Glycoproteins and Entry into Host Cells – Structure and Function of the KSHV fusion machinery

糖蛋白和进入宿主细胞 âKSHV 融合机制的结构和功能

• 批准号: 530009014
• 负责人: Dr. Alexander Hahn
• 金额: --
• 依托单位: Virologie Structurale
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/530009014?language=en
• 关键词: Glycoproteins; Entry; into; Host; Cells

Kaposi’s sarcoma herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma and two B cell malignancies. KSHV engages diverse cellular receptors to enter target cells via endocytosis. KSHV glycoproteins gB and gH/gL form the 'core fusion machinery' that induces merger of viral and endosomal membranes, allowing for the genome release into the cytosol. gB is the actual fusogen. The gH/gL complex regulates gB activity, in a still unknown manner. The fusion trigger for gB has not been identified either. In addition to its role in fusion, gB binds to integrins and DC-SIGN, and these interactions promote endocytosis. We discovered that the EphA receptors play an important role in KSHV entry by engaging gH/gL, and recently determined the structure of the gH/gL-EphA2 complex, but structural information on gB and complexes with its receptors is entirely missing. Since KSHV infection can occur independently of the known receptors, the list of the cellular receptors mentioned above is not exhaustive. This project will be carried out as a collaboration between Marija Backovic, a structural biologist specializing in viral glycoproteins, and Alexander Hahn, who is a molecular virologist and expert in genetic manipulation and biology of KSHV biology. Each partner is uniquely qualified and has a long-standing experience and technical know-how in different aspects of herpesvirus biology. Thus, we propose an integrated approach that relies on structural biology (X-ray crystallography and cryo-EM) and biophysics (Backovic), and functional studies to investigate virus entry, virus-receptor interactions, and host factor identification (Hahn). Our objectives are to 1) generate gB variants stabilized in the pre-fusion state, 2) search for new KSHV receptors by CRISPR/Cas9, 3) determine the interaction sites for gB bound to cellular receptors (known, and TIM-1 and NRP1, which were recently identified by Hahn group), 4) identify fusion trigger (pH and/or a receptor), 5) obtain a panel of neutralizing nanobodies (Nb) by immunizing llamas with stabilized gB and gH/gL, and 6) identify the best neutralizers and determine their structures in complexes with gB or gH/gL. The reason we plan to develop Nbs is the lack of monoclonal Abs (mAbs) against KSHV GPs. Thus, one of the most informative tools to study GP-mediated entry is largely missing in the field. Knowing the epitopes targeted by neutralizing Nbs will reveal vulnerabilities of the virus that can be exploited for the development of a prophylactic vaccine or therapeutic antibodies. Overall, these studies have a potential to significantly advance our understanding of the function of the fusion machinery and its interactions with cellular receptors, on a structural and on a functional level. The outcomes of this proposal will in addition inform us on the design of KSHV immunogens, opening roads to further studies focusing on the development of KSHV vaccine candidates.

卡波西肉瘤疱疹病毒(KSHV)是卡波西肉瘤和两种B细胞恶性肿瘤的病原体。KSHV与多种细胞受体结合，通过内吞作用进入靶细胞。KSHV糖蛋白gB和Gh/gl形成‘核心融合机制’，诱导病毒和内体膜的合并，允许基因组释放到细胞质中。GB是实际的FusoGen。Gh/Gl复合体以一种未知的方式调节GB的活动。GB核聚变的触发因素也尚未确定。除了在融合中的作用外，GB还与整合素和DC-SIGN结合，这些相互作用促进内吞作用。我们发现EphA受体通过与Gh/Gl结合在KSHV进入KSHV中发挥重要作用，并最近确定了Gh/Gl-EphA2复合体的结构，但关于Gb及其受体复合体的结构信息完全缺乏。由于KSHV感染可以独立于已知的受体发生，上述细胞受体的清单并不详尽。该项目将由专门研究病毒糖蛋白的结构生物学家Marija Backovic和分子病毒学家、KSHV生物学遗传操纵和生物学专家Alexander Hahn合作实施。每个合作伙伴都是唯一合格的，在疱疹病毒生物学的不同方面都有长期的经验和技术诀窍。因此，我们提出了一种综合的方法，依赖于结构生物学(X射线结晶学和低温EM)和生物物理学(Backovic)，以及功能研究来研究病毒入侵、病毒-受体相互作用和宿主因子识别(Hahn)。我们的目标是1)产生稳定在融合前状态的Gb变异体，2)通过CRISPR/Cas9寻找新的KSHV受体，3)确定Gb与细胞受体(已知，以及Hahn小组最近发现的Tim-1和Nrp1)的相互作用部位，4)识别融合触发器(pH和/或受体)，5)通过用稳定的Gb和Gh/gl免疫大羊驼获得一组中和纳米体(Nb)，以及6)确定最好的中和剂并确定它们在Gb或Gh/gl复合体中的结构。我们计划发展NBS的原因是缺乏抗KSHV GPs的单抗。因此，研究GP介导的条目的最具信息量的工具之一在该领域基本上是缺失的。了解中和NBS所针对的表位将揭示病毒的脆弱性，可用于开发预防性疫苗或治疗性抗体。总体而言，这些研究有可能显著提高我们对融合机制的功能及其与细胞受体在结构和功能水平上的相互作用的理解。这项提案的结果还将为我们提供有关KSHV免疫原设计的信息，为进一步研究KSHV候选疫苗的开发开辟道路。