Fusion glycoproteins of HIV-1 and the Ebola Virus: Structure, function, and inhibition with antibodies
Regula, Lauren K.
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There are two major topics covered in this thesis, together providing an analysis of the fusion glycoproteins of human immunodeficiency virus type-I (HIV-1) (gp41) and the Ebola Virus (GP2), class I fusion viruses. The first topic, HIV-1, is divided into two studies, one on the gp41 heptad-repeat (HR) regions and one on the gp41 membrane-proximal external region (MPER), both of which are highly conserved and targeted by broadly neutralizing antibodies (BNAbs). The first study uses minimalist diversity synthetic antibody (Ab) libraries to study the gp41 HR regions. Upon receptor binding, the HR regions form alpha-helical coiled coils to create a six-helix bundle which lowers the kinetic barrier for fusion. A phage-displayed Fab library with diversity restricted to tyrosine and serine (Y/S) was generated and panned against 5-helix (5H), a gp41 HR mimic structure and entry inhibitor. Y/S Fabs were identified that bound specifically to 5H with affinity comparable to a B cell-derived BNAb that targets 5H, however they did not show neutralizing activity. This study identified minimalist synthetic antibodies with specificity and high affinity for 5H which can be used as diagnostic, research, and therapeutic tools. The second study dissects the mechanism of HIV-1 neutralization by the MPER-targeting BNAb Z13e1. It was hypothesized that increasing the membrane-binding activity of Z13e1 would enhance its neutralization potency because the more broad and potent BNAb 4E10 binds to the MPER with comparable affinity to Z13e1 but possesses higher membrane-binding activity that is required for its neutralizing activity. Site-directed mutagenesis yielded several Z13e1 IgG mutants to test for neutralizing activity, however these mutants were unstable and the hypothesis could not be tested. Instead, a thorough review of the HIV-1 MPER, the BNAbs that interact with it, and potential mechanisms for their neutralizing activity are discussed.;The second major topic characterizes the Ebola virus GP2 MPER in its physiologically-relevant lipid environment. Peptides representing the MPER of two Ebolavirus species were synthesized and purified. A combination of circular dichroism, fluorescence, and nuclear magnetic resonance spectroscopy were performed in the absence and presence of micelle-forming detergents to determine the structure of the peptide in a membrane environment as well as the nature of these lipid interactions. It was found that the MPER-peptides are largely unstructured and become alpha-helical at their C-terminus upon micelle binding, and have the ability to moderately inhibit viral entry. However, they did not induce leakage in vesicles, a function that is observed for the gp41 MPER. The final chapter provides the conclusions drawn from the findings discussed throughout this thesis and how they tie together to give insight to the entry mechanism of class I fusion viruses and approaches that can be used to inhibit entry.
- Theses and Dissertations