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: 10889837
• 负责人: Eva-Maria Strauch
• 金额: $ 19.44万
• 依托单位: WASHINGTON UNIVERSITY
• 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 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10889837
• 关键词: Adaptor Signaling Protein; Address; Advanced Development; Affect; Affinity; Amino Acid Substitution; Antibodies; Architecture; Binding; Biological Assay; Biological Models; Biology; Cell-Matrix Junction; Cells; Chimeric Proteins; Clinical Trials; Complex; 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; Measles virus; 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; Scanning; Site; Structural Protein; Structure; Surface; System; Technology; Tissues; Viral; Viral Physiology; Viral Proteins; Virus; Virus Diseases; Virus Integration; Virus Receptors; Visualization; antibody engineering; anticancer treatment; cell type; delivery vehicle; design; 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; usability

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 在缺乏选择性递送的情况下，许多有前途的药物无法到达靶细胞，而是 另一方面，许多病毒已经掌握了识别技术。 例如，它们依赖于“局部”。 蛋白酶（有时是两种）来激活其融合蛋白。为了开发更好的靶向装置，我们的目标是 剖析病毒表面蛋白中嵌入的分子特性和功能，特别关注 受体相互作用、稳定性和融合触发（目标 I），我们将采用表面显示 我们将重点关注副粘病毒（PMV）的病毒表面机制。 大多数 PMV 都有分工，以保持宿主受体结合和细胞进入。 分开作为两个独立的功能编码成两个分子：一个四聚体蛋白质负责分子 识别和负责宿主和病毒膜融合的三聚体融合蛋白。 区室化使 PMV 成为重新利用的优秀模型系统，因为融合蛋白可以 保持不变，而识别过程可以重新设计。 我们将利用深度突变扫描来评估所有可能的氨基酸 通过获取每个基因的不同适应度景观来替代任何给定的基因选择。 讨论了分子特性，我们将能够解决有关生物学的有趣问题 病毒，例如其蛋白质化学背景下的突变耐受性，重要的是，适应性景观将会。 对输送设备的工程产生直接影响，因为它们将提供大致的蓝图 我们将使用获得的序列-功能-结构图来构建这些复杂机器的分子结构。 开发一种新的、适应性强的靶向递送平台，该平台将整合病毒表面 具有抗体片段的机器（目标 II）。关键点是开发一种接头分子 整合抗体片段，同时保持病毒识别机制的所有调节功能 通常表现出涉及构象变化的控制，以前的努力尚未成功。 在这里，我们将获得并利用一个宝贵的数据库。 病毒蛋白质结构以及我们新获得的序列功能知识，我们将其与 新技术——蛋白质设计——来推进这个看似简单但雄心勃勃的工程项目。 我们的目标是为新的靶向机制提供一个普遍适用的平台，其中包含这些 分子机制，同时还利用大量已识别和工程化的抗体。 通过将病毒感染机制的一部分与抗体片段和接头蛋白相结合，我们 预计我们将能够显着推进药物和基因传递系统的开发， 从而也为基因组工程提供了急需的新的精确靶向技术。