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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 FRET-FLIM analyses.(A) Schematic representation of the constructs used in this study. (B) Immunofluorescence analysis of the co-expression of CFP-p7 and YFP-NS2. U2OS cells were co-transfected with plasmids expressing CFP-p7 and YFP-NS2. At 24h post-transfection, the subcellular localization of the different proteins was assessed by confocal microscopy. (C) Western blot analysis of the expression of CFP-p7, YFP-NS2 and CFP-EYF. U2OS cells were transfected with plasmids expressing CFP-p7, YFP-NS2 or CFP-EYF. At 24h post-transfection, cells were lysed and protein expression was confirmed by SDS-PAGE followed by Western blotting. (D) FLIM analyses. Samples were subjected to FLIM and color coded maps were obtained. The regions where the FRET events are present are marked with squares. The colors represent the progression from minimum (yellow) to maximum (blue) fluorescence lifetime.
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ppat-1001278-g008: NS2 and p7 interact in FRET-FLIM analyses.(A) Schematic representation of the constructs used in this study. (B) Immunofluorescence analysis of the co-expression of CFP-p7 and YFP-NS2. U2OS cells were co-transfected with plasmids expressing CFP-p7 and YFP-NS2. At 24h post-transfection, the subcellular localization of the different proteins was assessed by confocal microscopy. (C) Western blot analysis of the expression of CFP-p7, YFP-NS2 and CFP-EYF. U2OS cells were transfected with plasmids expressing CFP-p7, YFP-NS2 or CFP-EYF. At 24h post-transfection, cells were lysed and protein expression was confirmed by SDS-PAGE followed by Western blotting. (D) FLIM analyses. Samples were subjected to FLIM and color coded maps were obtained. The regions where the FRET events are present are marked with squares. The colors represent the progression from minimum (yellow) to maximum (blue) fluorescence lifetime.

Mentions: To confirm the p7-NS2 interaction with another approach, we used the FRET-FLIM technique. FRET-FLIM requires the presence of two fluorophores (a donor and an acceptor) fused in frame to the studied proteins. If the two proteins interact, an energy transfer occurs between the two fluorophores and the fluorescence life time of the donor (a parameter of the energy emitted by the donor) will decrease. To measure p7-NS2 interactions by FRET-FLIM, Cerulean fluorescent protein (CFP) and Venus yellow fluorescent protein (YFP) were fused to the N-terminus of p7 and NS2, respectively (Figure 8A).


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 FRET-FLIM analyses.(A) Schematic representation of the constructs used in this study. (B) Immunofluorescence analysis of the co-expression of CFP-p7 and YFP-NS2. U2OS cells were co-transfected with plasmids expressing CFP-p7 and YFP-NS2. At 24h post-transfection, the subcellular localization of the different proteins was assessed by confocal microscopy. (C) Western blot analysis of the expression of CFP-p7, YFP-NS2 and CFP-EYF. U2OS cells were transfected with plasmids expressing CFP-p7, YFP-NS2 or CFP-EYF. At 24h post-transfection, cells were lysed and protein expression was confirmed by SDS-PAGE followed by Western blotting. (D) FLIM analyses. Samples were subjected to FLIM and color coded maps were obtained. The regions where the FRET events are present are marked with squares. The colors represent the progression from minimum (yellow) to maximum (blue) fluorescence lifetime.
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Related In: Results  -  Collection

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

ppat-1001278-g008: NS2 and p7 interact in FRET-FLIM analyses.(A) Schematic representation of the constructs used in this study. (B) Immunofluorescence analysis of the co-expression of CFP-p7 and YFP-NS2. U2OS cells were co-transfected with plasmids expressing CFP-p7 and YFP-NS2. At 24h post-transfection, the subcellular localization of the different proteins was assessed by confocal microscopy. (C) Western blot analysis of the expression of CFP-p7, YFP-NS2 and CFP-EYF. U2OS cells were transfected with plasmids expressing CFP-p7, YFP-NS2 or CFP-EYF. At 24h post-transfection, cells were lysed and protein expression was confirmed by SDS-PAGE followed by Western blotting. (D) FLIM analyses. Samples were subjected to FLIM and color coded maps were obtained. The regions where the FRET events are present are marked with squares. The colors represent the progression from minimum (yellow) to maximum (blue) fluorescence lifetime.
Mentions: To confirm the p7-NS2 interaction with another approach, we used the FRET-FLIM technique. FRET-FLIM requires the presence of two fluorophores (a donor and an acceptor) fused in frame to the studied proteins. If the two proteins interact, an energy transfer occurs between the two fluorophores and the fluorescence life time of the donor (a parameter of the energy emitted by the donor) will decrease. To measure p7-NS2 interactions by FRET-FLIM, Cerulean fluorescent protein (CFP) and Venus yellow fluorescent protein (YFP) were fused to the N-terminus of p7 and NS2, respectively (Figure 8A).

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