Hepatitis C Virus: Basics Ralf Bartenschlager - - PowerPoint PPT Presentation

Hepatitis C Virus: Basics

Ralf Bartenschlager

AREVIR-GenaFor-Meeting Bonn April 22 - 23, 2010

HCV Particle Properties

E1/E2 capsid RNA genome lipoproteins

• Hepacivirus, fam. Flaviviridae
• Enveloped particle
• Associated with lipoproteins
• ss (+)RNA genome

5´NTR IRES 3´NTR

(+) strand RNA genome translation, processing

E1 C E2

virus particle

p7

ion channel, assembly

3

4A

2

protease, assembly protease/ helicase NS3- cofactor

5B 4B 5A

membrane- vesicle replication, assembly polymerase structural non-structural proteins

5B 5A 4B

4A

3 2

p7

E2 E1 C

HCV Genome Organization

C+1 ?

Mini- cores

Replicase complex ‚Assembly module‘

HCV Replication Cycle

LD

Assembly membranous web RNA replication RF RI

Polyprotein processing

HCV replicons strictly intracellular replication

neo

5‘ E-I Lohmann et al., Science 1999

5B 5A 4B

4A

3 2

p7

E2 E1 C

3‘ 5‘ 3‘

Cell Culture Systems for Hepatitis C Virus

5B 5A 4B

4A

3

• 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

5B 5A 4B

4A

3 2

p7

E2 E1 C

3‘ 5‘

JFH-1

Wakita, Pietschmann et al., Nat Med 2005

Contr. 1 2 3 4 Infectious units (log10) JFH1/wt

α – NS3 JFH1 (GT 2a) J6 (2a)

Lindenbach et al., Science, 2005; Pietschmann et al., PNAS 2006

JFH-1: 10e3 TCID50/ml Jc1: 10e6 TCID50/ml

Jc1

5B 5A 4B

4A

3 2

p7

E2 E1 C

3‘ 5‘

HCV Entry into the Host Cell

LDLr GAGs SR-BI CD81 CLDN1 OCLN Clathrin- coated pit H+ Early endosome Fusion and genome release

Burlone & Budkowska, JGV 2009

The Hepatitis C Virus Replication Complex

Membranous Web

nucleus

LD LD

ER Rv?

nucleus

LD LD

ER Rv?

Membranous web in HCV-infected cell Comparison of Replication and Assembly Compartments in HCV- and DENV-Infected Huh7 Cells Membranous replication complex 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

Dengue Virus Hepatitis C Virus

Ve

Double-Membrane Vesicles in DENV and HCV Replication Complexes

HCV-Induced Membranous Web is Tightly Linked With and Contains Lipid Droplets

• A. Merz
• I. Romero-Brey

Hydrophobic core (esp. TAGs, sterol esters) phospholipid monolayer

PAT-proteins

• cytoplasmic storage of neutral lipids
• functionally linked to other organelles
• intracellular mobility
• complex ‘proteome’
• metabolically active structures
• de novo synthesis
• sites of HCV core and NS5A accumulation
• sites of HCV assembly

Properties of Lipid Droplets

LDs core NS5A merge

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

LD

Hypothetical Model for Nucleocapsid Formation and Envelopement

Rv

VLDL pathway

Huang et al., 2007; Chang et al., 2007; Gastaminza et al, 2008

• A. Merz
• G. Long

N-Terminal E2 Tagging Does Not Affect Assembly

Immuno-EM Analysis of Jc1E2Flag Particles

• Average diameter 73nm
• Capture with anti-apoE
• Neutralization with anti-apoE
• TCID50 : RNA : core : apoE

1 : 1 : ~1,000 : ~ 250

Sphingomyelin Phosphatidylcholine Cholesterylesters

The HCV Lipidome is Unique and Resembles (V)LDL

~ 45% of HCV particle lipids are cholesteryl esters problematic for lipid bilayer

1 1 1 2

1Wiesner et al., J Lipid Res 2008; 2Brügger et al., PNAS 2006

Tentative model of the HCV “Lipo-Viro-Particle”

• F. Penin, P. André

to exploit its host cell to escape from immunological control

HCV has evolved numerous strategies...

www.despair.com

control of IFN induction (NS3/4A protease) weak induction

• f IFN response

interference with acquired immune response protection of viral RNA in membranous RCs

