The Hepatitis B Virus Capsid Protein:
Biology and Trafficking
Life cycle of HBV – a Retroid Virus
Empty virion
11
(HBs+HBc) 10 /ml
P
P
P
P
Complete virion
9
(HBs+HBc+DNA) 10 /ml
IV
rc
II
P
III
P
P P
P
rc
integration
ccc
LS
S
TF/
HNF
HSP
pgRNA
P
P
P
NTCP
dsl
DNA
repair
PPase
SS DNA
P P
P
HBe
rc
L, M, S
P
Spheres/filaments
1014/ml
(HBs)
CDK/PKC
I
C
LS
S
PreC
X
P
C
X
L, M, S
PreC
Core Protein and Capsid Structure
NTD CTD + ++ + + 170!
162!
257!
259!
245!
239!
SPRRRTPSPRRRRSQSPRRRRSQSREPQC!
230!
232!
C-terminal domain (CTD):
• Arginine-rich
HBV • Phosphorylated
• NLS
• RNA packaging
DHBV • DNA synthesis
155!
P P P SKSRERRAPTPQRAGSPLPRSSSSHHRSPSPRK!
I.
Packaging of pgRNA and RT
Assembly of Nucleocapsids
Nucleocapsid Assembly in Hepadnavirus:
Recognition of RT-ε RNP by Assembling Core Subunits
C
C
C
C
P
cis
pgRNA
Cap
P
C
An
C
1
1
1
2
1
An
C
C
C C
Cap
ε
2
C
C
C
C
C
C
C
P
Requirements for RNA Packaging beyond
RT-pgRNA Interactions
RNP formation
RNA Packaging
H SP
RT
TP
CC
ε
HSP
P
u HBc CTD phosphorylation
u RNase H domain of RT
u Apical loop of ε RNA
RT
P
TP
P
RH
P
AAA(n)
u 5’ cap and short distance to ε
u How??
u Targeted by HBc (NTD) effectors
Hu & Seeger, 1996; Hu & Morgan, 2006 Hu et al, 1997; 2002; 2004
Jones et al, 2012, 2013a, 2013b II.
Regulation of Reverse Transcription
Dynamic Core Phosphorylation
Dephosphorylation of DHBV Core Protein
during Nucleocapsid Maturation-Mass Spectrometry
Immature
RNA NC
P
P
P
P
P
Mature
DNA NC
Virion NC
Perlman et al (2005)
S/T-P Phosphorylation Required for (-) Strand and
Dephosphorylation for (+) Strand DNA Synthesis!
Core!
S to A: no DNA synthesis
RC!
S to D: (-) DNA OK defect in (+) DNA accumulation
SS!
Basagoudanavar et al (2007) Core Phosphorylation Dynamics during
Nucleocapsid Maturation
P P
Cdk2
P P
RT
P P
P P
P
Complete minus DNA !
Initiation of plus DNA!
P
S/LS!
Cdk2
Cdk2
P
?
Cdk2, PKC
P
Cdk2
Cdk2
kinases?!
pgRNA!
packaging!
P
PPase?!
Plus DNA elongation!
NC Dephosphorylation!
?
Maturation signal!
emerges?!
Envelopment/!
secretion !
Perlman et al, 2005 Basagoudanavar et al, 2007 Ludgate et al, 2012 Manipulating
Core Functions
by Expressing
CTD in trans
Liu, et al, 2015
Sequestration of CDK2 (PPase) by CTD in trans
Inhibits Core Phosphorylation and Reverse Transcription
HCTD
PPase
?
(HCC141-AAAAAAA)
P
RC
Nucle
PPase
ar imp
ort
RC
?
CCC
SS
P
pgRNA
CDK2
HCTD
(HCTD141-AAAAAAA)
CDK2
Liu, et al, 2015
III.
cccDNA Formation
Nucleocapsid Disassembly (Uncoating)
and Nuclear Genome Delivery
Destabilization of Nucleocapsids
Associated with Maturation
Sensitivity
Sedimentation
Mobility
(Sucrose gradient) (Agarose gel) Protease Nuclease
Slowest
Slowest
Yes
Yes
Protease
-
+
M-3
RC DNA
’
Fast
Slow
Yes
No
M-2
’
+
Fast
Slow
No
No
M-1
’
Slow
Fast
No
No
IM
’
SS DNA
SS DNA
1
Cui, et al, 2013
M, Mature NC
IM, Immature NC
pgRNA
2
pgRNA
Sequestration of Importin (Others) by CTD in trans
Inhibits CCC DNA Formation
HCTD
?
