Mechanistic Studies of Viral Host Cell Recognition and Entry and their Implication for Protein Design of Molecular Delivery Devices

病毒宿主细胞识别和进入的机制研究及其对分子递送装置蛋白质设计的意义

• 批准号: 10527903
• 负责人: Eva-Maria Strauch
• 金额: $ 22.42万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-23 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10527903
• 关键词: Adaptor Signaling Protein; Address; Advanced Development; Affect; Affinity; Amino Acid Substitution; Antibodies; Architecture; Arts; Binding; Biological Assay; Biological Models; Biology; Cell-Matrix Junction; Cells; Chimeric Proteins; Clinical Trials; Complex; Crystallization; Data; Databases; Development; Devices; Drug Delivery Systems; Engineering; Equilibrium; Event; Exhibits; Family; Gene Delivery; Genome engineering; Goals; HIV; Head; Hereditary Disease; Homing; Human body; Immunoglobulin Fragments; Infection; Knowledge; Laboratories; Libraries; Lifting; Malignant Neoplasms; Maps; Membrane; Membrane Fusion; Membrane Fusion Activity; Membrane Proteins; Methodology; Molecular; Molecular Conformation; Mumps; Mutagenesis; Mutation; Parainfluenza; Paramyxovirus; Peptide Hydrolases; Pharmaceutical Preparations; Phenotype; Play; Process; Property; Protein Chemistry; Protein Engineering; Proteins; Reagent; Receptor Cell; Rewards; Scanning; Site; Structural Protein; Structure; Surface; System; Technology; Tissues; Viral; Viral Physiology; Viral Proteins; Virus; Virus Diseases; Virus Receptors; antibody engineering; anticancer treatment; base; cell type; delivery vehicle; design; exhaustion; fitness; gene delivery system; genetic selection; genome editing; high reward; high risk; human disease; improved; insight; member; molecular recognition; mutation screening; new technology; novel strategies; parainfluenza virus; pressure; protein structure; receptor; receptor binding; side effect; stem; targeted delivery; tool

Summary In the absence of selective delivery, many promising drugs do not reach the targeted cells, but rather cause toxic side effects. Many viruses, on the other hand, have mastered the art of identifying microenvironmental clues and selectively find and infect a specific cell. For instance, they depend on “local” proteases, sometimes two, to activate their fusion proteins. To develop better targeting devices, we aim to dissect molecular properties and functions embedded in viral surface proteins, specifically focusing on receptor interactions, stability, and fusion triggering (Aim I) for which we will employ surface display and infectivity assays. We will focus on the viral surface machinery of the paramyxoviruses (PMVs), specifically Parainfluenza virus 5 (PIV5). Most PMVs have a division of labor keeping host receptor binding and cell entry apart as two separate functions encoded into two molecules: one tetrameric protein responsible for molecular recognition and a trimeric fusion protein responsible for the merging of host and viral membranes. This compartmentalization makes the PMVs an excellent model system for repurposing as the fusion protein can remain untouched, while the recognition process can be re-engineered. We will take advantage of deep mutational scanning which allows us to evaluate all possible amino acid substitutions for any given genetic selection. By acquiring differential fitness landscapes for each of the aforementioned molecular properties, we will be able to address interesting questions about the biology of viruses, such as mutational tolerance in context of their protein chemistry. Importantly, fitness landscapes will have an immediate impact on engineering of delivery devices as they will provide rough blueprints of the molecular architecture of these complex machineries. We will use obtained sequence-function-structure maps for the development of a new, adaptable targeted delivery platform that will integrate viral surface machinery with antibody fragments (Aim II). The key point will be to develop an adapter molecule that integrates the antibody fragment while maintaining all regulatory function that the viral recognition machinery normally exhibits, which involves control of conformational changes. Previous efforts have not succeeded in developing an efficient, general delivery system. Here, we will obtain and leverage an invaluable database of virus protein structures together with our newly obtained sequence-function knowledge, which we combine with new technology – protein design – to advance this seemingly simple but ambitious engineering project. We aim to provide a generally applicable platform for a new targeting machinery that incorporates these molecular mechanisms while also taking advantage of the vast amount of identified and engineered antibodies. Through combining parts of the viral infection machinery with antibody fragments and adapter proteins, we anticipate that we will be able to significantly advance the development of drug and gene delivery systems and thereby also provide new and much needed precision targeting technology for genome engineering.

概括 在没有选择性递送的情况下，许多有前途的药物没有到达目标细胞，而是 导致有毒的副作用。另一方面，许多病毒已经掌握了识别的艺术 微环境线索并有选择地查找并感染了特定的细胞。例如，它们取决于“本地” 蛋白酶，有时两个，以激活其融合蛋白。为了开发更好的定位设备，我们的目标是 剖析病毒表面蛋白中的分子特性和功能，特别关注 受体相互作用，稳定性和融合触发（AIM I），我们将向其雇员表面显示 和感染分析。我们将重点关注帕托伏病毒（PMV）的病毒表面机械，特别是 Parainfluenza病毒5（PIV5）。大多数PMV的人工划分保持宿主受体结合和细胞进入 除以两个编码两个分子的两个独立功能：一个负责分子的四聚体蛋白 识别和三聚体融合蛋白负责宿主和病毒膜的合并。这 隔室化使PMVS成为重新利用的极好模型系统，因为融合蛋白可以 保持未触及，同时可以重新设计识别过程。 我们将利用深突变扫描，使我们能够评估所有可能的氨基酸 取代任何给定的遗传选择。通过获取差异化健身景观 关于分子特性，我们将能够解决有关生物学的有趣问题 病毒，例如在其蛋白质化学背景下突变耐受性。重要的是，健身景观将 对交付设备的工程有直接影响，因为它们将提供粗糙的蓝图 这些复杂机器的分子结构。我们将使用获得的序列函数结构图 为了开发新的，适应性的目标输送平台，该平台将整合病毒表面 具有抗体片段的机械（AIM II）。关键是要开发一个适配器分子 整合抗体片段，同时保持病毒识别机制的所有调节功能 通常展示，涉及控制构象变化。以前的努力尚未成功 开发一个高效的一般交付系统。在这里，我们将获得并利用一个宝贵的数据库 病毒蛋白结构以及我们新获得的序列功能知识，我们将其与之结合 新技术 - 蛋白质设计 - 推进这似乎是简单但雄心勃勃的工程项目。 我们旨在为新的定位机械提供一个普遍适用的平台，该平台合并这些 分子机制，同时还利用了大量已鉴定和工程抗体的优势。 通过将病毒感染机械的部分与抗体片段和衔接蛋白相结合，我们 预计我们将能够显着提高药物和基因输送系统的发展，以及 因此，还为基因组工程提供了新的急需的精确定位技术。