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)

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

• Doxycycline is a tetracycline antibiotic

• Feeding it to these cells increases FasL expression

Cells with HBx expression linked to a doxycycline promoter were used

HBx Induces Expression of the FasL Gene

• HBx and FasL expressed together (Fig. 1A)

• The -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 non-mutant 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

• 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

Without HBx, Egr's have little effect on transactivation (Fig.

7A)

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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