Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host-directed antivirals.
Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host-directed antivirals.
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DOI：
10.1073/pnas.2201208119
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发表时间：
2022-07-26
影响因子：
11.1
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中科院分区：
文献类型：
--
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拉萨病毒(LASV)是拉沙热(LF)的病原体，在西非是一个重要的公共卫生问题。目前还没有食品和药物管理局(FDA)批准的治疗干预措施来治疗LF。由于其有限的基因组编码能力，LASV蛋白通常是多功能的，并与细胞因子协调复杂的相互作用，以执行完成病毒生命周期所需的步骤。LASV聚合酶对病毒基因组的复制和表达是必不可少的，因此是抗病毒干预的一个有吸引力的目标。在这里，我们介绍了LASV聚合酶的宿主相互作用组，它可以指导识别新的可药物宿主细胞靶点，以开发具有成本效益的LF抗病毒治疗。拉萨病毒(LASV)生命周期的完成关键取决于病毒编码的、依赖于RNA的RNA聚合酶在感染细胞的细胞质中复制和转录病毒RNA基因组的活性。细胞蛋白在这些过程中的作用尚不清楚。在这里，我们应用邻近蛋白质组学来定义在重建LASV RNA合成的条件下细胞中LASV聚合酶的相互作用组。我们设计了一种LASV聚合酶-生物素连接酶(TurboID)融合蛋白，它保留了聚合酶活性，并成功地使近端蛋白质组生物素化，从而鉴定了42个高可信的LASV聚合酶相互作用。随后，我们进行了小干扰RNA(SiRNA)筛选，以确定在真正的LASV感染中具有功能作用的相互作用因素。作为原理的证明，我们鉴定了真核细胞肽链释放因子亚单位3a(eRF3a/GSPT1)，我们发现它是一个与LASV聚合酶物理上相关的前病毒因子。小分子候选药物CC-90009靶向降解GSPT1后，对培养细胞中LASV感染有较强的抑制作用。我们的工作证明了利用邻近蛋白质组学来阐明和表征尚未定义的宿主-病原体相互作用组的可行性，这将揭示新的生物学特性，并为开发抗高致病性RNA病毒的抗病毒药物提供新的靶点。
Lassa virus (LASV), the causative agent of Lassa fever (LF), represents an important public health problem in Western Africa. There is no Food and Drug Administration (FDA)-approved therapeutic intervention to treat LF. Because of their limited genome coding capacity, LASV proteins are often multifunctional and orchestrate complex interactions with cellular factors to execute steps required to complete the viral life cycle. LASV polymerase is essential for replication and expression of the viral genome and, thus, is an attractive target for antiviral intervention. Here, we present the host interactome of LASV polymerase that can guide identification of novel druggable host cellular targets for the development of cost-effective antiviral therapies for LF. Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded, RNA-dependent RNA polymerase in replication and transcription of the viral RNA genome in the cytoplasm of infected cells. The contribution of cellular proteins to these processes remains unclear. Here, we applied proximity proteomics to define the interactome of LASV polymerase in cells under conditions that recreate LASV RNA synthesis. We engineered a LASV polymerase-biotin ligase (TurboID) fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed the identification of 42 high-confidence LASV polymerase interactors. We subsequently performed a small interfering RNA (siRNA) screen to identify those interactors that have functional roles in authentic LASV infection. As proof of principle, we characterized eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we found to be a proviral factor that physically associates with LASV polymerase. Targeted degradation of GSPT1 by a small-molecule drug candidate, CC-90009, resulted in strong inhibition of LASV infection in cultured cells. Our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet-to-be-defined host-pathogen interactome, which can reveal new biology and uncover novel targets for the development of antivirals against highly pathogenic RNA viruses.
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