Hepatitis B Virus X Protein Induces Expression of Fas Ligand Gene through Enhancing Transcriptional Activity of Early Growth Response Factor

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Hepatitis B Virus X Protein Induces Expression of
Fas LigandGene through Enhancing Transcriptional
Activity of EarlyGrowth Response Factor

• by Young-Gun Yoo and Mi-Ock Lee (Sejong
  University, ROK)

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Hepatitis B Virus (its 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!

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HBV Survival in Hepatocytes

• HBV evades T-cells by inducing apoptosis in
  T-cells that contact infected hepatoctyes!

How?
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Fas-mediated Apoptotic Pathway

• Goldsby, R.A., Kindt, T.J., Osborne, B.A., and
  Kuby, J. (2003) Immunology, 5th ed. Freeman, New
  York, 551p

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When Fas binds FasL, FADD (Fas-associated protein
with death domain) is recruited and binds to Fas
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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

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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

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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

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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)

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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...

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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

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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)

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Comparison of mutant and non-mutant Egr binding
sites (Figs. 3C, 3D)

• Activity levels differ greatly (P/I is a positive
  control)

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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)

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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)

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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)

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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)

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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

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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)

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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)

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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

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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

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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 doesnt seem to interact with
  FasL promoter)

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Further research?

• Egr's could be a target for future therapeutic
  treatments of viral diseases

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I'm Not Here To Make You Fall Asleep...
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I'm Not Here To Make You Fall Asleep...
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I'm Just Spoon Feeding You Info