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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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Subcellular localization of NS2 in assembly deficient mutants in NS5A protein.(A, B) Phenotype of the NS5A mutants. Virus infectivity (A), extra (black bars) and intracellular (light grey bars) core determination (B) were performed as described in Figure 2. The following viruses were analyzed: JFH-HA (WT), JFH-S/A-HA (S/A), JFH-3BS/A-HA (3BS/A) and JFH-S/D-HA (S/D). In addition, JFH-HA-PP (PP) was also analyzed in parallel. (C) Analysis of the expression and phosphorylation of the NS5A mutants. The hyperphosphorylated form of NS5A is indicated by an asterisk. The presence of core and NS5A was confirmed by Western blotting and the actin content was also analyzed to verify that equal amounts of cell lysates have been loaded. (D) Effect of mutations on the accumulation of NS2 in dotted structures. Huh-7 cells electroporated with JFH-HA RNA (WT) or mutant genomes were grown on coverslips and fixed at 72h post-electroporation. The subcellular localization of HA-NS2 was analyzed by immunofluorescence using anti-HA (red) and anti-NS5A (green) antibodies. The nuclei were stained with DAPI. Representative confocal images of NS2 and NS5A labelings are shown in grey, and the merge images in color. Bar, 10 µm. (E) Mutations in NS5A protein drastically decrease the number of cells presenting NS2 dotted structures. Huh-7 cells electroporated with JFH-HA RNA or the indicated mutants were grown on coverslips, fixed at 72 h post-electroporation and labeled with anti-HA and anti-NS5A antibodies. Cells showing at least 3 dots NS2/NS5A positive were considered positive for NS2 dotted structures. The results were expressed as percentage of total counted cells (at least 228). Error bars indicate SD from at least two independent experiments.
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ppat-1001278-g009: Subcellular localization of NS2 in assembly deficient mutants in NS5A protein.(A, B) Phenotype of the NS5A mutants. Virus infectivity (A), extra (black bars) and intracellular (light grey bars) core determination (B) were performed as described in Figure 2. The following viruses were analyzed: JFH-HA (WT), JFH-S/A-HA (S/A), JFH-3BS/A-HA (3BS/A) and JFH-S/D-HA (S/D). In addition, JFH-HA-PP (PP) was also analyzed in parallel. (C) Analysis of the expression and phosphorylation of the NS5A mutants. The hyperphosphorylated form of NS5A is indicated by an asterisk. The presence of core and NS5A was confirmed by Western blotting and the actin content was also analyzed to verify that equal amounts of cell lysates have been loaded. (D) Effect of mutations on the accumulation of NS2 in dotted structures. Huh-7 cells electroporated with JFH-HA RNA (WT) or mutant genomes were grown on coverslips and fixed at 72h post-electroporation. The subcellular localization of HA-NS2 was analyzed by immunofluorescence using anti-HA (red) and anti-NS5A (green) antibodies. The nuclei were stained with DAPI. Representative confocal images of NS2 and NS5A labelings are shown in grey, and the merge images in color. Bar, 10 µm. (E) Mutations in NS5A protein drastically decrease the number of cells presenting NS2 dotted structures. Huh-7 cells electroporated with JFH-HA RNA or the indicated mutants were grown on coverslips, fixed at 72 h post-electroporation and labeled with anti-HA and anti-NS5A antibodies. Cells showing at least 3 dots NS2/NS5A positive were considered positive for NS2 dotted structures. The results were expressed as percentage of total counted cells (at least 228). Error bars indicate SD from at least two independent experiments.

Mentions: Recruitment of core protein to LDs was reported to be essential for a productive assembly process [12], [13]. The proline residues 138 and 143 in domain D2 of the core protein are crucial for virus production and core recruitment to LDs [13], [41]. Furthermore, the mutation of these proline residues has been previously shown to prevent the core induced recruitment of NS5A to the LDs [12]. Therefore, we introduced these mutations in the context of JFH-HA virus (Figure 6, JFH-HA-PP). As previously reported [13], the mutation prevented the production of infectious virions (Figure 9A), and the core protein was not redistributed to LDs, which in turn remained spread in the cytoplasm rather than the perinuclear localization induced by a functional core protein (Figure S4). As shown in Figure 6, in the context of this mutation, NS2 protein maintained the capacity to accumulate in dotted structures that colocalized with NS5A. In contrast to the wild-type, NS2 dotted structures were not found in the vicinity of LDs in the context of the PP mutation, suggesting that NS2 does not have the signals to localize by itself around the LDs (Figure S4). Importantly, in this context, the number of cells presenting NS2 dotted structures increased tremendously in comparison to the wild-type (Figure 6B). These observations suggest that the PP mutation induces a block in the assembly process, which favors the accumulation of NS2 protein in the dotted structures.


