| Abstract |
Human cytomegalovirus (HCMV) causes severe clinical disease in AIDS patients. Although highly active antiretroviral therapy ( HAART) has reduced the mortality rate due to HCMV disease in HIV/AIDS patients, HCMV disease remains a problem in untreated, poorly-responding patients, and even some patients on HAART. Treatment of HCMV infections is currently limited to drugs that target viral DNA synthesis, which, although efficient, are relatively toxic, have high relapse rates, and are losing their potency due to mounting drug resistance. Thus, there is a clear need for new, selective and less toxic drugs, especially those that target a different step in the viral life cycle. Targeting HCMV cell entry as well as cell-cell spread using a structure-based drug-design strategy is one attractive approach. Such an approach requires detailed structural information on all components of the HCMV entry/cell spread machinery.
Glycoprotein B (gB) is absolutely essential for HCMV cell entry and plays several fundamental roles in infection, including receptor binding, membrane fusion, and activation of signal transduction pathways. gB thus represents a new and attractive target for anti-HCMV therapeutics. This application focuses on one very important facet of gB function: its interaction with cellular receptors, integrins, that allow HCMV to enter cells. Once we understand the molecular mechanism of gB interaction with integrins, we would be able to design efficient inhibitors of that interaction. Since integrins are present on the surface of a wide range of cells - possibly explaining the very broad tropism of HCMV - inhibiting gB/integrin interaction would help prevent HCMV entry into different cell types.
No structural or mechanistic information is currently available on HCMV gB, and how it interacts with its integrin receptors is completely unknown. Moreover, although the wide repertoire of cellular integrins capable of binding gB has been narrowed down to three, it's unclear which integrins gB binds preferentially. Our long-term goal is to determine the atomic-level structural mechanisms of HCMV entry into host cells as well as cell-cell spread and to use this knowledge to design effective inhibitors of both processes for preventative and therapeutic purposes. The objective of this application is to initiate structural studies on gB and integrin, its cellular receptor. Therefore, our specific aims are 1) to express, purify and crystallize the extracellular domain of HCMV gB; and 2) to express the ligand-binding "headpiece" portions of integrins a 2 b 1, a 6 b 1, and a V b 1 in an appropriate heterologous system and to identify specific integrin heterodimers that bind directly to the disintegrin-like domain (DLD) of gB. The integrin with the highest affinity for the disintegrin-like domain of gB will be used to assemble integrin/DLD complexes for structural studies.
This project should be considered an initial HIV/AIDS award, because the principal investigator is new to the AIDS research field, and the project is a new endeavor within the principal investigator's laboratory.
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