Determining how EBV episome maintenance is regulated by TIMELESS function
Lead Research Organisation:
University of Sussex
Department Name: Sch of Life Sciences
Abstract
Epstein-Barr virus (EBV) causes 200,000 cancers per year and is responsible for the non-cancerous but debilitating disease, glandular fever. EBV infects the B lymphocytes of the immune system resulting in life long latent infection of the cells where the viral DNA is maintained in cells by a small number of viral proteins. One such viral protein is EBNA1 which binds to the viral DNA and promotes its replication and the continued growth of the cells. Understanding how EBNA1 functions will help us understand how its function can be counteracted and virus infected cells eliminated. One cellular factor that appears to be linked to EBNA1 function is the DNA replication protein TIMELESS (TIM). TIM is required to both prevent the process of DNA replication from causing DNA damage and also to promote the repair of DNA damage detected during DNA replication. Since loss of TIM has been observed to cause breakage of the EBV circular DNA genome in latently infected cells, it appears that TIM works to somehow prevent viral DNA breakage. However, it is unknown which of TIMs several functions in DNA replication and repair are involved in EBV maintenance in infected cells. Understanding how TIM functions in EBV DNA maintenance will not only provide us with a better understanding of how this virus manipulates cellular proteins to maintain infection, but may also lead to new clinical strategies or therapies. TIM is known to work with other DNA repair proteins whose function has being successfully targeted in the clinic. If TIM works with these proteins to maintain EBV infection in cells, we maybe able to repurpose these drugs for use in the treatment of EBV-associated diseases.
Therefore, in this proposal we will;
1. Genetically manipulate TIM protein expression in cells so that the cell expresses different TIM proteins. Individually these proteins lack the ability to carry out specific functions of TIM while allowing other functions to occur. We will use these modified proteins to identify which functions of TIM are required for continued viral infection in cells.
2. Since loss of TIM is associated with DNA breakage of the viral DNA, we will investigate when and where TIM linked DNA damage occurs and how TIM prevents the accumulation of broken DNA.
3. Since TIM is associated with a number of DNA repair pathways, we will examine how loss of these and linked DNA repair factors affect EBV infection. This will allow us to describe the broader context of how TIM functions are linked to the wider response to DNA damage in cells.
Therefore, in this proposal we will;
1. Genetically manipulate TIM protein expression in cells so that the cell expresses different TIM proteins. Individually these proteins lack the ability to carry out specific functions of TIM while allowing other functions to occur. We will use these modified proteins to identify which functions of TIM are required for continued viral infection in cells.
2. Since loss of TIM is associated with DNA breakage of the viral DNA, we will investigate when and where TIM linked DNA damage occurs and how TIM prevents the accumulation of broken DNA.
3. Since TIM is associated with a number of DNA repair pathways, we will examine how loss of these and linked DNA repair factors affect EBV infection. This will allow us to describe the broader context of how TIM functions are linked to the wider response to DNA damage in cells.
Technical Summary
Epstein-Barr virus (EBV) infection of B lymphocytes causes 200 000 cancers a year. During latent infection the virally encoded protein EBNA 1 binds to the oriP region of the viral episome where it promotes initiation, replication fork pausing and resolution of the viral chromosome. The cellular multifunctional DNA replication and repair protein TIMELESS (TIM) is required for EBV episome maintenance. It is also closely linked to EBNA1 at oriP where it appears to prevent DNA breakage in the viral episome. In cells TIM functions in several distinct pathways that both prevent and repair DNA damage generated during DNA replication. Interestingly, TIM is known to prevent the DNA damage generated when a replication fork replicates through a DNA protein covalent complex (DPC). Interestingly EBNA1 has recently been found to form a DPC at oriP, suggesting that TIM could be preventing DNA breakage when a replication fork collides with this complex.
In this proposal we will investigate which of the functions of TIM is most important for EBV episome maintenance and how TIM prevents the accumulation of DNA breakage in viral DNA. Specifically, we will;
1) Use cellular depletion/expression systems to express specific N and C terminal truncations of TIM that are only proficient for a subset of TIM functions. We will then use these systems to determine which of TIM's functions are necessary for viral genome maintenance.
2) We will investigate where, when and how DNA breakage occurs on the viral episome and how this is regulated by TIM using END-SEQ, agarose gel assays for single strand and double strand breaks on the viral episome and DNA checkpoint and DNA repair factor inhibitors.
3) We will perform an siRNA screen of DNA repair and replication factors to determine which cellular repair pathways are co-opted by EBV to maintain the viral episome.
In this proposal we will investigate which of the functions of TIM is most important for EBV episome maintenance and how TIM prevents the accumulation of DNA breakage in viral DNA. Specifically, we will;
1) Use cellular depletion/expression systems to express specific N and C terminal truncations of TIM that are only proficient for a subset of TIM functions. We will then use these systems to determine which of TIM's functions are necessary for viral genome maintenance.
2) We will investigate where, when and how DNA breakage occurs on the viral episome and how this is regulated by TIM using END-SEQ, agarose gel assays for single strand and double strand breaks on the viral episome and DNA checkpoint and DNA repair factor inhibitors.
3) We will perform an siRNA screen of DNA repair and replication factors to determine which cellular repair pathways are co-opted by EBV to maintain the viral episome.
