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Virus Hepatitis Research Group I. Department of Internal Medicine
Center of Internal MedicineUniversity Medical Center Hamburg-Eppendorf
Hamburg, Germany
Maura Dandri
Biology of HBV infection
Fondation Merieux Conference CenterVeyrier-du-Lac France
(2-4 May 2016)
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- ca. 250 million people are chronically infected with HBV worldwide
- HBV polymerase inhibitors efficiently suppress viral replication
Chronic Infection with hepatitis B Virus
Limited knowledge about - mechanisms leading to infection establishment- cccDNA formation, stability & regulation- virus-host interactions (evasion mechanisms)
- Prevention of HBV infection is achievable (effective vaccine)
But no cure for CHB due to persistence of cccDNA
& weak immune responses
invisible or
active evasion?
HBV95% Acute self-limiting infection
5% Persistent infection (but high rates in perinatal age)
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Hepatitis B Virus: structure
HBV
(adapted from Neuvet et al., J. Hepatol. 2010)
• smallest DNA enveloped animal virus (3,2 Kbp)(4 overlapping ORFs)
• Genome in circulating virions:- relaxed circular partially double-stranded DNA (rcDNA)- covalently linked to viral polymerase (RT)
• Capsid: core protein (HBcAg), icosahedral arrangement
• Envelope: Large (L), Middle (M), Small (S) proteins(host derived-lipids)
•High species-specific: humans, chimpanzees(Tupaia)
• High tissue-specific: liver tropism
HBV extremely adapted to its host!
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(S. Urban, R. Bartenschlager, R. Kubitz, F. Zoulim, Gastroenterol. 2014)
Hepatitis B Virus: structure
AGL (=antigen loop)
Binding to HSPG
neutr. Abs(HBIG)
myristoylation
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HBV infection: early entry steps
(Urban et al. Gastroenterology 2014)
Attachment: 1) Reversible interaction with HSPGs
(Schulze, Hepatol. 2007)
2) Irreversible binding to NTCP (Yan, Elife 2012)
NTCP: sodium taurocholate co-transporting polypeptide is localized to the basolateral membrane of highly
differentiated primary hepatocytes it mediates most of the hepatocellular uptake of bile salts Binding is mediated by the preS1 domain of the HBV
envelope protein
HBV HDV
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HBV infection: early entry steps
Pre-screening of new antiviral compounds & elucidation of unclear infection steps
The recognition of NTCP as the bona fide HBV and HDV entry receptor has enabled theestablishment of human cell lines supporting HBV and HDV infection
HBVHBV
PHHs(primary human
hepatocytes)
dHepaRG(hepatocyte-like cells
+ biliary-like cells)
hepatomacell lines
(HepG2, Huh7)
h-NTCPHBV
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Implications of HBV receptor discovery
hNTCP Transgenic mice(immune competent HBV/HDV
infection models)
h-NTCP
aa 84-87
m-NTCP
(Yan et al, Elife 2012)
HDV-δAg in HepG2-hNTCPHBV-HBsAg in HepG2-hNTCP
(Bhadra et al. unpublished)
HDV displays higher infection efficacy!
Improved in vitro infection systems
Improved in vivo infection systems ?
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hNTCP transgenic primary murine hepatocytes (in vivo & in vitro) are not HBV permissive(Li et al. Cell. & Mol. Immunol.2014, Yan et al. J.Virol.2013)
HBVHBV
Low efficiency and Temporary HDV infection could be shown in hNTCP transgenic mice(He et al. PLOS Pathogens 2015)
Implications of HBV receptor discovery
Unknown species-specific factors hinder HBV infection establishment in mice
A mouse liver cell line (AML12) supports HBV entry upon hNTCP expression(HBV RNA and proteins production, rcDNA – virion productivity – barely detectable)
(Lempp J Virol 2016)HBcAg
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Steps involved in HBV infection establishment
Attachment:1) Interaction with HSPGs (Schulze, Hepatol. 2007)
2) Binding to NTCP (Yan, Elife 2012)
Internalization:By endocytosis mediated by host factors- caveolin-1 (Macovei, J.Virol.2010)
- clathrin- Rab proteins (GTPases) (Macovei, J.Virol.2013)
Uncoating
Transport to the nucleus along microtubules
(Urban et al. Gastroenterology 2014)
?