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)

Subcellular localization of NS2 in assembly deficient mutants in NS5A protein.(A, B) Phenotype of the NS5A mutants. Virus infectivity (A), extra (black bars) and intracellular (light grey bars) core determination (B) were performed as described in Figure 2. The following viruses were analyzed: JFH-HA (WT), JFH-S/A-HA (S/A), JFH-3BS/A-HA (3BS/A) and JFH-S/D-HA (S/D). In addition, JFH-HA-PP (PP) was also analyzed in parallel. (C) Analysis of the expression and phosphorylation of the NS5A mutants. The hyperphosphorylated form of NS5A is indicated by an asterisk. The presence of core and NS5A was confirmed by Western blotting and the actin content was also analyzed to verify that equal amounts of cell lysates have been loaded. (D) Effect of mutations on the accumulation of NS2 in dotted structures. Huh-7 cells electroporated with JFH-HA RNA (WT) or mutant genomes were grown on coverslips and fixed at 72h post-electroporation. The subcellular localization of HA-NS2 was analyzed by immunofluorescence using anti-HA (red) and anti-NS5A (green) antibodies. The nuclei were stained with DAPI. Representative confocal images of NS2 and NS5A labelings are shown in grey, and the merge images in color. Bar, 10 µm. (E) Mutations in NS5A protein drastically decrease the number of cells presenting NS2 dotted structures. Huh-7 cells electroporated with JFH-HA RNA or the indicated mutants were grown on coverslips, fixed at 72 h post-electroporation and labeled with anti-HA and anti-NS5A antibodies. Cells showing at least 3 dots NS2/NS5A positive were considered positive for NS2 dotted structures. The results were expressed as percentage of total counted cells (at least 228). Error bars indicate SD from at least two independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037360&req=5

ppat-1001278-g009: Subcellular localization of NS2 in assembly deficient mutants in NS5A protein.(A, B) Phenotype of the NS5A mutants. Virus infectivity (A), extra (black bars) and intracellular (light grey bars) core determination (B) were performed as described in Figure 2. The following viruses were analyzed: JFH-HA (WT), JFH-S/A-HA (S/A), JFH-3BS/A-HA (3BS/A) and JFH-S/D-HA (S/D). In addition, JFH-HA-PP (PP) was also analyzed in parallel. (C) Analysis of the expression and phosphorylation of the NS5A mutants. The hyperphosphorylated form of NS5A is indicated by an asterisk. The presence of core and NS5A was confirmed by Western blotting and the actin content was also analyzed to verify that equal amounts of cell lysates have been loaded. (D) Effect of mutations on the accumulation of NS2 in dotted structures. Huh-7 cells electroporated with JFH-HA RNA (WT) or mutant genomes were grown on coverslips and fixed at 72h post-electroporation. The subcellular localization of HA-NS2 was analyzed by immunofluorescence using anti-HA (red) and anti-NS5A (green) antibodies. The nuclei were stained with DAPI. Representative confocal images of NS2 and NS5A labelings are shown in grey, and the merge images in color. Bar, 10 µm. (E) Mutations in NS5A protein drastically decrease the number of cells presenting NS2 dotted structures. Huh-7 cells electroporated with JFH-HA RNA or the indicated mutants were grown on coverslips, fixed at 72 h post-electroporation and labeled with anti-HA and anti-NS5A antibodies. Cells showing at least 3 dots NS2/NS5A positive were considered positive for NS2 dotted structures. The results were expressed as percentage of total counted cells (at least 228). Error bars indicate SD from at least two independent experiments.
Mentions: Recruitment of core protein to LDs was reported to be essential for a productive assembly process [12], [13]. The proline residues 138 and 143 in domain D2 of the core protein are crucial for virus production and core recruitment to LDs [13], [41]. Furthermore, the mutation of these proline residues has been previously shown to prevent the core induced recruitment of NS5A to the LDs [12]. Therefore, we introduced these mutations in the context of JFH-HA virus (Figure 6, JFH-HA-PP). As previously reported [13], the mutation prevented the production of infectious virions (Figure 9A), and the core protein was not redistributed to LDs, which in turn remained spread in the cytoplasm rather than the perinuclear localization induced by a functional core protein (Figure S4). As shown in Figure 6, in the context of this mutation, NS2 protein maintained the capacity to accumulate in dotted structures that colocalized with NS5A. In contrast to the wild-type, NS2 dotted structures were not found in the vicinity of LDs in the context of the PP mutation, suggesting that NS2 does not have the signals to localize by itself around the LDs (Figure S4). Importantly, in this context, the number of cells presenting NS2 dotted structures increased tremendously in comparison to the wild-type (Figure 6B). These observations suggest that the PP mutation induces a block in the assembly process, which favors the accumulation of NS2 protein in the dotted structures.

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