Limits...
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.

Show MeSH

Related in: MedlinePlus

Subcellular localization of NS2 mutants in TM region.(A) and (B) 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 following viruses were analyzed: JFH-HA (WT), JFH-?TM12-HA (?TM12), JFH-A16-HA (A16) and JFH-A41-HA (A41). The subcellular localization of HA-NS2 was analyzed by immunofluorescence using anti-HA (red), anti-NS5A (green) (Panel A) and anti-E2 (green) (Panel B) 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 micro µm. (C) Mutations in NS2 transmembrane region drastically decrease the number of cells presenting NS2 dotted structures. Huh-7 cells electroporated with JFH-HA RNA were grown on coverslips and fixed at different time points post-electroporation and labeled with anti-HA and anti-NS5A antibodies. Cells showing at least 3 NS2/NS5A dots were considered positive for NS2 dotted structures. The results were expressed as percentage of total counted cells. At least 250 infected cells were counted. Error bars indicate SD from at least two independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3037360&req=5

ppat-1001278-g005: Subcellular localization of NS2 mutants in TM region.(A) and (B) 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 following viruses were analyzed: JFH-HA (WT), JFH-?TM12-HA (?TM12), JFH-A16-HA (A16) and JFH-A41-HA (A41). The subcellular localization of HA-NS2 was analyzed by immunofluorescence using anti-HA (red), anti-NS5A (green) (Panel A) and anti-E2 (green) (Panel B) 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 micro µm. (C) Mutations in NS2 transmembrane region drastically decrease the number of cells presenting NS2 dotted structures. Huh-7 cells electroporated with JFH-HA RNA were grown on coverslips and fixed at different time points post-electroporation and labeled with anti-HA and anti-NS5A antibodies. Cells showing at least 3 NS2/NS5A dots were considered positive for NS2 dotted structures. The results were expressed as percentage of total counted cells. At least 250 infected cells were counted. Error bars indicate SD from at least two independent experiments.

Mentions: The next obvious step was to determine the subcellular localization of NS2 for the different mutants defective in assembly. In the case of ΔTM12, NS2 localized in confined structures which did not colocalize with NS5A and they were not associated with the core protein or LD (Figure 5, panels A and C and data not shown). Moreover, there was no colocalization between the truncated NS2 and the E2 glycoprotein (Figure 5B), which correlates with the lack of interaction between the two proteins (Figure 2C). Then, we analyzed the subcellular localization of NS2 for the alanine insertion mutants. While A16 mutant presented NS2 dotted structures as wild-type, A41 mutation induced a drastic decrease in the percentage of cells with NS2 dots (Figure 5, panels A and C). These data suggest that NS2 transmembrane region is an important localization determinant.


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 mutants in TM region.(A) and (B) 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 following viruses were analyzed: JFH-HA (WT), JFH-?TM12-HA (?TM12), JFH-A16-HA (A16) and JFH-A41-HA (A41). The subcellular localization of HA-NS2 was analyzed by immunofluorescence using anti-HA (red), anti-NS5A (green) (Panel A) and anti-E2 (green) (Panel B) 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 micro µm. (C) Mutations in NS2 transmembrane region drastically decrease the number of cells presenting NS2 dotted structures. Huh-7 cells electroporated with JFH-HA RNA were grown on coverslips and fixed at different time points post-electroporation and labeled with anti-HA and anti-NS5A antibodies. Cells showing at least 3 NS2/NS5A dots were considered positive for NS2 dotted structures. The results were expressed as percentage of total counted cells. At least 250 infected cells were counted. Error bars indicate SD from at least two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001278-g005: Subcellular localization of NS2 mutants in TM region.(A) and (B) 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 following viruses were analyzed: JFH-HA (WT), JFH-?TM12-HA (?TM12), JFH-A16-HA (A16) and JFH-A41-HA (A41). The subcellular localization of HA-NS2 was analyzed by immunofluorescence using anti-HA (red), anti-NS5A (green) (Panel A) and anti-E2 (green) (Panel B) 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 micro µm. (C) Mutations in NS2 transmembrane region drastically decrease the number of cells presenting NS2 dotted structures. Huh-7 cells electroporated with JFH-HA RNA were grown on coverslips and fixed at different time points post-electroporation and labeled with anti-HA and anti-NS5A antibodies. Cells showing at least 3 NS2/NS5A dots were considered positive for NS2 dotted structures. The results were expressed as percentage of total counted cells. At least 250 infected cells were counted. Error bars indicate SD from at least two independent experiments.
Mentions: The next obvious step was to determine the subcellular localization of NS2 for the different mutants defective in assembly. In the case of ΔTM12, NS2 localized in confined structures which did not colocalize with NS5A and they were not associated with the core protein or LD (Figure 5, panels A and C and data not shown). Moreover, there was no colocalization between the truncated NS2 and the E2 glycoprotein (Figure 5B), which correlates with the lack of interaction between the two proteins (Figure 2C). Then, we analyzed the subcellular localization of NS2 for the alanine insertion mutants. While A16 mutant presented NS2 dotted structures as wild-type, A41 mutation induced a drastic decrease in the percentage of cells with NS2 dots (Figure 5, panels A and C). These data suggest that NS2 transmembrane region is an important localization determinant.

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