Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key Role as organizer of virion assembly

• Verfasst von: Jirasko, Vlastimil [VerfasserIn]
• Verfasst von: Montserret, Roland [VerfasserIn]
• Verfasst von: Lee, Ji Young [VerfasserIn]
• Verfasst von: Gouttenoire, Jérôme [VerfasserIn]
• Verfasst von: Moradpour, Darius [VerfasserIn]
• Verfasst von: Penin, Francois [VerfasserIn]
• Verfasst von: Bartenschlager, Ralf [VerfasserIn]
• Titel: Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key Role as organizer of virion assembly
• Verf.angabe: Vlastimil Jirasko, Roland Montserret, Ji Young Lee, Jérôme Gouttenoire, Darius Moradpour, Francois Penin, Ralf Bartenschlager
• E-Jahr: 2010
• Jahr: December 16, 2010
• Umfang: 22 S.
• Fussnoten: Gesehen am 28.08.2023
• Titel Quelle: Enthalten in: Public Library of SciencePLoS pathogens
• Ort Quelle: Lawrence, Kan. : PLoS, 2005
• Jahr Quelle: 2010
• Band/Heft Quelle: 6(2010), 12, Artikel-ID e1001233, Seite 1-22
• ISSN Quelle: 1553-7374
• DOI: doi:10.1371/journal.ppat.1001233
• Datenträger: Online-Ressource
• Sprache: eng
• Sach-SW: Genomics
• Sach-SW: Hepatitis C virus
• Sach-SW: Insertion mutation
• Sach-SW: Microbial mutation
• Sach-SW: Protein interactions
• Sach-SW: RNA structure
• Sach-SW: Substitution mutation
• Sach-SW: Transfection
• K10plus-PPN: 1858103606

Abstract

Non-structural protein 2 (NS2) plays an important role in hepatitis C virus (HCV) assembly, but neither the exact contribution of this protein to the assembly process nor its complete structure are known. In this study we used a combination of genetic, biochemical and structural methods to decipher the role of NS2 in infectious virus particle formation. A large panel of NS2 mutations targeting the N-terminal membrane binding region was generated. They were selected based on a membrane topology model that we established by determining the NMR structures of N-terminal NS2 transmembrane segments. Mutants affected in virion assembly, but not RNA replication, were selected for pseudoreversion in cell culture. Rescue mutations restoring virus assembly to various degrees emerged in E2, p7, NS3 and NS2 itself arguing for an interaction between these proteins. To confirm this assumption we developed a fully functional JFH1 genome expressing an N-terminally tagged NS2 demonstrating efficient pull-down of NS2 with p7, E2 and NS3 and, to a lower extent, NS5A. Several of the mutations blocking virus assembly disrupted some of these interactions that were restored to various degrees by those pseudoreversions that also restored assembly. Immunofluorescence analyses revealed a time-dependent NS2 colocalization with E2 at sites close to lipid droplets (LDs) together with NS3 and NS5A. Importantly, NS2 of a mutant defective in assembly abrogates NS2 colocalization around LDs with E2 and NS3, which is restored by a pseudoreversion in p7, whereas NS5A is recruited to LDs in an NS2-independent manner. In conclusion, our results suggest that NS2 orchestrates HCV particle formation by participation in multiple protein-protein interactions required for their recruitment to assembly sites in close proximity of LDs.