The topology of the BST-2 Mediated Restriction of HIV-1
Kathleen Fitzpatrick, University of California, San Diego
Advisor: John Guatelli
Basic Biomedical Sciences
Dissertation Award
2009
Bone marrow stromal cell antigen-2 (BST-2) is an antiviral cellular protein found at the surface of some host cells. BST-2 can be expressed by all cells and is found at the surface of cells treated with compounds produced by the immune system called interferons. BST-2 is now known to inhibit viral release, called “tethering”. These findings led to the conclusion that BST-2 is an effector of the immune response against viruses such as HIV-1.
Inhibition of viral release and the functional interaction of BST-2 with the HIV accessory protein VPU are recent concepts. Virus containing a functional vpu gene readily escapes from cells that express BST-2, but virus that lacks a functional vpu remains stuck or “tethered” to the cell surface. Additionally, the expression of VPU causes removal of BST-2 from the cell surface. These results indicate a mechanism utilized by HIV to counteract the host’s response as mediated by BST-2.
Structural data on BST-2 are minimal. Based on computer modeling, BST-2 likely contains a single membrane-spanning domain, an extracellular region, and ends in a chemical membrane anchor. Additionally, the extracellular domain of BST-2 contains two sites for sugar addition as well as three sites for bonds with other proteins. BST-2 forms disulfide-linked dimers (where a complex consists of two BST-2 proteins); however, this is not the only possible means of interaction: computer modeling suggests that two BST-2 complexes could form a larger BST-2 complex.
To test the topological model of tethering, I propose to use a number of approaches. Changes within the BST-2 ectodomain will be created and the mutant proteins will be analyzed for tethering activity and as well as the ability to form multi-protein complexes as explained below. These mutations will target the sugar modification sites, the binding sites, and the predicted regions where BST-2 could interact with other BST-2 proteins. To test the necessity of a specific extracellular protein shape, the activity of a naturally occurring protein which is similar to BST-2, but which lacks a similar extracellular region will be tested. Finally, interaction experiments will be used to illustrate whether dimerization occurs under varying conditions or in the case of altered protein sequence. These methods will verify and characterize a extracellular interactions or provide evidence for an alternative structure. Upon completion of this project, the mechanism by which BST-2 tethers HIV-1 to the surface of cells will be known. The exact nature of this mechanism, with regards to specific structure within the extracellular domain will be illustrated.
