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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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Related in: MedlinePlus

Subcellular localization of NS2.(A) NS2 accumulates in dotted structures. Huh-7 cells electroporated with JFH-HA RNA were grown on coverslips and fixed at 48h and 72h post-electroporation. The subcellular localization of HA-NS2 was analyzed by immunofluorescence using an anti-HA antibody. (B) Colocalization of NS2 dotted structures with cellular and viral markers. JFH-HA electroporated cells grown on coverslips were fixed at 72h post-electroporation and processed for double-label immunofluorescence for HA-NS2 (red) and the ER marker calreticulin (CRT in green) or the LDs stained with BODIPY 493/503 (green). JFH-HA electroporated cells were further stained for HA-NS2 (red) and HCV proteins NS3 or E2 (green). The nuclei were stained with DAPI. Representative confocal images of individual cells are shown in grey and the merge images in color. Zoomed views of the indicated areas are shown in the right column. Bar, 10 µm.
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ppat-1001278-g003: Subcellular localization of NS2.(A) NS2 accumulates in dotted structures. Huh-7 cells electroporated with JFH-HA RNA were grown on coverslips and fixed at 48h and 72h post-electroporation. The subcellular localization of HA-NS2 was analyzed by immunofluorescence using an anti-HA antibody. (B) Colocalization of NS2 dotted structures with cellular and viral markers. JFH-HA electroporated cells grown on coverslips were fixed at 72h post-electroporation and processed for double-label immunofluorescence for HA-NS2 (red) and the ER marker calreticulin (CRT in green) or the LDs stained with BODIPY 493/503 (green). JFH-HA electroporated cells were further stained for HA-NS2 (red) and HCV proteins NS3 or E2 (green). The nuclei were stained with DAPI. Representative confocal images of individual cells are shown in grey and the merge images in color. Zoomed views of the indicated areas are shown in the right column. Bar, 10 µm.

Mentions: To obtain more insight into the assembly defects of our viral mutants, we decided to determine their potential effect on the subcellular localization of NS2. To this aim, we first characterized the subcellular localization of the wild-type NS2 in the context of an infectious virus, which has never been reported before. NS2 presented an ER-like reticulated pattern, and in some cells, accumulated in dotted structures (Figure 3A and data not shown). The number of these NS2-positive dot-like structures increased over time, suggesting a transition from an initial reticulate pattern to these dotted structures. At 72 hours post-electroporation, 34±15% of infected cells from 8 different electroporations displayed NS2 dots. The size of these structures was 0.84±0.38 µm (mean±SD, n = 402).


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.(A) NS2 accumulates in dotted structures. Huh-7 cells electroporated with JFH-HA RNA were grown on coverslips and fixed at 48h and 72h post-electroporation. The subcellular localization of HA-NS2 was analyzed by immunofluorescence using an anti-HA antibody. (B) Colocalization of NS2 dotted structures with cellular and viral markers. JFH-HA electroporated cells grown on coverslips were fixed at 72h post-electroporation and processed for double-label immunofluorescence for HA-NS2 (red) and the ER marker calreticulin (CRT in green) or the LDs stained with BODIPY 493/503 (green). JFH-HA electroporated cells were further stained for HA-NS2 (red) and HCV proteins NS3 or E2 (green). The nuclei were stained with DAPI. Representative confocal images of individual cells are shown in grey and the merge images in color. Zoomed views of the indicated areas are shown in the right column. Bar, 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001278-g003: Subcellular localization of NS2.(A) NS2 accumulates in dotted structures. Huh-7 cells electroporated with JFH-HA RNA were grown on coverslips and fixed at 48h and 72h post-electroporation. The subcellular localization of HA-NS2 was analyzed by immunofluorescence using an anti-HA antibody. (B) Colocalization of NS2 dotted structures with cellular and viral markers. JFH-HA electroporated cells grown on coverslips were fixed at 72h post-electroporation and processed for double-label immunofluorescence for HA-NS2 (red) and the ER marker calreticulin (CRT in green) or the LDs stained with BODIPY 493/503 (green). JFH-HA electroporated cells were further stained for HA-NS2 (red) and HCV proteins NS3 or E2 (green). The nuclei were stained with DAPI. Representative confocal images of individual cells are shown in grey and the merge images in color. Zoomed views of the indicated areas are shown in the right column. Bar, 10 µm.
Mentions: To obtain more insight into the assembly defects of our viral mutants, we decided to determine their potential effect on the subcellular localization of NS2. To this aim, we first characterized the subcellular localization of the wild-type NS2 in the context of an infectious virus, which has never been reported before. NS2 presented an ER-like reticulated pattern, and in some cells, accumulated in dotted structures (Figure 3A and data not shown). The number of these NS2-positive dot-like structures increased over time, suggesting a transition from an initial reticulate pattern to these dotted structures. At 72 hours post-electroporation, 34±15% of infected cells from 8 different electroporations displayed NS2 dots. The size of these structures was 0.84±0.38 µm (mean±SD, n = 402).

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