Transcript Wk12-DeanApop

Hepatitis B Virus X Protein
Induces Expression of Fas
Ligand
Gene through Enhancing
Transcriptional Activity of Early
Growth Response Factor
by Young-Gun Yoo and Mi-Ock Lee
(Sejong University, ROK)
Hepatitis B Virus (it’s very bad
for you…)
• HBV(Orthohepadnovirus) is a dsDNA virus
• Transmission through bodily fluids
(transfusions, sex, etc.)
• Liver inflammation, cirrhosis, liver cancer
• Infection is very widespread (1000 US
deaths in 1999)
• But you should have had shots already, or
they wouldn't have let you into CSUS!
HBV Survival in Hepatocytes
HBV evades T-cells by inducing
apoptosis in T-cells that contact
infected hepatoctyes!
How?
Fas-mediated Apoptotic Pathway
Goldsby, R.A., Kindt, T.J., Osborne, B.A., and Kuby, J.
(2003) Immunology, 5th ed. Freeman, New York, 551p
When Fas binds FasL, FADD
(Fas-associated protein with
death domain) is recruited and
binds to Fas
This cleaves procaspase-8 to
form active caspase-8, leading
the cell on two possible
PATHWAYS TO DOOM:
• Bid-mediated mitochondrial death pathway
• Cascade of caspases ultimately resulting in
apoptosis
The HBx Protein Is Encoded By
the HBV Genome
• Found in cytosol and nucleus of infected
cells
• Modulates host-cell transcriptional activity
• Previously linked to induction of FasL
expression
HBx Induces Expression of the
FasL Gene
Cells with HBx expression linked to a
doxycycline promoter were used
• Doxycycline is a tetracycline antibiotic
• Feeding it to these cells increases FasL
expression
HBx Induces Expression of the
FasL Gene
• HBx and FasL
expressed together
(Fig. 1A)
• The a-tubulin positive
control was not
affected
• FasL promoter
cotransfected with
HBx greatly increased
promoter activity (Fig.
1B)
Delineation of HBx-responsive
cis-Acting Elements in the FasL
Promoter
• Serially deleted FasL
promoter was used in
luciferase-linked
reporter genes (Fig.
2A)
• This involved cutting
out pieces from the
promoter until it no
longer worked...
Delineation of HBx-responsive
cis-Acting Elements in the FasL
Promoter
• Luciferase activity
was detected in
everything but the 205 to -2 fragment
(Fig. 2B)
• This indicates that
HBx must be affecting
something in the -271
to -205 range
This Region Contains Egr
Binding Sites!
• Egr's (Early Growth
Response factor) are
transcription factors
• Mutations (Fig. 3A) in
the Egr binding site
resulted in loss of
promoter activity (Fig.
3B)
Comparison of mutant and nonmutant Egr binding sites (Figs.
3C, 3D):
• Activity levels differ
greatly (P/I is a
positive control)
Induction of FasL by HBx Is
Mediated by Egr-2 and Egr-3
• FasL expression
correlates with Egr-3
expression (Fig. 4A),
but not with Egr-1
(Fig. 4B)
Induction of FasL by HBx Is
Mediated by Egr-2 and Egr-3
• Blocking Egr function
reduces transcriptional
activity (Fig. 4C)
• Antisense Egr's
resulted in decreased
activity (Fig. 4D)
HBx Induces Expression as Well
as Transactivation Function of
Egr-2 and Egr-3
• Doxycycline enhances
expression of all Egr's
(Fig. 5A) at the
transcription level
• This increase is
abolished by the Egr
inhibitor cyclosporin A
(Fig. 5B)
HBx Induces Expression as Well
as Transactivation Function of
Egr-2 and Egr-3
• HBx binds Egr-2 and
Egr-3 in vivo (Fig.
6A)
• Egr-1 also bound by
HBx despite its far
lesser effects on FasL
transactivation (Fig.
6B)
HBx Increases Transactivation
By Recruiting CBP
Without HBx, Egr's
have little effect on
transactivation (Fig.
7A)
• CBP is a co-activator
binding to Egr's
• HBx drastically
increases Egr-3
binding to CBP (Fig.
7B)
• Effect on EGR-1 is
much weaker
The COOH-terminal Region of HBx Is Sufficient to
Induce Transcriptional Activity of Egr
• Truncated
mutants (with
either the
amino or
carboxyl
terminus
region) were
constructed to
determine
function (Fig.
8A)
The COOH-terminal Region of HBx Is Sufficient to
Induce Transcriptional Activity of Egr
• Amino terminus
did not activate
transcription,
but carboxyl
terminus did
(Fig. 8B)
• Egr-3
expression was
increased by
carboxyl
terminus, but
not by amino
terminus (Fig.
8C)
The COOH-terminal Region of HBx Is Sufficient to
Induce Transcriptional Activity of Egr
• In short, the carboxyl terminus of the HBx protein
is necessary and sufficient for
transcription/activation
Overall conclusions:
HBx induces FasL expression by
inducing EGR-2 and -3 expression
and recruiting CBP to enable the
Egr's to increase transcription of FasL
to thwart Fas-expressing T-cells
Further research? Other factors
may be involved in HBx/FasL
• Nuclear factors of activated T-cells (NFAT):
evidence for FasL regulatory role
• Interferon response factor (IGF): binds FasL
promoter
• Nur77: regulates FasL expression in
presence of HBx (even though it doesn’t
seem to interact with FasL promoter)
Further research?
• Egr's could be a target for future therapeutic
treatments of viral diseases
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