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

HIV-1表面糖蛋白Env与宿主细胞受体结合，包括CCR5或CXCR4 趋化因子受体，以驱动必要的蛋白质重排，介导病毒进入细胞。 阻断HIV-1Env与趋化因子受体相互作用的疗法在临床上是有效的，强调了它们的 重要性。该方案利用深度突变扫描新技术，全面 确定CCR5、CXCR4和Env中的序列-功能关系。深度突变扫描联合收割机 通过体外进化和深度测序，无偏见的、多样化的突变文库，使其有可能 在一次实验中确定数千种突变的相对表型。由此 史无前例的突变数据，蛋白质的序列适合性图景可以通过实验绘制，从 可以推断稳定离散构象的功能位点和重要残基。这个 序列适合度图景还揭示了突变，这些突变可以结合在一起来设计具有新的或 增强的特性。深度突变扫描主要局限于表达在 噬菌体、细菌或酵母，但在本提案中，包含所有单一氨基酸替换的文库 在人类细胞中表达的CCR5、CXCR4和Env将被进化。这项建议的具体目的是 1.通过深层突变扫描确定CCR5和CXCR4的寡聚结构。什么时候 对CCR5和CXCR4的文库进行排序，以获得识别静止构象的抗体的高亲和力， 序列适合性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，这将用生化方法进行验证 方法：研究方法。来自突变扫描的残基守恒分数将指导对 二聚态。目的2：全面绘制CCR5和CXCR4的序列适宜性图谱 向激动剂发出反应信号。细胞分选机将适用于连续混合和分选钙离子- 趋化因子指示剂染色文库。趋化因子相互作用的关键残基，G蛋白偶联， 而采用主动构象将在序列适应度景观中保持保守。目标3：实现 通过深层突变扫描研究趋化因子受体与HIV-1gp120-CD4之间的相互作用。 CCR5和CXCR4序列适合与gp120-CD4紧密结合的环境将揭示相似性和 R5和X4 HIV-1毒株如何接触这些趋化因子受体的差异，以及马拉韦罗如何- 抗性环境克隆改变了CCR5相互作用足迹。目标四：全面确定 HIV-1包膜蛋白与可溶性CD4和广谱中和抗体相互作用的序列适合性图谱 VRC01和PG16。这些蛋白质配体识别不同的Env四级结构，尽管CD4和 VRC01共享一个共同的结合位点。深度突变扫描，覆盖17,000多个Env突变，将 指导三聚体环境工程以预先稳定构象，并对免疫原设计产生影响。

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.