genotype 2a, accession number AB047639) and Con1 (genotype 1b, AJ238799). Amino acids are numbered with respect to NS2 and the HCV JFH1 polyprotein (top row). Second. struct., secondary structure deduced from the NMR analyses of NS2 synthetic peptides from Con1 strain [19] (Jirasko et.al. the 16th international Conference on HCV and Related Viruses, Nice, October 3–7, 2009); c = coil, h = helix; capital letters indicate canonical helix structure. Predicted TM, consensus transmembrane (TM) segment predictions were deduced from a set of 6 available web-based algorithms prediction methods (DAS, TOPPRED2, TMHMM, SOSUI, TMPRED, PHD-TM) and represented by stretches of “T”. Arrows indicate the positions of the various alanine insertions. (C) Ribbon representations of the molecular homology model of NS2[1–27] of JFH1 (left) and the theoretical model for alanine insertion mutant A16 (right). An Ala insertion (shown in magenta) twists the helix by 110°. The N-terminal part of the model is shown in the same orientation as in the left model to highlight the distortion of residue positions on the C-terminal part of the helix. The side chains of indicated residues are shown to highlight this distortion. These models were constructed by using the NMR structure of Con1 NS2[1–27] ([19]; PDB entry, 2JY0) as template and the Swiss-PdbViewer program (http://www.expasy.ch/swissmod/). Residues are colored based on the chemical properties of their side chains: hydrophobic (gray) and polar (yellow). Acidic (Asp) and basic (Arg, Lys) residues are red and blue, respectively. His is cyan, and Gly is light gray. The membrane interfaces and hydrophobic core are schematically represented.
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ppat-1001278-g001: Rationale for mutagenesis in NS2 transmembrane region.(A) Schematic representation of the topology of E2, p7 and NS2 proteins. (B) Position of the inserted alanine residues in the putative N-terminal membrane domain of NS2. Alignments of NS2 membrane domain sequences from HCV strains JFH1 (genotype 2a, accession number AB047639) and Con1 (genotype 1b, AJ238799). Amino acids are numbered with respect to NS2 and the HCV JFH1 polyprotein (top row). Second. struct., secondary structure deduced from the NMR analyses of NS2 synthetic peptides from Con1 strain [19] (Jirasko et.al. the 16th international Conference on HCV and Related Viruses, Nice, October 3–7, 2009); c = coil, h = helix; capital letters indicate canonical helix structure. Predicted TM, consensus transmembrane (TM) segment predictions were deduced from a set of 6 available web-based algorithms prediction methods (DAS, TOPPRED2, TMHMM, SOSUI, TMPRED, PHD-TM) and represented by stretches of “T”. Arrows indicate the positions of the various alanine insertions. (C) Ribbon representations of the molecular homology model of NS2[1–27] of JFH1 (left) and the theoretical model for alanine insertion mutant A16 (right). An Ala insertion (shown in magenta) twists the helix by 110°. The N-terminal part of the model is shown in the same orientation as in the left model to highlight the distortion of residue positions on the C-terminal part of the helix. The side chains of indicated residues are shown to highlight this distortion. These models were constructed by using the NMR structure of Con1 NS2[1–27] ([19]; PDB entry, 2JY0) as template and the Swiss-PdbViewer program (http://www.expasy.ch/swissmod/). Residues are colored based on the chemical properties of their side chains: hydrophobic (gray) and polar (yellow). Acidic (Asp) and basic (Arg, Lys) residues are red and blue, respectively. His is cyan, and Gly is light gray. The membrane interfaces and hydrophobic core are schematically represented.

Mentions: NS2 is a polytopic transmembrane protein containing 3 putative transmembrane segments [19](Figure 1). The p7 polypeptide and E1E2 heterodimer, which are putative partners of NS2, are also membrane proteins that contain transmembrane segments [24], [25]. Due to their respective topologies (Figure 1A), it is expected that interactions between these three proteins would involve helix-helix contacts in their transmembrane segments. Furthermore, helix-helix interactions between transmembrane segments of NS2 are also likely to take place.


NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly.

