Applying the principle of synthetic lethality to virus-host protein-protein interactions as a novel approach for antiviral development

 描述（由申请方提供）：所有病毒的生命周期都依赖于宿主细胞;因此，鉴定每种病毒所需的宿主因子可以揭示抗病毒开发的新靶点。我们最近报道了一种新的基于定量蛋白质组学的策略，用于以高置信度的分子病毒-宿主相互作用进行全面询问，并用登革热病毒（DV）验证了该管道。DV是一种重要的蚊媒病毒，对世界大部分地区造成重大的经济和公共卫生负担，并且由于其蚊媒最近的地理扩张，也是对美国的潜在威胁。DV基因组仅编码10种蛋白质;因此，DV依赖于这几种蛋白质来招募许多宿主因子来复制并完成其感染和繁殖周期。我们专注于两个DV的非结构蛋白，是必不可少的复制，NS 5和NS 3。我们的策略确定了约850个与NS 5，NS 3或两者相互作用的独特的高置信度候选者。因此，这些蛋白质是参与DV复制的宿主因子的有希望的候选者。挑战是扩展我们先前发现的这些相互作用的蛋白质，以有效地确定哪些宿主蛋白质对DV感染至关重要，以及它们如何发挥其功能。我们将使用合并的shRNA基因敲低方法全面功能验证我们的相互作用数据集。这项工作将产生一系列宿主因子，这些因子先前通过它们与NS 5/NS 3的相互作用而被鉴定，这些因子对于DV感染是重要的。我们接下来将测试高度创新的概念，即病毒蛋白质和宿主蛋白质之间的相互作用降低宿主蛋白质的功能，产生病毒诱导的“亚型”（vi-亚型）。因此，这些vi-亚型的合成致死/合成病态（SL）相互作用伴侣也是潜在的治疗靶标，预测其抑制选择性地杀死感染的疟原虫。使用比较基因组学策略，我们将预测和测试从酵母到人类的SL相互作用的保守性。作为原理证明，我们将通过靶向这些推定的vi亚型的SL配偶体并确定表达病毒诱饵的细胞是否被选择性杀死来测试病毒诱饵的存在是否降低其宿主配偶体的功能。我们在这里提出的探索性方法将使我们能够对DV建立有利于感染的环境的分子机制进行详细的机制研究，使这种应用非常适合R21资助机制。此外，我们的新SL为基础的方法将有可能对开发新的治疗方法对广泛的细胞内病原体的广泛影响。

 DESCRIPTION (provided by applicant): All viruses depend on host cells for their life cycle; thus, identifying the host factors required by each virus can reveal novel targets for antiviral development. We recently reported a novel quantitative proteomics-based strategy for comprehensively interrogating with high confidence molecular viral-host interactions and validated this pipeline with dengue virus (DV). DV is an important mosquito-borne virus that poses significant economic and public health burdens on much of the world and is also a potential threat to the US due to the recent geographical expansion of its mosquito vectors. The DV genome encodes only 10 proteins; thus, DV relies on these few proteins to recruit numerous host factors to replicate and complete its cycle of infection and reproduction. We focused on two DV nonstructural proteins that are essential for replication, NS5 and NS3. Our strategy identified ~850 unique high confidence candidates that interacted with NS5, NS3, or both. These proteins are thus promising candidates for host factors involved in DV replication. The challenge is to extend our previous discovery of these interacting proteins to efficiently determine which host proteins are critical for DV infection and how they perform their functions. We will comprehensively functionally validate our interaction datasets using a pooled shRNA gene knockdown approach. This work will yield a list of host factors, previously identified through thei interactions with NS5/NS3, which are important for DV infection. We will next test the highly innovative concept that interactions between viral proteins and host proteins reduce the functions of host proteins, generating viral-induced "hypomorphs" (vi-hypomorphs). The synthetic lethal/synthetic sick (SL) interaction partners of these vi-hypomorphs are thus also potential therapeutic targets, the inhibition of which is predicted to selectively kill infected cels. Using a comparative genomics strategy, we will predict and test the conservation of SL interactions from yeast to humans. As proof-of-principle, we will test whether the presence of the viral bait reduces the function of its host partners by targeting the SL partners of these putative vi- hypomorphs and determining whether cells expressing the viral bait are selectively killed. The exploratory approach we propose here will allow us to launch into detailed mechanistic studies of the molecular mechanisms by which DV establishes an environment favorable for infection, making this application highly suitable for the R21 funding mechanism. Moreover, our novel SL-based approach will potentially have a broad impact on the development of novel therapeutics against a wide range of intracellular pathogens.