Hepatitis C Virus: Basics Ralf Bartenschlager AREVIR-GenaFor-Meeting Bonn April 22 - 23, 2010
HCV Particle Properties • Hepacivirus, fam. Flaviviridae • Enveloped particle RNA genome • Associated with lipoproteins • ss (+)RNA genome capsid E1/E2 lipoproteins
HCV Genome Organization (+) strand RNA genome 5´NTR 3´NTR IRES Mini- translation, processing cores C+1 ? structural non-structural proteins C E1 E2 2 3 4A 4B 5A 5B p7 C E1 E2 p7 2 3 4A 4B 5A 5B ion channel, protease/ membrane- polymerase virus particle assembly helicase vesicle protease, NS3- replication, assembly cofactor assembly ‚Assembly module‘ Replicase complex
HCV Replication Cycle LD Assembly RF RI Polyprotein processing RNA replication membranous web
Cell Culture Systems for Hepatitis C Virus HCV replicons strictly intracellular replication C E1 E2 2 3 4B 5A 5B 5‘ p7 4A 3‘ 3 4A 4B 5A 5B neo 3‘ 5‘ E-I Lohmann et al., Science 1999 • Efficient RNA replication requires replication enhancing mutations (REMs) • REMs interfere with virus production • Virus genomes without REMs release infectious virus, but replication in cell culture is too low for detailed studies Pietschmann et al., PLoS Pathogens 2009
Cell Culture Systems for Hepatitis C Virus JFH-1 C E1 E2 p7 2 3 4A 4B 5A 5B 3‘ 5‘ α – NS3 JFH1/wt Contr. 0 1 2 3 4 Infectious units (log10) Wakita, Pietschmann et al., Nat Med 2005 J6 (2a) JFH1 (GT 2a) Jc1 C E1 E2 2 3 4A 4B 5A 5B 3‘ 5‘ p7 Jc1: 10e6 TCID 50 /ml JFH-1: 10e3 TCID 50 /ml Lindenbach et al., Science, 2005; Pietschmann et al., PNAS 2006
HCV Entry into the Host Cell OCLN CLDN1 CD81 SR-BI GAGs LDLr Clathrin- coated pit Early endosome H + Fusion and genome release Burlone & Budkowska, JGV 2009
The Hepatitis C Virus Replication Complex LD Rv? LD ER nucleus Membranous Web
Comparison of Replication and Assembly Compartments in HCV- and DENV-Infected Huh7 Cells LD Rv? LD ER nucleus Membranous web Membranous replication complex in HCV -infected cell in Dengue Virus -infected cell
EM Tomography of DENV-Infected Cells Reveals a Network of Interconnected Membranes
EM Tomography of DENV-Induced Vesicles Welsch, Miller et al., Cell HM, 2009 S. Welsch A. Merz I. Romero-Brey
3D Reconstruction of the Dengue Virus Replication Complex: A Model for Hepatitis C Virus? Welsch, Miller et al., Cell HM, 2009
Double-Membrane Vesicles in DENV and HCV Replication Complexes Dengue Virus Hepatitis C Virus Ve
HCV-Induced Membranous Web is Tightly Linked With and Contains Lipid Droplets A. Merz I. Romero-Brey
Properties of Lipid Droplets Hydrophobic core (esp. TAGs, sterol esters) • cytoplasmic storage of neutral lipids PAT-proteins • functionally linked to other organelles • intracellular mobility core LDs • complex ‘proteome’ • metabolically active structures • de novo synthesis • sites of HCV core and NS5A accumulation NS5A merge • sites of HCV assembly phospholipid monolayer Moradpour et al., 1996; Barba et al., 1997; Shi et al., 2002; Sato et al., 2006, Miyanari et al., 2007; Boulant et al., 2007; Shavinskaya et al., 2007; Appel et al., 2008 N. Appel M. Zayas
Hypothetical Model for Nucleocapsid Formation and Envelopement Rv VLDL pathway LD Huang et al., 2007; Chang et al., 2007; Gastaminza et al, 2008
N-Terminal E2 Tagging Does Not Affect Assembly A. Merz G. Long
Immuno-EM Analysis of Jc1E2 Flag Particles • Average diameter 73nm • Capture with anti-apoE • Neutralization with anti-apoE • TCID50 : RNA : core : apoE 1 : 1 : ~1,000 : ~ 250
The HCV Lipidome is Unique and Resembles (V)LDL Sphingomyelin Cholesterylesters Phosphatidylcholine 1 1 1 2 ~ 45% of HCV particle lipids are cholesteryl esters problematic for lipid bilayer 1 Wiesner et al., J Lipid Res 2008; 2 Brügger et al., PNAS 2006
Tentative model of the HCV “Lipo-Viro-Particle” F. Penin, P. André
HCV has evolved numerous strategies... to exploit its host cell to escape from immunological control
www.despair.com
Strategies of HCV Persistence interference with acquired immune response control of IFN induction (NS3/4A protease) weak induction of IFN response protection of viral RNA in membranous RCs M. Binder V. Lohmann