Popescu CI, Callens N, Trinel D, Roingeard P, Moradpour D, Descamps V, Duverlie G, Penin F, Héliot L, Rouillé Y, Dubuisson J - PLoS Pathog. (2011)

Rationale for mutagenesis in NS2 transmembrane region.(A) Schematic representation of the topology of E2, p7 and NS2 proteins. (B) Position of the inserted alanine residues in the putative N-terminal membrane domain of NS2. Alignments of NS2 membrane domain sequences from HCV strains JFH1 (genotype 2a, accession number AB047639) and Con1 (genotype 1b, AJ238799). Amino acids are numbered with respect to NS2 and the HCV JFH1 polyprotein (top row). Second. struct., secondary structure deduced from the NMR analyses of NS2 synthetic peptides from Con1 strain [19] (Jirasko et.al. the 16th international Conference on HCV and Related Viruses, Nice, October 3–7, 2009); c = coil, h = helix; capital letters indicate canonical helix structure. Predicted TM, consensus transmembrane (TM) segment predictions were deduced from a set of 6 available web-based algorithms prediction methods (DAS, TOPPRED2, TMHMM, SOSUI, TMPRED, PHD-TM) and represented by stretches of “T”. Arrows indicate the positions of the various alanine insertions. (C) Ribbon representations of the molecular homology model of NS2[1–27] of JFH1 (left) and the theoretical model for alanine insertion mutant A16 (right). An Ala insertion (shown in magenta) twists the helix by 110°. The N-terminal part of the model is shown in the same orientation as in the left model to highlight the distortion of residue positions on the C-terminal part of the helix. The side chains of indicated residues are shown to highlight this distortion. These models were constructed by using the NMR structure of Con1 NS2[1–27] ([19]; PDB entry, 2JY0) as template and the Swiss-PdbViewer program (http://www.expasy.ch/swissmod/). Residues are colored based on the chemical properties of their side chains: hydrophobic (gray) and polar (yellow). Acidic (Asp) and basic (Arg, Lys) residues are red and blue, respectively. His is cyan, and Gly is light gray. The membrane interfaces and hydrophobic core are schematically represented.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037360&req=5

ppat-1001278-g001: Rationale for mutagenesis in NS2 transmembrane region.(A) Schematic representation of the topology of E2, p7 and NS2 proteins. (B) Position of the inserted alanine residues in the putative N-terminal membrane domain of NS2. Alignments of NS2 membrane domain sequences from HCV strains JFH1 (genotype 2a, accession number AB047639) and Con1 (genotype 1b, AJ238799). Amino acids are numbered with respect to NS2 and the HCV JFH1 polyprotein (top row). Second. struct., secondary structure deduced from the NMR analyses of NS2 synthetic peptides from Con1 strain [19] (Jirasko et.al. the 16th international Conference on HCV and Related Viruses, Nice, October 3–7, 2009); c = coil, h = helix; capital letters indicate canonical helix structure. Predicted TM, consensus transmembrane (TM) segment predictions were deduced from a set of 6 available web-based algorithms prediction methods (DAS, TOPPRED2, TMHMM, SOSUI, TMPRED, PHD-TM) and represented by stretches of “T”. Arrows indicate the positions of the various alanine insertions. (C) Ribbon representations of the molecular homology model of NS2[1–27] of JFH1 (left) and the theoretical model for alanine insertion mutant A16 (right). An Ala insertion (shown in magenta) twists the helix by 110°. The N-terminal part of the model is shown in the same orientation as in the left model to highlight the distortion of residue positions on the C-terminal part of the helix. The side chains of indicated residues are shown to highlight this distortion. These models were constructed by using the NMR structure of Con1 NS2[1–27] ([19]; PDB entry, 2JY0) as template and the Swiss-PdbViewer program (http://www.expasy.ch/swissmod/). Residues are colored based on the chemical properties of their side chains: hydrophobic (gray) and polar (yellow). Acidic (Asp) and basic (Arg, Lys) residues are red and blue, respectively. His is cyan, and Gly is light gray. The membrane interfaces and hydrophobic core are schematically represented.
Mentions: NS2 is a polytopic transmembrane protein containing 3 putative transmembrane segments [19](Figure 1). The p7 polypeptide and E1E2 heterodimer, which are putative partners of NS2, are also membrane proteins that contain transmembrane segments [24], [25]. Due to their respective topologies (Figure 1A), it is expected that interactions between these three proteins would involve helix-helix contacts in their transmembrane segments. Furthermore, helix-helix interactions between transmembrane segments of NS2 are also likely to take place.

Bottom Line: Our data demonstrate molecular interactions between NS2 and p7 and E2.We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization.Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein.

View Article: PubMed Central - PubMed

Affiliation: Inserm U1019, CNRS UMR8204, Center for Infection & Immunity of Lille (CIIL), Institut Pasteur de Lille, Université Lille Nord de France, Lille, France.

ABSTRACT
Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.

Show MeSH
Related in: MedlinePlus