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 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 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

I'm Not Here To Make You Fall Asleep...

I'm Just Spoon Feeding You Info