Mechanistic studies of human immunodeficiency virus entry

Envelope glycoprotein, composed of the subunits gp120 and gp41, mediates HIV entry into target cells. gp 120 recognizes the target cells by interacting with a receptor, CD4, and undergoes conformational changes that expose a coreceptor binding site. Then, gp 120 binds to a coreceptor, CCR5 or CXCR4, and undergoes additional conformational changes. These changes propagate from gp 120 to gp41 via the gp 120-gp41 interface, ultimately exposing gp41 and allowing it to form the six-helix bundle structure that provides a driving force for membrane fusion.; Before forming the six-helix bundle structure, gp41 forms the transient prehairpin intermediate structure, in which the N-peptide region forms the N-trimer without the C-peptide region. Despite extensive efforts, a potent and broadly neutralizing antibody against the N-trimer has been elusive. In Chapter 2, we fuse a C-peptide inhibitor, which binds to the N-trimer, to cargo proteins of various sizes to examine the steric accessibility of the N-trimer. These inhibitors show a progressive loss of inhibitory potency with increasing cargo protein size and a partial recovery of inhibition with increasing linker length. These results demonstrate that the critical hairpin-forming machinery of gp41 is sterically protected and suggest new strategies to generate neutralizing Abs that overcome this steric block.; There are many unanswered questions concerning the gp120-gp41 interface, such as (1) the specific regions of gp41 and gp120 involved, (2) the mechanism by which receptor and coreceptor binding-induced conformational changes in gp120 are communicated to gp41, (3) how six-helix bundle formation is prevented in the prefusogenic gp120-gp41 complex, and ultimately, (4) the structure of the prefusion gp120-gp41 complex. In Chapter 3, we develop a biochemical model system that mimics a key portion of the gp120-gp41 interface in the prefusogenic state. We find that a gp41 fragment containing the disulfide bond loop and the C-peptide region binds primarily to the gp120 CS region and that this interaction prevents six-helix bundle formation in the prefusogenic gp120-gp41 complex. These findings provide a foundation for future structural and mechanistic studies on the gp120-gp41 complex to understand HIV entry at the molecular level and develop new HIV therapeutics.