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
基本信息
- 批准号:9348071
- 负责人:
- 金额:$ 37.77万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AdoptedAffinityAgonistAlgorithmsAlpha CellAmino Acid SequenceAmino Acid SubstitutionAntibodiesAntibody AffinityAntibody ResponseAntigensBacteriaBacteriophagesBig DataBindingBinding SitesBiochemicalBiologyCCR5 geneCXCR4 geneCell membraneCellsChimeric ProteinsClinicalCommunitiesComplementComputer SimulationCouplingCrystallizationDataDimerizationDirected Molecular EvolutionDistalEngineeringEvolutionFlow CytometryFluorescenceG-Protein-Coupled ReceptorsG-substrateGTP-Binding ProteinsHIVHIV Envelope Protein gp120HIV resistanceHIV-1HumanImmunoprecipitationIn VitroIncubatedIntegral Membrane ProteinLibrariesLigandsMammalian CellMapsMediatingMembrane GlycoproteinsMethodsModelingMolecular ConformationMutagenesisMutationMutation AnalysisPharmaceutical PreparationsPhenotypePhysiologicalPilot ProjectsPropertyProtein RegionProteinsReceptor CellResistanceRestSignal TransductionSiteSorting - Cell MovementStaining methodStainsStructureSurfaceVariantVirusYeastsbasechemokinechemokine receptordeep sequencingdesigndimerenv Gene Productsenv Glycoproteinsexperimental studyextracellularfitnessgenome editingimprovedinsightmutantmutation screeningneutralizing antibodynew technologynext generation sequencingprotein protein interactionprotein structurereceptorresistant strainresponsestructural biology
项目摘要
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Env接合宿主细胞受体,包括CCR 5或CXCR 4
趋化因子受体,驱动介导病毒进入细胞的必要蛋白质重排。
阻断HIV-1 Env与趋化因子受体相互作用的疗法在临床上是有效的,这强调了其
重要性该方案利用深度突变扫描新技术,
确定CCR 5、CXCR 4和Env.深度突变扫描结合了
无偏见的,多样化的突变库与体外进化和深度测序,使其有可能
在一次实验中确定数千种突变的相对表型。从这个
前所未有的突变数据,蛋白质的序列适应度景观可以通过实验绘制,从
可以推断出稳定离散构象的功能位点和重要残基。的
序列适应度景观还揭示了突变,可以组合以工程改造具有新的或
增强性能。深度突变扫描主要局限于表达于大肠杆菌中的蛋白质。
噬菌体、细菌或酵母,但在本提案中,包含噬菌体、细菌或酵母的所有单个氨基酸取代的库
在人类细胞中表达的CCR 5、CXCR 4和Env将被进化。该提案的具体目标是
1:通过深度突变扫描确定CCR 5和CXCR 4的寡聚体结构。当
分选CCR 5和CXCR 4文库对识别静息构象的抗体的高亲和力,
序列适合度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,这将通过生物化学方法来验证。
方法.来自突变扫描的残基保守性得分将指导突变的计算建模。
二聚态目的2:全面绘制CCR 5和CXCR 4的序列适应度景观,
对激动剂的信号应答。细胞分选机将适用于连续混合和分选Ca 2 +-
用趋化因子指示剂染色的文库。趋化因子相互作用,G蛋白偶联,
并且采用活性构象将在序列适合度景观中被保守。目标3:
通过深度突变扫描表征趋化因子受体与HIV-1 gp 120-CD 4之间的相互作用。
CCR 5和CXCR 4与gp 120-CD 4紧密亲和力的序列适应度景观将揭示相似性,
这些趋化因子受体如何与R5和X4 HIV-1毒株结合的差异,以及马拉韦罗-
抗性Env克隆改变了CCR 5相互作用足迹。目标4:全面确定
HIV-1 Env与可溶性CD 4和广泛中和抗体相互作用的序列适合度图谱
VRC 01和PG 16。这些蛋白质配体识别不同的Env四级结构,尽管CD 4和
VRC 01具有共同的结合位点。深度突变扫描,覆盖超过17,000个Env突变,
指导三聚体Env的工程化以预稳定构象,并对免疫原设计产生影响。
项目成果
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