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Intracytoplasmic transport via microtubules
capsids dissociate frommicrotubules (unknown mechanisms)
attach to importin α/β
pass the Nuclear Pore (NPC)
Capsids dissociate to core protein dimers
Genome is released
cccDNA formation
(Blondot, Bruss, Kann, J Hepatol. 2016
Intracellular transport
Steps involved in HBV infection establishment
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cccDNA formation
- HBV Polymerase removal mediated by host DNA repair enzymes like TDP2 (tyrosyl-DNA-phosphodiesterase)
- Removal of a short terminal redundancy- & small RNA primer on + strand- Completion of the plus strand- Ligation
(adapted from Königer et al. PNAS, 2014)
Conversion of rcDNA to cccDNA is a largely unknown multistep process involving different host enzymes
PCAFPCAF
p300p300
acetylated histones core protein
The cccDNA assembles with histone and non-histone proteins to form a stable minichromosome in hepatocyte nuclei
(Newbold; Levrero et al. J.Hepatol.2009; Lucifora et al. J.Hepatol. 2011; Tropberger PNAS 2015)
HBx
(≈ damaged dsDNA) ( ≈ plasmid)
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cccDNA
Endocytosis & uncoating
(Modified from Dandri, Locarnini, Gut 2012)
NTCP
HBV productive infection = cccDNA establishment & transcription
NPC
rcDNA
HBV
The cccDNA minichromosome serves as transcription template for all HBV RNAs
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cccDNA
AAApgRNA
AAA
AAA
AAApreS/S
LMS
envelopeproteins
core proteins HBV
polymerase
X mRNA
transcription
SVPs
Endocytosis & uncoating
Re-entry
HBV Virion
(Modified from Dandri, Locarnini, Gut, 2012)
NTCP
HBsAg
HBV replication cycle
subgenomic RNAs
NUCs efficiently suppress HBV replication but not cccDNA-driven RNA transcription(no reduction of viral proteins and circulating HBsAg)
NPC
rcDNA
MVB
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cccDNA transcription can be reduced by IFNα
(Belloni et al., J.Clin.Invest. 2012)
IFNα can inhibit cccDNA transcription by reducing the acetylation status of histones bound to the cccDNA
human IFNa
Ac-Histone H4on cccDNA
Re
lFo
ldin
du
ctio
n
+IFNaNT
0.2
0.4
0.6
0.8
1.0
Peg-IFN treateduntreated
Hu-CK18 HBcAg
(Allweiss et al., J. Hepatology 2014)
12 weeks of peg-IFNα treatment in humanized micelacking adaptive immune responses led to significantsuppression of viremia and antigen production
HBV-infectedhuman liver chimeric
uPA/SCID mice
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I) Serological parameters: viremia changesIFNα & LTβR activation can promote cccDNA destabilization
(Lucifora, et al. Science 2014)
IFNα-treatment & activation of thelymphotoxin β receptor can induceAPOBEC-mediated cccDNA deaminationand partial cccDNA degradation
peg-IFNa
IFN-a induced epigeneticsuppression of the cccDNAand strong reduction of viralantigen production
Ac-Histone H4
Re
lFo
ldin
du
ctio
n
+IFNaNT0
0.2
0.4
0.6
0.8
1.0
(Belloni, JCI 2012; Allweiss, J.Hepatology2014) (Adapted from Thimme & Dandri, J.Hepatol. 2012)
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Studies aiming at understanding cccDNA biology / dynamics & HBV-host interactions
cccDNA
AAApgRNA
AAA
AAA
AAApreS/S
LMS
envelopeproteins
core proteins HBV
polymerase
X mRNA
transcription
SVPs
Endocytosis & uncoating
Re-entry
HBV Virion
(Modified from Dandri, Locarnini, Gut, 2012)
NTCP
HBsAg
Use of preS1-derived Lipopeptide Myr-B
Human liver chimeric mice
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Preclinical studies with HBV entry inhibitors
Chemically synthesized lipopeptides derivedfrom the preS1 domain of HBV envelopeblock de novo HBV infection in vitro and invivo
(Petersen, Dandri, Urban, Nature Biotech.2008)
+Myrcludex-B
3 6 9weeks
Log
