Deep mutational scanning of the HIV-1 Env protein and HIV-targeted host chemokine receptors

HIV-1 Env 蛋白和 HIV 靶向宿主趋化因子受体的深度突变扫描

• 批准号: 9348071
• 负责人: Erik Procko
• 金额: $ 37.77万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9348071
• 关键词: Adopted; Affinity; Agonist; Algorithms; Alpha Cell; Amino Acid Sequence; Amino Acid Substitution; Antibodies; Antibody Affinity; Antibody Response; Antigens; Bacteria; Bacteriophages; Big Data; Binding; Binding Sites; Biochemical; Biology; CCR5 gene; CXCR4 gene; Cell membrane; Cells; Chimeric Proteins; Clinical; Communities; Complement; Computer Simulation; Coupling; Crystallization; Data; Dimerization; Directed Molecular Evolution; Distal; Engineering; Evolution; Flow Cytometry; Fluorescence; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Proteins; HIV; HIV Envelope Protein gp120; HIV resistance; HIV-1; Human; Immunoprecipitation; In Vitro; Incubated; Integral Membrane Protein; Libraries; Ligands; Mammalian Cell; Maps; Mediating; Membrane Glycoproteins; Methods; Modeling; Molecular Conformation; Mutagenesis; Mutation; Mutation Analysis; Pharmaceutical Preparations; Phenotype; Physiological; Pilot Projects; Property; Protein Region; Proteins; Receptor Cell; Resistance; Rest; Signal Transduction; Site; Sorting - Cell Movement; Staining method; Stains; Structure; Surface; Variant; Virus; Yeasts; base; chemokine; chemokine receptor; deep sequencing; design; dimer; env Gene Products; env Glycoproteins; experimental study; extracellular; fitness; genome editing; improved; insight; mutant; mutation screening; neutralizing antibody; new technology; next generation sequencing; protein protein interaction; protein structure; receptor; resistant strain; response; structural biology; Adopted; Affinity; Agonist; Algorithms; Alpha Cell; Amino Acid Sequence; Amino Acid Substitution; Antibodies; Antibody Affinity; Antibody Response; Antigens; Bacteria; Bacteriophages; Big Data; Binding; Binding Sites; Biochemical; Biology; CCR5 gene; CXCR4 gene; Cell membrane; Cells; Chimeric Proteins; Clinical; Communities; Complement; Computer Simulation; Coupling; Crystallization; Data; Dimerization; Directed Molecular Evolution; Distal; Engineering; Evolution; Flow Cytometry; Fluorescence; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Proteins; HIV; HIV Envelope Protein gp120; HIV resistance; HIV-1; Human; Immunoprecipitation; In Vitro; Incubated; Integral Membrane Protein; Libraries; Ligands; Mammalian Cell; Maps; Mediating; Membrane Glycoproteins; Methods; Modeling; Molecular Conformation; Mutagenesis; Mutation; Mutation Analysis; Pharmaceutical Preparations; Phenotype; Physiological; Pilot Projects; Property; Protein Region; Proteins; Receptor Cell; Resistance; Rest; Signal Transduction; Site; Sorting - Cell Movement; Staining method; Stains; Structure; Surface; Variant; Virus; Yeasts; base; chemokine; chemokine receptor; deep sequencing; design; dimer; env Gene Products; env Glycoproteins; experimental study; extracellular; fitness; genome editing; improved; insight; mutant; mutation screening; neutralizing antibody; new technology; next generation sequencing; protein protein interaction; protein structure; receptor; resistant strain; response; structural biology

