Multiplex discovery of synthetic host-protein combinations that inhibit HIV

项目摘要 几十年的艾滋病毒研究已经产生了一组有效的艾滋病毒药物，但我们需要新方法的帮助 来消除全球艾滋病的流行。现代生物技术开辟了新的途径， 针对病毒生命周期中剩余漏洞的工程蛋白质。新型蛋白质组合 将宿主衍生的HIV结合肽与可标记HIV蛋白降解的效应结构域连接 可以有效地干扰感染而不引起主要的细胞毒性。不幸的是，有数百个 成千上万的组合需要测试，这几乎是不可能使用传统的，一个接一个的 接近。 我们建议将尖端的合成生物学工具应用于蛋白质和细胞工程，利用多重， 基于文库的测定以鉴定最有前途的设计。在第一个目标中，我们将进行大规模的 将已知的HIV结合肽与不同的细胞泛素连接酶效应结构域成对融合， 找出具有最佳治疗潜力的组合。第二个目标是使用一个无偏见的， 一种全基因组转座子插入诱变方法，用于发现能够抑制HIV的新型结合剂 当与泛素连接酶结构域融合时复制。这两种方法都将利用新的组合， 已经在人类中发现和表达的蛋白质序列，避免对非自身肽的免疫反应 抗原这项工作将产生新的领先疗法，推进合理的蛋白质设计，并提供生物学上的 深入了解HIV蛋白靶点的化学计量和工程化的多聚化 蛋白酶体降解机制提供了最精确和特异的抗病毒应答。

PROJECT SUMMARY Decades of HIV research have yielded a panel of potent HIV drugs, but we need the help of new approaches to eliminate the global HIV epidemic. Modern biotechnologies have opened new avenues toward developing engineered proteins that target the remaining vulnerabilities in the virus life-cycle. Novel protein combinations linking host-derived HIV binding peptides with effector domains that can mark the HIV protein for degradation may potently interfere with infection without causing major cellular toxicity. Unfortunately, there are hundreds to thousands of combinations that need to be tested, which is nearly impossible using traditional, one-by-one approaches. We propose to apply cutting edge synthetic biology tools for protein and cell engineering, harnessing multiplex, library-based assays to identify the most promising designs. In the first aim, we will perform large-scale pairwise fusions of known HIV binding peptides with different cellular ubiquitin ligase effector domains, to identify the combinations that have the best therapeutic potentials. The second aim uses an unbiased, genome-wide transposon insertional mutagenesis approach to uncover novel binders capable of inhibiting HIV replication when fused to a ubiquitin ligase domain. Both approaches will harness novel combinations of protein sequences already found and expressed in humans, avoiding immune responses to non-self peptide antigens. This work will yield new lead therapeutics, advance rational protein design, and provide biological insight into how the stoichiometries of HIV protein targets and the multimerization of the engineered proteasomal degradation machinery provides the most accurate and specific antiviral responses.