Role of nonstructural protein 2 (NS2) for replication and assembly of infectious hepatits C virus
Final Report Abstract
The major aim of this project was the characterization of the hepatitis C virus (HCV) nonstructural (NS) protein 2 with respect to its role in formation of infectious virus particles. This protein is composed of a highly hydrophobic N-terminal transmembrane domain and a cytosolic C-terminal domain containing a cysteine protease that is responsible for liberation of the C-terminus of NS2 from the viral polyprotein precursor. During the second funding period of this project we pursued two subprojects. The first aimed to unravel the structure of the N-terminal NS2 trans-membrane domain. We were able to determine the NMR structure of this domain and established a model for full length NS2 with respect to its membrane topology. By using intensive reverse and forward genetic approaches, we established a detailed genetic map of NS2 and demonstrated that this protein is a central organizer of the HCV assembly process by pulling together the viral replicase with the envelope glycoproteins via a p7-containing complex. This multi-protein complex likely coordinates the different steps of HCV assembly. In the second subproject we determined the proteome of NS2-containing complexes by using a fully functional HCV genome that contains a tandem-tagged NS2. We used mass spectrometry and identified several NS2 interactants that were validated by pull-down assays. In addition, we probed the relevance of identified NS2 interactants by using RNA interference-mediated knockdown approaches. We identified one host cell factor restricting HCV assembly by a NS2 interaction-dependent manner. Currently we determine the molecular mechanism by which this host cell factor affects formation of infectious HCV particles.
Publications
- 2009. Cyclosporine A inhibits hepatitis C virus nonstructural protein 2 through cyclophilin A. Hepatology 50:1638-45
Ciesek S, Steinmann E, Wedemeyer H, Manns MP, Neyts J, Tautz N, Madan V, Bartenschlager R, von Hahn T, Pietschmann T
- 2009. Investigation of a role for lysine residues in non-structural proteins 2 and 2/3 of the hepatitis C virus for their degradation and virus assembly. J Gen Virol. 90:1071-80
Welbourn S., Jirasko V., Breton V., Reiss S., Penin F., Bartenschlager R., Pause A.
- 2010. DEB025 (Alisporivir) inhibits hepatitis C virus replication by preventing a cyclophilin A induced cis-trans isomerisation in domain II of NS5A. PLoS One. 5(10):e13687
Coelmont L, Hanoulle X, Chatterji U, Berger C, Snoeck J, Bobardt M, Lim P, Vliegen I, Paeshuyse J, Vuagniaux G, Vandamme AM, Bartenschlager R, Gallay P, Lippens G, Neyts J
- 2010. Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key role as organizer of virion assembly. PLoS Pathog. 16;6(12):e1001233
Jirasko V, Montserret R, Lee JY, Gouttenoire J, Moradpour D, Penin F, Bartenschlager R
- 2011. A concerted action of hepatitis C virus p7 and nonstructural protein 2 regulates core localization at the endoplasmic reticulum and virus assembly. PLoS Pathog. 7(7):e1002144
Boson B, Granio O, Bartenschlager R, Cosset FL
- 2012. MAP-kinase regulated cytosolic phospholipase A2 activity is essential for production of infectious hepatitis C virus particles. PLoS Pathog. 8(7):e1002829
Menzel N, Fischl W, Hueging K, Bankwitz D, Frentzen A, Haid S, Gentzsch J, Kaderali L, Bartenschlager R, Pietschmann T
(See online at https://doi.org/10.1371/journal.ppat.1002829) - 2012. Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLoS Pathog. 8(12):e1003056
Romero-Brey I, Merz A, Chiramel A, Lee JY, Chlanda P, Haselman U, Santarella- Mellwig R, Habermann A, Hoppe S, Kallis S, Walther P, Antony C, Krijnse-Locker J, Bartenschlager R
(See online at https://doi.org/10.1371/journal.ppat.1003056)
