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

 描述(由申请人提供)：所有病毒的生命周期都依赖于宿主细胞；因此，识别每种病毒所需的宿主因素可以揭示抗病毒开发的新靶点。我们最近报道了一种基于定量蛋白质组学的新策略，用于全面询问高置信度分子病毒与宿主的相互作用，并用登革病毒(DV)验证了这一途径。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，而且由于其蚊子媒介最近在地理上的扩张，也是对美国的潜在威胁。DV基因组只编码10种蛋白质；因此，DV依靠这几种蛋白质招募大量宿主因子进行复制，并完成其感染和繁殖周期。我们重点研究了两种对复制至关重要的DV非结构蛋白，NS5和NS3。我们的策略确定了大约850个与NS5和/或NS3互动的唯一高信心候选人。因此，这些蛋白是参与DV复制的宿主因子的有希望的候选者。挑战是扩大我们之前发现的这些相互作用的蛋白，以有效地确定哪些宿主蛋白对DV感染至关重要，以及它们如何发挥功能。我们将使用汇集的shRNA基因敲除方法从功能上全面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素，这些因素以前是通过与NS5/NS3的相互作用而确定的，这些因素对DV感染很重要。接下来，我们将测试一个高度创新的概念，即病毒蛋白和宿主蛋白之间的相互作用会降低宿主蛋白的功能，产生病毒诱导的“亚态”(vi-亚态)。因此，这些vi亚型的合成致死/合成致病(SL)相互作用伙伴也是潜在的治疗靶点，其抑制被预测选择性地杀死受感染的细胞。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。作为原则证明，我们将测试病毒诱饵的存在是否通过靶向这些假定的vi亚型的SL伙伴并确定表达病毒诱饵的细胞是否被选择性地杀死来降低其宿主伙伴的功能。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展开详细的机制研究，使这一应用非常适合R21资助机制。此外，我们基于SL的新方法将潜在地对针对广泛的细胞内病原体的新疗法的开发产生广泛的影响。

 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.