Importin α
DCTD/HCTD141-ATASASS
?
RC
Nucle
ar imp
ort
RC
CCC
P
P
rtin α
o
p
Im
SS
P
P
P
P
pgRNA
P
P
Liu, et al, 2015
IV.
Virion Secretion
Nucleocapsid Envelopment
Mature (DS DNA) But Not Immature Nucleocapsids
Are Enveloped and Secreted
Virion
DS DNA
Classical
Retroviruses
(RNA viruses)
SS DNA
RT
pgRNA
RT
Mature NC
Immature NC
Envelopment
and secretion
“Maturation Signal”
Secretion of Empty (Genome-free) HBV Virions
1011/ml
109/ml
1014/ml
HBe
HBe
rc
DNA
(viremia)
HBsAg
S/M/L
rc
dsl
rc
NRTI
integration
ccc
pgRNA
LS
S
C
LS
S
PreC
Ning et al (2011)
Negative vs. Positive Signal for Virion Secretion
A. SS blocking model: Nega;ve signal induced by SS RNA or SS DNA Env!
core Env!
B. Matura;on signal model: Secre;on (posi;ve) signal induced by DS DNA Env!
core pgRNA (-­‐) DNA (+) DNA blocking signal secre7on signal Ning et al (2011)
What Can We Do with
Empty Virions?
1. Serum Empty Virions as a Peripheral
Marker to Monitor Hepatic CCC DNA Levels
Change in Complete/Empty Virions and HBsAg
During Antiviral (TFV) Therapy
1014 1011 109
Complete
Virion
(HBV DNA)
Empty
Virion
(HBcAg)
HBsAg
Luckenbaugh et al (2014)
Changes in Complete/Empty Virions and HBsAg
During Antiviral (TFV) Therapy
1014 1011 109
Complete
Virion
(HBV DNA)
Empty
Virion
(HBcAg)
HBsAg
Luckenbaugh et al (2014)
Serum Empty Virions As a Marker for Hepatic CCC DNA
During Antiviral Therapy
rc
1. HBV DNA
(complete virions)
decreases rapidly
with therapy.
S/M/L
2. HBsAg
produced from
both integrated
& CCC DNA.
3. HBeAg lost in
HBeAg(-) phase.
4. Empty virions
driven by CCC
DNA – better
surrogate marker
for CCC DNA.
HBe
HBe
rc
dsl
rc
integration
NRTI
LS
S
ccc
pgRNA
C
LS
S
PreC
2. Empty Virions as a Vaccine Candidate
HBV Vaccines
Filament (S/M/L)
sphere
1st generation:
Human plasma derived spheres/filaments
sphere
2nd generation:
Yeast expressed (S protein only)
Empty Virions: 3rd Generation Vaccine?
1. Empty virions can have all three
HBs proteins (L, M, S) –
addressing vaccine escape and
failure
2. Spatial arrangement (40 structure)
and potentially 30 structure of Env
more similar to complete virions
than HBsAg spheres/filaments
3. Also have the internal HBc
- all major structural proteins
- HBc: major targets of CTL
4. Safe (no genome)
- completely non-replicative
5. Prophylactic & therapeutic
Multiple Roles of HBc in the Viral Life Cycle
Empty virion
11
(HBs+HBc) 10 /ml
P
P
P
P
Complete virion
9
(HBs+HBc+DNA) 10 /ml
IV
rc
II
P
III
P
P P
P
rc
integration
ccc
LS
S
TF/
HNF
HSP
pgRNA
P
P
P
NTCP
dsl
DNA
repair
PPase
SS DNA
P P
P
HBe
rc
L, M, S
P
Spheres/filaments
1014/ml
(HBs)
CDK/PKC
I
C
LS
S
PreC
X
P
C
X
L, M, S
PreC
The Multi-functional HBV Core Protein
1. Nucleocapsid assembly remains ill-understood.
Likely multiple interactions of HBc with RT, pgRNA, and host factors?
2. Nucleocapsid maturation controlled by dynamic CTD
phosphorylation and dephosphorylation.
3. Nucleocapsid disassembly (uncoating) facilitated by maturationassociated destabilization (priming) – role of CTD as well as
NTD.
4. Nucleocapsid envelopment regulated by both (+) and (-) signals:
1) Genome-free as well DS DNA virions, but not SS DNA or RNA.
2) Empty virions as biomarkers, and 3rd generation vaccine?