La Jolla Institute for Immunology, La Jolla, California, USA.
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, California, USA.
mBio. 2019 Jul 23;10(4):e00734-19. doi: 10.1128/mBio.00734-19.
The filoviruses are etiological agents of life-threatening hemorrhagic fever with high mortality rate and risk of potential outbreak. Among members of this family, the Ebola (EBOV), Sudan (SUDV), and Marburg (MARV) viruses are considered the most pathogenic for humans. The ebolavirus nucleoprotein (NP) is the most abundant protein in infected cells and is essential for viral transcription and replication; thus, it represents an attractive target for therapeutic intervention. Here, we present the structure of SUDV NP in complex with the amino-terminal portion of the phosphoprotein VP35 at 2.3 Å. This structure captures VP35 chaperoning SUDV NP in a monomeric and RNA-free state. This transient state has been proposed to be key to maintaining a pool of monomeric and RNA-free NPs prior to NP-NP polymerization and encapsidation of the viral RNA genome. This structure also reveals a newly visualized interaction between NP and VP35, a well-defined beta sheet that is not present in previous structures. Affinity binding assays demonstrate that this beta sheet is essential for maintaining the high-affinity interaction between VP35 and a hydrophobic pocket on SUDV NP, and electron microscopy indicates the importance of this binding interaction to the oligomeric state and assembly of NP in human cells. Complementary structure-directed mutagenesis identifies critical residues conserved across the filovirus family that could be targeted by broadly effective antivirals. Outbreaks of the filoviruses can be unpredictable in timing, location, and identity of the causative virus, with each of Ebola virus, Sudan virus, Bundibugyo virus, and Marburg virus reemerging in the last several years to cause human disease with 30 to 90% lethality. The 2014-2016 outbreak in particular, with nearly 30,000 patients, highlighted the ability of these viruses to emerge unexpectedly and spread rapidly. Two ebolavirus outbreaks have emerged this year, yet we still lack FDA-approved drugs with pan-filovirus activity to treat existing and emergent ebolaviruses. For all filoviruses, the interaction between the nucleoprotein and the phosphoprotein is essential for the virus life cycle and is a potential target for therapeutic intervention. In this report, we describe the crystal structure of the SUDV nucleoprotein with the interacting domain of the viral phosphoprotein, and we identify residues critical for high-affinity interaction and for control of the oligomeric state of the nucleoprotein. Structural comparison of this heterodimer with other members of the filovirus family allowed us to find conserved and essential atomic features that will facilitate understanding of the virus life cycle and the rational design of antivirals.
丝状病毒是具有高死亡率和潜在爆发风险的危及生命的出血热的病因。在该家族成员中，埃博拉病毒（EBOV）、苏丹病毒（SUDV）和马尔堡病毒（MARV）被认为对人类最具致病性。埃博拉病毒核蛋白（NP）是感染细胞中最丰富的蛋白质，对病毒转录和复制至关重要；因此，它是治疗干预的一个有吸引力的靶点。在这里，我们展示了 SUDV NP 与病毒磷蛋白 VP35 的氨基末端部分在 2.3Å 的复合物结构。该结构以单体和无 RNA 的状态捕获 VP35 对 SUDV NP 的伴侣作用。这种瞬态状态被认为是在 NP-NP 聚合和病毒 RNA 基因组包装之前维持单体和无 RNA NP 池的关键。该结构还揭示了 NP 和 VP35 之间新发现的相互作用，这是一个以前的结构中不存在的明确的β片层。亲和结合测定表明，该β片层对于维持 VP35 与 SUDV NP 上一个疏水口袋之间的高亲和力相互作用至关重要，电子显微镜表明该结合相互作用对于 NP 在人细胞中的寡聚状态和组装很重要。互补的结构导向突变分析确定了丝状病毒家族中保守的关键残基，这些残基可能成为广谱有效的抗病毒药物的靶点。丝状病毒的爆发在时间、地点和致病病毒的身份方面可能是不可预测的，埃博拉病毒、苏丹病毒、邦迪布焦病毒和马尔堡病毒在过去几年中都重新出现，导致人类疾病的死亡率为 30%至 90%。特别是 2014-2016 年的疫情，有近 3 万名患者，突显了这些病毒出人意料地迅速传播的能力。今年又爆发了两起埃博拉病毒疫情，但我们仍然缺乏经美国食品和药物管理局批准的、可治疗现有和新出现的埃博拉病毒的泛丝状病毒活性药物。对于所有的丝状病毒，核蛋白与磷蛋白之间的相互作用对于病毒的生命周期至关重要，是治疗干预的一个潜在靶点。在本报告中，我们描述了 SUDV 核蛋白与病毒磷蛋白相互作用结构域的晶体结构，并确定了高亲和力相互作用和控制核蛋白寡聚状态的关键残基。该异二聚体与丝状病毒家族其他成员的结构比较使我们能够找到保守且必需的原子特征，这将有助于理解病毒的生命周期和抗病毒药物的合理设计。