Blockage of IFN Production by NS3/4A Protease IFN- β virus IFNAR JAK-1 / TYK-2 vRNA NS3/4A dsRNA dsRNA RIG-I Relevance in vivo? TLR3 Antiviral genes MAVS TRIF Yes for MAVS IFN- β ? For TRIF ISGF-3 ISRE ISG IFN- β Adapted from Haller et al., Virology, 2005
Block of the effector phase by HCV proteins? endogenous exogenous IFN- β IFNAR IFN- α / β JAK-1 / TYK-2 dsRNA HCV proteins X Mx ISG20 (core) X X OAS PKR STAT-1 -2 IRF-7 -3 E2, NS5A Reduction of ISG expression IRF-9 by HCV-mediated activation of PKR* IFN- α ISGF-3 IFN- β ISRE ISG *Garaigorta & Chisari, 2010
High IFN- α Sensitivity of HCV Replicons and Virus neo 4A 3‘ 5‘ 3 4B 5A 5B E-I IC 50 ~ 2-5U/ml (GT-1b, 1a, 2a) hyg 4A 3‘ 5‘ 3 4B 5A 5B IC 50 ~ 1 U/ml (GT-1b, 1a, 2a) neo p7 Do we have the right systems 4A E-I 3‘ C E1 E2 2 3 4B 5A 5B 5‘ IC 50 ~ 2-5U/ml (GT-1b, 1a, 2a) to study HCV persistence in cell culture? p7 4A 5‘ 3‘ C E1 E2 2 3 4B 5A 5B IC 50 ~ 2-5U/ml (JFH-1; GT2a) • independent from ISDR signature in NS5A • in different cell systems (Huh-7, HuH6, HeLa, Hepa1-6, 293, PHHs) • no effect on IFN-induced transcriptome
The Huh7 dif Culture System day 0 day 44 day 14 day 16 day 18 differentiation sampling DMSO HCV IFN infection 10 6 95 quantil 10 5 10 4 5 quantil TCID 50 /ml 10 3 no treatment 10 2 100 U/ml IFN- α 10 1 median 10 0 day 18 day 23 day 30 day 37 day 44 Huh-7 dif (day 14)
Properties of the Huh7 dif Culture System • Long term viremia (> 4 weeks) • Persistent infection in the face of IFN-response • Relapse of viral RNA upon IFN withdrawal • Preferential HCV replication in cells with low IFN-response Number of HCV infected cells 10 20 30 40 50 0 MxA high MxA low O. Bauhofer
Possible Strategies of HCV Persistence • infection of cells responding poorly to type 1 IFN (exploiting the ‚stochastics‘ of the IFN system) IFN refractory (HCV permissive) cells time point Y time point X • low-level inhibition of type 1 IFN-induced signalling • inhibition of (expression of) IFN-induced effectors
The Intimate HCV – Host Cell Interaction Exploit: Tight junctions Defend & Eliminate Transport system Stress response Lipid pathways Apoptosis VLDL Interferons … T-cells B-cells HCV Persist: poor RIG-I activation Antiviral therapy blocking IFN induction Host exploit stochastics? high genetic variability …
Department Infectious Diseases Molecular Virology Gualtiero Alvisi Nicole Appel* Oliver Bauhofer Marco Binder Carola Berger Abhilash Chiramel Marie-Sophie Hiet Ulrike Herian Stephanie Kallis J. Krijnse-Locker Gang Long Andreas Merz Sven Miller* Marion Pönisch Ines Romero-Brey Torsten Schaller* Ilka Wörz Margarita Zayas Volker Lohmann & Team Stephan Urban & Team
reagents & tools collaborations NIID, Tokyo T. Wakita, N. Kato Debiopharma Twincore Hannover G. Vuagniaux, R. Crabé T. Pietschmann, S. Haid, S. Ciesek, E. Steinmann Rockefeller University New York Lyon (different) C.M. Rice, T. Tellinghuisen F. Penin & Team University of Lausanne P. André, V. Lotteau D. Trono F.-L. Cosset, D. Lavilette University of Lille G. Lippens, X. Hanouille University of Lausanne D. Moradpour, G. Gouttenoire EMBL Heidelberg S. Welsch, P. Chlanda, J. Briggs, C. Antony University of Ulm funding P. Walther University of Strasbourg T. Baumert DFG University Ghent, Belgium EU G. Leroux-Roels, P. Meuleman BMBF University of Geneva CHS Foundation T. Pertel, J. Luban
Hepatitis C 1 – 3 2 – 10 decades years persistent HCV liver cirrhosis hepatocellular carcinoma (~70%) (10 - 20%) (1 - 5% per year) • Major cause of liver disease • ~170 mio infected individuals • a main indication for liver transplantation • no approved selective drugs • no vaccine
NS5A and CypA are not Major Components of Infectious HCV Particles
The Intimate HCV – Host Cell Interaction Exploit: Tight junctions Defend & Eliminate Transport system Stress response Lipid pathways Apoptosis VLDL Interferons … T-cells B-cells HCV Persist: Counteracting innate Antiviral therapy And adaptive defense Host
Membrane Topology of HCV Proteins Moradpour et al., Nat Rev Microbiol 2007
Efficient Affinity Capture of Infectious HCV Particles
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