Regulation of hepatitis C virus RNA synthesis by viral nonstructural proteins and an essential host factor
Final Report Abstract
Hepatitis C virus (HCV) is a positive strand RNA virus belonging to the family of Flaviviridae. Despite the availability of cell culture models allowing the analysis of HCV replication, still little is known about the regulation of RNA synthesis by viral and cellular factors. During the first funding period we have identified critical determinants in the polymerase NS5B of HCV strain JFH1 governing highly efficient replication. We furthermore laid the ground for an in vitro model of viral RNA synthesis based on purified components. Finally we gained insight into the essential role of a lipid kinase (PI4KIIIα) in viral replication and discovered that this cellular enzyme is activated by nonstructural protein NS5A. In the continuation grant we focused on the mechanistic role of viral and host proteins in HCV RNA replication and on an in depth characterization of the mechanisms governing PI4KIIIα interactions with HCV proteins. We aimed to use purified nonstructural protein complexes to dissect the function of nonstructural proteins and cis acting replication elements in the initiation of viral negative strand synthesis. While we found stimulating effects of the viral helicase NS3 and NS5A on the polymerase activity of NS5B, several subaims could not be achieved due to technical challenges and lack of robustness of the model. Therefore, we put more efforts into the characterization of PI4KIIIα. PI4KIIIα converts phosphatidylinositol to phosphatidylinositol 4-phosphate (PI4P) at the endoplasmic reticulum (ER) and at the plasma membrane. The enzymatic activity of PI4KIIIα is critically involved in the formation of viral induced membrane alterations harboring the sites of viral genome replication. We have shown that the viral nonstructural proteins NS5A and NS5B recruit PI4KIIIα to the HCV replication sites and activate the lipid kinase, resulting in strongly elevated intracellular PI4P levels. We have identified the PI4KIIIα functional interaction site in NS5A and mapped critical domains in PI4KIIIα required for HCV replication. We furthermore found an unexpected impact of PI4KIIIα on phosphorylation of NS5A and identified several novel phosphorylation sites in this important regulatory viral protein. Our work thereby provided insights into the complex interplay of viral and cellular proteins governing HCV RNA synthesis. It furthermore helped to define the membranous HCV replication compartment as a viral achilles’ heel for therapeutic interventions that can be targeted by diverse classes of direct acting antivirals as well as host targeting antivirals with clinical proof of concept.
Publications
- 2009. Structural and functional analysis of hepatitis C virus strain JFH1 polymerase. J. Virol. 83:11926-11939
Simister, P., M. Schmitt, M. Geitmann, O. Wicht, U. H. Danielson, R. Klein, S. Bressanelli, and V. Lohmann
- 2010. Identification of hepatoprotective flavonolignans from silymarin. Proc. Natl. Acad. Sci. USA 107:5995-5999
Polyak, S. J., C. Morishima, V. Lohmann, S. Pal, D. Y. Lee, Y. Liu, T. N. Graf, and N. H. Oberlies
- 2010. Multiple effects of silymarin on the hepatitis C virus lifecycle. Hepatology 51: 1912-1921
Wagoner J, Negash A, Kane OJ, Martinez LE, Nahmias Y, Bourne N, Owen DM, Grove J, Brimacombe C, McKeating JA, Pecheur EI, Graf TN, Oberlies NH, Lohmann V, Cao F, Tavis JE, Polyak SJ
- 2010. Role of annexin A2 in the production of infectious hepatitis C virus particles. J. Virol. 84: 5775-5789
Backes, P., D. Quinkert, S. Reiss, M. Binder, M. Zayas, U. Rescher, V. Gerke, R. Bartenschlager, and V. Lohmann
- 2011. A Comprehensive Structure-Function Comparison of Hepatitis C Virus Strain JFH1 and J6 Polymerases Reveals a Key Residue Stimulating Replication in Cell Culture across Genotypes. J Virol. 85: 2565-2581
Schmitt M, Scrima N, Radujkovic D, Caillet-Saguy C, Simister PC, Friebe P, Wicht O, Klein R, Bartenschlager R, Lohmann V, Bressanelli S
- 2011. Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment. Cell Host Microbe 9: 32-45
Reiss S, Rebhan I, Backes P, Romero-Brey I, Erfle H, Matula P, Kaderali L, Poenisch M, Blankenburg H, Hiet MS, Longerich T, Diehl S, Ramirez F, Balla T, Rohr K, Kaul A, Buhler S, Pepperkok R, Lengauer T, Albrecht M, Eils R, Schirmacher P, Lohmann V, Bartenschlager R
- 2013. Analysis of hepatitis C virus resistance to silibinin in vitro and in vivo points to a novel mechanism involving nonstructural protein 4B. Hepatology 57:953-963
Esser-Nobis, K., I. Romero-Brey, T. M. Ganten, J. Gouttenoire, C. Harak, R. Klein, P. Schemmer, M. Binder, P. Schnitzler, D. Moradpour, R. Bartenschlager, S. J. Polyak, W. Stremmel, F. Penin, C. Eisenbach, and V. Lohmann
(See online at https://doi.org/10.1002/hep.26260) - 2013. Hepatitis C virus RNA replication. Curr. Top. Microbiol. Immunol. 369:167-198
Lohmann V
- 2013. The Lipid Kinase Phosphatidylinositol-4 Kinase III Alpha Regulates the Phosphorylation Status of Hepatitis C Virus NS5A. PLoS Pathog. 9:e1003359
Reiss S, Harak C, Romero-Brey I, Radujkovic D, Klein R, Ruggieri A, Rebhan I, Bartenschlager R, Lohmann V
(See online at https://doi.org/10.1371/journal.ppat.1003359) - 2014. Daclatasvir-like Inhibitors of NS5A Block Early Biogenesis of HCV-induced Membranous Replication Factories, Independent of RNA Replication. Gastroenterology. 147:1094-110
Berger C, Romero-Brey I, Radujkovic D, Terreux R, Zayas M, Paul D, Harak C, Hoppe S, Gao M, Penin F, Lohmann V, Bartenschlager R
(See online at https://doi.org/10.1053/j.gastro.2014.07.019) - 2014. Mapping of functional domains of the lipid kinase phosphatidylinositol 4-kinase type III alpha involved in enzymatic activity and hepatitis C virus replication. J Virol. 88(17):9909-26
Harak C, Radujkovic D, Taveneau C, Reiss S, Klein R, Bressanelli S, Lohmann V
(See online at https://doi.org/10.1128/JVI.01063-14) - 2015. Novel perspectives for hepatitis A virus therapy revealed by comparative analysis of hepatitis C virus and hepatitis A virus RNA replication. Hepatology. 62:397-408
Esser-Nobis K, Harak C, Schult P, Kusov Y, Lohmann V
(See online at https://doi.org/10.1002/hep.27847)
