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Fusion glycoproteins of HIV-1 and the Ebola Virus: Structure, function, and inhibition with antibodies.

机译：HIV-1和埃博拉病毒的融合糖蛋白：抗体的结构，功能和抑制作用。

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摘要

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.
机译：本文涵盖两个主要主题，共同分析了人类I型免疫缺陷病毒（HIV-1）（gp41）和埃博拉病毒（GP2）I类融合病毒的融合糖蛋白。第一个主题是HIV-1，分为两项研究，一项是关于gp41七肽重复（HR）区域的研究，另一项是关于gp41膜近端外部区域（MPER）的研究，这两项研究都是高度保守的，可以通过广泛中和来靶向抗体（BNAb）。第一项研究使用极简多样性合成抗体（Ab）文库来研究gp41 HR区域。受体结合后，HR区域形成α-螺旋盘绕线圈，形成六螺旋束，降低了融合的动力学势垒。生成了噬菌体展示的Fab库，其多样性仅限于酪氨酸和丝氨酸（Y / S），并针对5-螺旋（5H），gp41 HR模拟结构和进入抑制剂进行了淘选。鉴定出Y / S Fab特异性结合5H，其亲和力与靶向5H的B细胞来源的BNAb相当，但是它们不显示中和活性。这项研究确定了对5H具有特异性和高亲和力的极简合成抗体，可以用作诊断，研究和治疗工具。第二项研究剖析了以MPER为目标的BNAb Z13e1中和HIV-1的机制。假设增加Z13e1的膜结合活性将增强其中和效能，因为更广泛和有效的BNAb 4E10以与Z13e1相当的亲和力与MPER结合，但具有中和活性所需的更高的膜结合活性。定点诱变产生了几个Z13e1 IgG突变体以测试中和活性，但是这些突变体不稳定并且无法检验该假设。相反，讨论了对HIV-1 MPER，与其相互作用的BNAb以及其中和活性的潜在机制的详尽综述。第二个主要主题是埃博拉病毒GP2 MPER在与生理相关的脂质环境中的特征。合成并纯化了代表两种埃博拉病毒种的MPER的肽。在不存在形成胶束的去污剂的情况下，将圆二色性，荧光和核磁共振波谱结合起来进行测定，以确定膜环境中肽的结构以及这些脂质相互作用的性质。已发现，MPER肽在胶束结合后在很大程度上未结构化并且在其C末端变成α-螺旋，并且具有适度抑制病毒进入的能力。但是，它们没有引起囊泡渗漏，这是针对gp41 MPER观察到的功能。最后一章提供了从整个论文中讨论的发现得出的结论，以及它们如何结合在一起以提供对I类融合病毒的进入机制的洞察力以及可用于抑制进入的方法。

著录项

  • 作者

    Regula, Lauren K.;

  • 作者单位

    Yeshiva University.;

  • 授予单位 Yeshiva University.;
  • 学科 Chemistry Biochemistry.;Health Sciences Immunology.;Biology Virology.
  • 学位 Ph.D.
  • 年度 2013
  • 页码 165 p.
  • 总页数 165
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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