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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

Model of NS2 role in the assembly process.Upon viral polyprotein translation and processing, three viral modules are formed: the core protein (C), the replication complex (RC) and the E1E2p7NS2 complex. E1E2p7NS2 complex assembles through the interaction of E1E2 heterodimer and p7NS2 unit (4) and migrates close to the RC independently of core protein due to signals present in p7NS2 and E2 (3). The core protein localizes around the LDs (1) where it recruits the RC by core-NS5A interaction (2).
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ppat-1001278-g010: Model of NS2 role in the assembly process.Upon viral polyprotein translation and processing, three viral modules are formed: the core protein (C), the replication complex (RC) and the E1E2p7NS2 complex. E1E2p7NS2 complex assembles through the interaction of E1E2 heterodimer and p7NS2 unit (4) and migrates close to the RC independently of core protein due to signals present in p7NS2 and E2 (3). The core protein localizes around the LDs (1) where it recruits the RC by core-NS5A interaction (2).

Mentions: In our current view, the assembly process would involve several steps. Upon viral genome translation and polyprotein processing, formation of different complexes occurs: the E1E2 native heterodimer, the p7NS2 unit and the RC. The core protein and other viral proteins (e.g. NS4B) create the LD-ER microenvironment by redistribution of the LDs and intracellular membranes. The LDs surrounded by core protein are recruited to the RC. E1E2 complex interacts with p7NS2 unit and E1E2p7NS2 arrives to NS5A positive membranes in the proximity of LDs due to a combination of signals in p7, NS2 and E2 proteins. NS5A switches from the replication to assembly mode by phosphorylation, which stabilizes the presence of NS2 in dotted structures favoring the assembly process (Figure 10). Finally, our data indicate a crucial role played by NS2 in the assembly process and highlight the complexity of the mechanism of its action. In conclusion, NS2 emerges as an essential mediator between the structural and non-structural proteins in HCV assembly process.


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)

Model of NS2 role in the assembly process.Upon viral polyprotein translation and processing, three viral modules are formed: the core protein (C), the replication complex (RC) and the E1E2p7NS2 complex. E1E2p7NS2 complex assembles through the interaction of E1E2 heterodimer and p7NS2 unit (4) and migrates close to the RC independently of core protein due to signals present in p7NS2 and E2 (3). The core protein localizes around the LDs (1) where it recruits the RC by core-NS5A interaction (2).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001278-g010: Model of NS2 role in the assembly process.Upon viral polyprotein translation and processing, three viral modules are formed: the core protein (C), the replication complex (RC) and the E1E2p7NS2 complex. E1E2p7NS2 complex assembles through the interaction of E1E2 heterodimer and p7NS2 unit (4) and migrates close to the RC independently of core protein due to signals present in p7NS2 and E2 (3). The core protein localizes around the LDs (1) where it recruits the RC by core-NS5A interaction (2).
Mentions: In our current view, the assembly process would involve several steps. Upon viral genome translation and polyprotein processing, formation of different complexes occurs: the E1E2 native heterodimer, the p7NS2 unit and the RC. The core protein and other viral proteins (e.g. NS4B) create the LD-ER microenvironment by redistribution of the LDs and intracellular membranes. The LDs surrounded by core protein are recruited to the RC. E1E2 complex interacts with p7NS2 unit and E1E2p7NS2 arrives to NS5A positive membranes in the proximity of LDs due to a combination of signals in p7, NS2 and E2 proteins. NS5A switches from the replication to assembly mode by phosphorylation, which stabilizes the presence of NS2 in dotted structures favoring the assembly process (Figure 10). Finally, our data indicate a crucial role played by NS2 in the assembly process and highlight the complexity of the mechanism of its action. In conclusion, NS2 emerges as an essential mediator between the structural and non-structural proteins in HCV assembly process.

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