Strategies of HCV Persistence

• M. Binder
• V. Lohmann

virus

IFN-β

dsRNA vRNA

IFN-β

TLR3

TRIF RIG-I MAVS

IFN-β

Adapted from Haller et al., Virology, 2005 ISG ISRE

IFNAR

JAK-1 / TYK-2 ISGF-3

dsRNA

Antiviral genes

Blockage of IFN Production by NS3/4A Protease

NS3/4A

Relevance in vivo? Yes for MAVS ? For TRIF

IFN-β

ISG ISRE

IFNAR dsRNA

IFN-α IFN-β

IFN-α/β

JAK-1 / TYK-2 STAT-1 -2 IRF-9 ISGF-3 Mx IRF-7 -3 PKR OAS ISG20

Block of the effector phase by HCV proteins?

endogenous exogenous HCV proteins (core)

E2, NS5A

X X X

Reduction of ISG expression by HCV-mediated activation

• f PKR*

*Garaigorta & Chisari, 2010

4A

5B 5A 4B 3 neo

3‘ 5‘

4A

5B 5A 4B 3 hyg

3‘ 5‘

5B 5A 4B

4A

3 2

p7

3‘

E2 E1 C

5‘ E-I

neo

E-I

IC50 ~ 2-5U/ml (GT-1b, 1a, 2a) IC50 ~ 1 U/ml (GT-1b, 1a, 2a) IC50 ~ 2-5U/ml (GT-1b, 1a, 2a)

High IFN-α Sensitivity of HCV Replicons and Virus

5B 5A 4B

4A

3 2

p7

3‘

E2 E1 C

5‘

IC50 ~ 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

Do we have the right systems to study HCV persistence in cell culture?

day 0 day 14 HCV infection DMSO differentiation day 18 sampling day 44

TCID50/ml 100 101 102 103 104 105 106 no treatment day 18 day 23 day 30 day 37 day 44 median

95 quantil 5 quantil

Huh-7dif (day 14)

day 16 IFN

100 U/ml IFN-α

The Huh7dif 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

Properties of the Huh7dif Culture System

• O. Bauhofer

Number of HCV infected cells

10 20 30 40 50

MxA high MxA low

Possible Strategies of HCV Persistence

• infection of cells responding poorly to type 1 IFN

(exploiting the ‚stochastics‘ of the IFN system)

• low-level inhibition of type 1 IFN-induced signalling
• inhibition of (expression of) IFN-induced effectors

IFN refractory (HCV permissive) cells

time point X time point Y

The Intimate HCV – Host Cell Interaction

Exploit: Tight junctions Transport system Lipid pathways VLDL … Persist: poor RIG-I activation blocking IFN induction exploit stochastics? high genetic variability … Defend & Eliminate Stress response Apoptosis Interferons T-cells B-cells Antiviral therapy

HCV Host

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

Department Infectious Diseases Molecular Virology

Twincore Hannover

• T. Pietschmann, S. Haid, S. Ciesek, E. Steinmann

Lyon (different)

• F. Penin & Team
• P. André, V. Lotteau

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

• P. Walther

University of Strasbourg

• T. Baumert

University Ghent, Belgium

• G. Leroux-Roels, P. Meuleman

University of Geneva

• T. Pertel, J. Luban

NIID, Tokyo

• T. Wakita, N. Kato

Debiopharma

• G. Vuagniaux, R. Crabé

Rockefeller University New York C.M. Rice, T. Tellinghuisen University of Lausanne

• D. Trono

collaborations reagents & tools funding

DFG EU BMBF CHS Foundation

Hepatitis C

• Major cause of liver disease
• ~170 mio infected individuals
• a main indication for liver transplantation
• no approved selective drugs
• no vaccine

persistent HCV (~70%) liver cirrhosis (10 - 20%) hepatocellular carcinoma (1 - 5% per year) 1 – 3 decades 2 – 10 years

NS5A and CypA are not Major Components

• f Infectious HCV Particles

The Intimate HCV – Host Cell Interaction

Exploit: Tight junctions Transport system Lipid pathways VLDL … Persist: Counteracting innate And adaptive defense Defend & Eliminate Stress response Apoptosis Interferons T-cells B-cells Antiviral therapy

HCV Host

Moradpour et al., Nat Rev Microbiol 2007

Membrane Topology of HCV Proteins

Efficient Affinity Capture of Infectious HCV Particles