|M.Sc Student||Kechker Peter|
|Subject||Studying the Role of the Adenovirus E4orf4 Protein in|
Silencing the DNA Damage Response in the Host Cell
|Department||Department of Medicine||Supervisor||Professor Tamar Kleinberger|
|Full Thesis text - in Hebrew|
Following replication of the adenoviral genome the host cell is flooded with a large quantity of short double-strand DNA molecules. This might activate the Double-strand break (DSB) DNA damage response (DDR). Additionally, the replication of the adenoviral genome includes a stage where single-strand DNA fragments are formed, which may activate the Single-strand break (SSB) DDR mechanism. Activation of the DDR serves as an anti-viral defense mechanism, since the final outcome is cell cycle arrest and generation of concatamers. Concatamerization inhibits viral replication and encapsidation. Proteins encoded by the adenovirus E4 and E1B genes are used by the virus to counteract the DDR.
The response of the cells to DNA damage is mediated by PI3-like kinases. DSBs activate the Ataxia telangiectasia mutated protein (ATM) kinase, and SSBs activate the Ataxia telangiectasia Rad-3-related protein (ATR) kinase. These kinases activate a signaling cascade leading to DNA repair. Another DDR protein, PARP-1, is a major factor in the DDR whose cellular activity causes the synthesis of PAR chains required for the recruitment of key proteins to the damage site.
In our work, infection with an E4 mutant adenovirus activated the DDR mechanism leading to high levels of phosphorylation in the cascade proteins. However, the phosphorylation was reduced in the presence of the adenovirus E4 open-reading-frame 4 (E4orf4) protein. We further found that the presence of the phosphatase 2A (PP2A) regulatory sub-unit B/B55α and the ability of E4orf4 to bind it were necessary for the inhibitory function of E4orf4.
In contrast, downregulating expression of another E4orf4 partner, ACF1 sub-unit of the ACF chromatin remodeling complex, was discovered to enhance the inhibitory role of E4orf4 in the DDR pathway.
Additionally, our research suggested a connection between the function of E4orf4 in inhibiting the DDR and the biological activity of the enzyme PARP-1. We found that like the other DDR proteins, the B55a sub-unit of PP2A has a specific recognition sequence for PAR chains. This sequence may allow PP2A-B55a to recruit E4orf4 to the damage site where it will regulate PP2A activity towards DDR proteins.
The importance of the inhibition of the DDR pathways by the Adenoviridae family was shown in experiments in which we compared the efficiency of infection between normal cells and cells lacking ATM. The infection was at least ten times more efficient in ATM-deficient cells. Additionally, the presence of E4orf4 improved infection efficiency both in the presence of ATM and in its absence.