טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentNebenzahl Sharon Keren
SubjectInhibition of the DNA Damage Response by the Adenovirus
E4orf4 Protein
DepartmentDepartment of Medicine
Supervisor Professor Tamar Kleinberger
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

The adenovirus E4 open-reading-frame 4 protein (E4orf4) is a multifunctional viral regulator. Within the context of the virus, E4orf4 contributes to regulation of the progression from the early to the late stage of viral infection by down-regulating cellular and early viral gene expression, facilitating alternative splicing of adenovirus mRNAs and regulating protein translation. When expressed alone, E4orf4 induces cell death, which is p53-and caspase-independent and is more efficient in cancer than in normal cells.

PP2A is one of the main cellular Serine/Threonine Phosphatases and is a major E4orf4 partner. Its interaction with E4orf4 was shown to contribute to all E4orf4 functions known to date during viral infection and induction of cell death.

The cellular DNA damage response (DDR) includes several mechanisms to detect and signal the presence of damaged DNA or replication stress, resulting in cell cycle checkpoint activation and DNA repair, or if the damage is too extensive, resulting in senescence or cell death. Formation of DNA lesions is recognized by sensor proteins, including, Ku proteins, the MRN complex, and Poly (ADP-ribose) polymerase-1 (PARP-1). PARP-1 synthesizes poly ADP-ribose (PAR) chains on itself and on other proteins and the PAR chains recruit additional DDR proteins to the damage site.

Various DDR branches are regulated by kinases of the phosphatidylinositol 3-kinase-like protein kinase family, including ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK).

Following replication of the adenoviral genome, the host cell is flooded with an enormous quantity of short linear double-strand DNA molecules whose ends may activate the DDR.  Because “repair” by ligation of viral genomes is inhibitory to virus replication, DNA viruses evolved ways to inhibit the DDR.

To date we know several mechanisms used by members of the Adenovirus family to counteract the DDR. The adenovirus E4orf6 protein with E1B55kDa causes degradation of the damage sensor MRN complex and of p53 protein, while the E4orf3 protein causes the removal of MRN components from viral replication foci.

In this work we report that E4orf4 provides an additional mechanism to inhibit the DDR during adenovirus infection.

A protein interaction screen for E4orf4 partners revealed that E4orf4 interacts with several DDR-related proteins, and this work validates and characterizes some of those interactions and their functional consequences. Furthermore we show that E4orf4 is recruited to local microirradiation-induced DNA damage sites, most likely via direct interaction with PAR chains, and that its recruitment is PARP-1 and DNA-PK dependent. At the damage site, E4orf4 acts to decrease phosphorylation levels of DDR proteins belonging to both ATM and ATR regulated pathways, and reduces total parylation levels in the cell. Inhibition of those signaling pathways, and E4orf4 expression enhance the efficiency of adenovirus replication, indicating that inhibition of the DDR at various levels by E4orf4 contributes to adenovirus replication.

These findings also provide one possible explanation for the cancer-specificity of E4orf4 induced cell death as many tumors have DDR deficiencies leading to increased reliance on the remaining intact DDR pathways and to enhanced susceptibility to DDR inhibitors such as E4orf4.