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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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NS2 and p7 interact in a co-immunoprecipitation assay.(A) Schematic representation of the constructs used in this study. NS2-GFP corresponds to the transmembrane domain of NS2 in fusion with GFP, whereas NS2-GTM corresponds to the transmembrane domain of VSV-G protein in fusion with the cytosolic domain of NS2. All the proteins contain a HA tag at their N-terminus. (B) HA-NS2 co-immunoprecipitates with p7-Flag. 293T cells were transfected with plasmids expressing p7-Flag, HA-NS2 from different genotypes, HA-NS2 mutants or control plasmids. At 24h post-transfection cells were lysed and immunoprecipitations with an anti-Flag antibody were performed. The immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with an anti-HA antibody to identify the presence of co-immunoprecipitated HA-NS2. The presence of HA-NS2 in transfected cells was confirmed by Western blotting. The actin content was also analyzed to verify that equal amounts of cell lysates have been used.
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ppat-1001278-g007: NS2 and p7 interact in a co-immunoprecipitation assay.(A) Schematic representation of the constructs used in this study. NS2-GFP corresponds to the transmembrane domain of NS2 in fusion with GFP, whereas NS2-GTM corresponds to the transmembrane domain of VSV-G protein in fusion with the cytosolic domain of NS2. All the proteins contain a HA tag at their N-terminus. (B) HA-NS2 co-immunoprecipitates with p7-Flag. 293T cells were transfected with plasmids expressing p7-Flag, HA-NS2 from different genotypes, HA-NS2 mutants or control plasmids. At 24h post-transfection cells were lysed and immunoprecipitations with an anti-Flag antibody were performed. The immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with an anti-HA antibody to identify the presence of co-immunoprecipitated HA-NS2. The presence of HA-NS2 in transfected cells was confirmed by Western blotting. The actin content was also analyzed to verify that equal amounts of cell lysates have been used.

Mentions: The above data suggest a possible interaction between p7 and NS2. We therefore explored this putative interaction in a biochemical assay, by analyzing p7-NS2 association in a co-immunoprecipitation assay. Due to the difficulties in analyzing p7-NS2 interactions in the context of an infectious virus, we analyzed these interactions by co-transfecting cells with plasmids expressing these two proteins only. In this approach, the p7 polypeptide and NS2 were tagged with a Flag or a HA epitope, respectively (Figure 7A, p7-Flag and HA-NS2).


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)

NS2 and p7 interact in a co-immunoprecipitation assay.(A) Schematic representation of the constructs used in this study. NS2-GFP corresponds to the transmembrane domain of NS2 in fusion with GFP, whereas NS2-GTM corresponds to the transmembrane domain of VSV-G protein in fusion with the cytosolic domain of NS2. All the proteins contain a HA tag at their N-terminus. (B) HA-NS2 co-immunoprecipitates with p7-Flag. 293T cells were transfected with plasmids expressing p7-Flag, HA-NS2 from different genotypes, HA-NS2 mutants or control plasmids. At 24h post-transfection cells were lysed and immunoprecipitations with an anti-Flag antibody were performed. The immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with an anti-HA antibody to identify the presence of co-immunoprecipitated HA-NS2. The presence of HA-NS2 in transfected cells was confirmed by Western blotting. The actin content was also analyzed to verify that equal amounts of cell lysates have been used.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037360&req=5

ppat-1001278-g007: NS2 and p7 interact in a co-immunoprecipitation assay.(A) Schematic representation of the constructs used in this study. NS2-GFP corresponds to the transmembrane domain of NS2 in fusion with GFP, whereas NS2-GTM corresponds to the transmembrane domain of VSV-G protein in fusion with the cytosolic domain of NS2. All the proteins contain a HA tag at their N-terminus. (B) HA-NS2 co-immunoprecipitates with p7-Flag. 293T cells were transfected with plasmids expressing p7-Flag, HA-NS2 from different genotypes, HA-NS2 mutants or control plasmids. At 24h post-transfection cells were lysed and immunoprecipitations with an anti-Flag antibody were performed. The immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with an anti-HA antibody to identify the presence of co-immunoprecipitated HA-NS2. The presence of HA-NS2 in transfected cells was confirmed by Western blotting. The actin content was also analyzed to verify that equal amounts of cell lysates have been used.
Mentions: The above data suggest a possible interaction between p7 and NS2. We therefore explored this putative interaction in a biochemical assay, by analyzing p7-NS2 association in a co-immunoprecipitation assay. Due to the difficulties in analyzing p7-NS2 interactions in the context of an infectious virus, we analyzed these interactions by co-transfecting cells with plasmids expressing these two proteins only. In this approach, the p7 polypeptide and NS2 were tagged with a Flag or a HA epitope, respectively (Figure 7A, p7-Flag and HA-NS2).

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