The HIV-1 surface glycoprotein Env engages host cell receptors, including either the CCR5 or CXCR4 chemokine receptors, to drive the necessary protein rearrangements that mediate virus entry into the cell. Therapies blocking HIV-1 Env interactions with chemokine receptors are clinically effective, underscoring their importance. This proposal uses the new technology of deep mutational scanning to comprehensively determine sequence-function relationships in CCR5, CXCR4 and Env. Deep mutational scanning combines unbiased, diverse libraries of mutations with in vitro evolution and deep sequencing, making it possible to determine the relative phenotypes of many thousands of mutations in a single experiment. From this unprecedented mutational data, a protein's sequence-fitness landscape can be experimentally mapped, from which functional sites and important residues for stabilizing discrete conformations can be inferred. The sequence-fitness landscape also reveals mutations that can be combined to engineer variants with new or enhanced properties. Deep mutational scanning has primarily been limited to proteins that are expressed in phage, bacteria or yeast, but in this proposal, libraries encompassing all single amino acid substitutions of CCR5, CXCR4 and Env expressed in human cells will be evolved. The specific aims of this proposal are Aim 1: To determine the oligomeric organization of CCR5 and CXCR4 by deep mutational scanning. When libraries of CCR5 and CXCR4 are sorted for high affinity to antibodies recognizing resting conformations, conserved residues in the sequence-fitness landscapes map to transmembrane surfaces of the receptors. We hypothesize that these conserved surfaces are dimerization sites, which will be validated using biochemical methods. Residue conservation scores from the mutational scans will guide computational modeling of the dimeric states. Aim 2: To comprehensively map the sequence-fitness landscapes of CCR5 and CXCR4 during signaling responses to agonists. A cell sorter will be adapted for continuous mixing and sorting of Ca2+- indicator stained libraries with chemokines. Critical residues for chemokine interactions, G protein coupling, and adopting an active conformation will be conserved in the sequence-fitness landscapes. Aim 3: To characterize the interaction between chemokine receptors and HIV-1 gp120-CD4 by deep mutational scanning. CCR5 and CXCR4 sequence-fitness landscapes for tight affinity to gp120-CD4 will reveal similarities and differences in how these chemokine receptors are engaged by R5 and X4 HIV-1 strains, and how maraviroc- resistant Env clones have altered CCR5 interaction footprints. Aim 4: To comprehensively determine the sequence-fitness landscape of HIV-1 Env interacting with soluble CD4 and broadly neutralizing antibodies VRC01 and PG16. These protein ligands recognize distinct Env quaternary structures, despite CD4 and VRC01 sharing a common binding site. Deep mutational scanning, covering over 17,000 Env mutations, will guide engineering of trimeric Env to pre-stabilize conformations, with implications for immunogen design.

HIV-1表面糖蛋白EV与宿主细胞受体接合，包括CCR5或CXCR4 趋化因子受体，以驱动介导病毒进入细胞的必要蛋白质重排。 阻断HIV-1环境与趋化因子受体相互作用的疗法在临床上有效，强调了它们 重要性。该建议使用深层突变扫描的新技术进行全面 确定CCR5，CXCR4和Env中的序列功能关系。深突变扫描结合了 具有体外进化和深度测序的突变的公正，多样化的库，使得成为可能 在一个实验中确定数千种突变的相对表型。由此 空前的突变数据，可以从实验映射蛋白质的序列拟合景观。 可以推断哪些功能位点和稳定离散构象的重要残基。这 序列拟合景观还揭示了可以与新的或具有新的或 增强的特性。深突变扫描主要仅限于在 噬菌体，细菌或酵母 在人类细胞中表达的CCR5，CXCR4和ENV将进化。该提议的具体目的是目标 1：通过深突变扫描来确定CCR5和CXCR4的寡聚组织。什么时候 CCR5和CXCR4的库被分类以高亲和力，以识别静止构象的抗体， 序列拟合景观中的保守残基映射到受体的跨膜表面。我们 假设这些保守的表面是二聚化位点，将使用生化验证 方法。突变扫描中的残留保护分数将指导的计算建模 二聚体状态。目的2：全面绘制CCR5和CXCR4的序列拟合景观 对激动剂的信号反应。细胞分类器将被调整以连续混合和分类Ca2+ - 指标用趋化因子染色文库。趋化因子相互作用，G蛋白偶联的关键残基， 并采用主动构象将在序列拟合景观中保存。目标3：到 通过深突变扫描来表征趋化因子受体与HIV-1 GP120-CD4之间的相互作用。 CCR5和CXCR4序列拟合景观与GP120-CD4的紧密亲和力将揭示相似性和 这些趋化因子受体如何通过R5和X4 HIV-1菌株参与的差异，以及如何maraviroc- 抗性ENV克隆改变了CCR5相互作用的足迹。目标4：全面确定 HIV-1 Env的序列拟合度与可溶性CD4相互作用并广泛中和抗体 VRC01和PG16。尽管CD4和 VRC01共享一个共同的绑定位点。深度突变扫描，涵盖17,000多个环境的突变，将 三聚合物Env的指导工程，以预先稳定构象，对免疫原设计产生影响。