Posttransplant lymphoproliferative disorder (PTLD) is a common and severe complication of both solid organ transplantation such as heart, lung transplants as well as bone marrow transplantation with high mortality rates. Epstein-Barr virus (EBV) infection is a major risk factor for developing PTLD. EBV is closely associated with the PTLD and virtually all PTLDs are infected with EBV. Thus, the prevention of EBV infection and the subsequent reinfection that occurs from EBV reactivation from latency in transplant patients provides an important therapeutic strategy for PTLD prevention and treatment. Entry into target cells is essential for EBV infection. Understanding the EBV entry mechanism will allow for the identification of targets to aid in the design and development of drugs which can prevent EBV infection and resulting PTLD. The core EBV fusion machinery consists of gB and gH/gL complex which play key roles in fusion and entry of both B cells and epithelial cells. gp42 is only required for B cell fusion and entry. The focus of this proposal is to investigate the detailed role of gH/gL and gB in EBV entry and utilize the crystal structure of these essential glycoproteins to identify the molecular mechanism of fusion and entry. In aim 1, I will determine if blocking access to the large groove between domain I and domain II of gH/gL that is important for fusion function may inhibit virus induced membrane fusion and subsequent entry. I plan to use Fast Rigid Exhaustive Docking (FRED) software to select peptides that can engage the large groove of gH/gL and test their inhibitory effect on virus fusion and entry. In aim 2, I will investigate if the gB cytoplasmic tail domain (CTD) contributes to the conformation and/or fusion function of the ectodomain of gB and that may be important for the interaction of gB with gH/gL. In my preliminary data, I find that gB dissociates with gH/gL after fusion indicating that gB may have two different conformations. We previously reported that truncation mutants of the gB CTD domain alter gB fusion activity. I plan to use FlAsH, a bis-arsenical analogue of fluorescein that binds Cys-Cys-X-X-Cys-Cys in linear protein sequences and when two Cys-Cys motifs are in close proximity in the secondary structure of proteins, to distinguish the conformation of gB before and after fusion and to test whether the CTD domain may be important for conformational changes of the gB ectodomain and for the interaction of gB with gH/gL.
|Effective start/end date||1/1/14 → 12/31/14|
- American Heart Association Midwest Affiliate (14POST18600021)