HB
V D
NA
/ml
5
6
7
8
9
Myc
lud
exco
ntr
ol
HBcAg
(Volz et al. J. Hepatology 2013)
HBV
0 6weeks 3 9
Myrcludex-B
Myrcludex-B efficiently blocked HBV spreading post-infection in the setting of constant viral exposure
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Antiviral efficacy of Myrcludex-B administration on intrahepatic cccDNA amplification
weeks3 9 9
0.1
1
10
0.001
0.01
Log cccDNA copies / cell
untreated mice
Myrcludex-B
(Volz et al. J.Hepatol 2013)
Myrcludex-B hindered the increase of intrahepatic / intracellular cccDNA loads
cccDNA
AAAAAA
Myrcludex-B
NTCP
Intracellular cccDNA amplification seems to be inefficient in PHHs,
explaining lower cccDNA/cell found in patients
Virus-specific differences among hepadnaviruses (DHBV vs. HBV)
(Kock et al. PloS Pathpgens 2010
HBV
0 6weeks
3 9
Myrcludex-B
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Chronic HBV infection has been associated with alterations in lipid and cholesterol metabolism
HBV and host metabolism
cccDNA
AAAAAA
NTCP
NTCP mediates 80% of the hepatocellular uptake of bile acids(Stieger, Handb. Exp. Pharmacol.2011)
(Bar-Yishay Liver Int 2011; Hsu et al., J. Viral Hepatitis 2012)
Studies in humanized mice indicated thatHBV alters the profile of various genes relatedto host metabolism
(Oehler, et al. Hepatol.2014)
Use of preS1-derived lipopeptide to explore virus-host interactions
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CYP7A1 is strongly enhanced in HBV- infected mice
HBV infecteduninfected
Hu-Cyp7A1
Hu-AAT
hCYP7A1 RNA
uninfected HBV0.0001
0.001
0.01
0.1
1
Cholesterol 7 alpha-hydroxylase
<0.0001 ***
Strong enhancement of CYP7A1, the rate-limiting enzymepromoting conversion of cholesterol to bile acids
CYP7A1 increase was confirmed also in liver biopsies fromCHB patients
hCYP7A1 8h-24h
c 8h Myr 24h Myr0.0001
0.001
0.01
0.1
1
CY
P7A
1 e
xp
ressio
n
Myrcludex-B enhanced CYP7A1 expression in uninfectedmice, thus identifying the preS1-domain as the viralcomponent triggering CYP7A1 induction
(Oehler, et al. Hepatol.2014)
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uninfected HBV-infected(Oehler, et al. Hepatology 2014)
Nuclear localization of the bile acidsensor FXR was strongly reduced inHBV-infected cells
HBV and host metabolism
Binding of HBV to NTCP limitsthe uptake of bile salts,HBV hinders FXR nucleartranslocation and alters theexpression of bile acidmetabolic genes
(Ni et al. Gastroenterology 2014;Oehler, et al. Hepatology 2014)
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cccDNA
AAApgRNA
AAA
AAA
AAApreS/S
LMS
envelopeproteins
core proteins
HBeAgHBV
polymerase
X mRNA
transcription
SVPs
Endocytosis & uncoating
Re-entry
HBV Virion
(Modified from Dandri, Locarnini, Gut, 2012)
NTCP
HBsAg
subgenomic RNAs
SUMMARY
Entry inhibition strategies (Myr-B ) block HBV spreading & cccDNA accumulation
cccDNA targeting:Silencing / destabilization
HBV can alter the profile of various genes related to host metabolism
Interference with innate immune responses?
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AG Virushepatitis:
Marc LütgehetmannTassilo Volz
Lena AllweissKatja Giersch
Janine Kah
Claudia DettmerAnne Groth
Roswitha Reusch
Jan-Hendrik BockmannNicola OehlerOliver BhadraSebastian LuftPhilipp Fritzke
AW Lohse(Head of Dept. Int. Medicine
UKE
J. M. Pollok(UKB, Bonn)
L. Fischer(UKE, Hamburg)
S. Urban(Univ. of Heidelberg)
U. Protzer(TUM, Munich)
M. Levrero(Univ. of Rome)
Acknowledgement
M. Buti(Hospital Vall d'Hebron
Barcelona, Spain)
T. PollicinoG. Raimondo
(Univ. Messina, Italy)
T. Baumert(Inserm, Strasbourg)
J. M. Taylor(Fox Chase Cancer Center
Philadelphia, US)
A. Bertoletti(A*Star, Duke, Singapore)
D. Glebe(RKI, Giessen)
C. Sureau(Inserm, Paris)
Jörg Petersen(IFI Institute, Hamburg)