Identification of Flavivirus Nucleocapsid Core-Envelope Glycoprotein Interactions

项目总结/摘要 黄病毒（一个由90多种已知病毒组成的家族，包括寨卡病毒、登革热和西尼罗河病毒）是重要的 人类病原体每年影响31亿人，其中大多数包膜病毒没有任何 有效的疫苗或抗病毒药物能够阻止其传播。当前黄病毒抗病毒药物的主要目的 设计是为了破坏黄病毒生命周期中对病毒生存至关重要的特定机制，包括病毒 附着宿主细胞、病毒内吞、基因组脱壳、基因组复制和病毒成熟 穿过高尔基体这些潜在的药物机制已经在我们的研究中进行了非常详细的探索。 实验室和其他地方。然而，对于驱动感染性病毒颗粒的组装机制知之甚少 阵由于所有黄病毒之间的高度结构同源性， 机制将为病毒增殖的治疗干预提供普遍存在的靶点。 在其他包膜病毒系统中，组装依赖于病毒核心蛋白与脂质的相互作用 膜或膜结合糖蛋白以产生感染性病毒颗粒。通过单次探测 颗粒冷冻电镜重建显示，未成熟寨卡病毒的核衣壳核心（NC）在 与病毒脂质双层内侧的包膜糖蛋白非常接近。由于这一密切 由于NC与包膜糖蛋白之间的相互作用，我假设NC在病毒组装过程中与包膜糖蛋白相互作用。我进一步 这些相互作用发生在衣壳蛋白（CP）和跨膜螺旋之间。 前体膜（prM）蛋白和包膜（E）蛋白。 由于目前还没有关于依赖于CP-prM/E相互作用的病毒组装的信息，这些相互作用 有潜力被开发为能够抑制黄病毒增殖的新药物靶标。 为此，我正在研究两种独立的策略来验证假设的NC-prM/E 使用登革病毒血清型2（DENV 2）作为模型系统的相互作用。库恩实验室特别擅长于 使用DENV 2的诱变研究，这就是为什么决定使用这种病毒作为我们的模型系统 而不是寨卡病毒首先，在prM/E跨膜螺旋内的氨基酸被认为是关键的， DENV 2和其他黄病毒的组装过程已经或将要突变，以研究它们在DENV 2和其他黄病毒组装过程中的作用。 促进颗粒组装。第二，DENV 2 prM和E跨膜螺旋相互作用的能力 通过使用SMA脂质纳米颗粒内的重构prM和E蛋白来检查CP。 由于所有黄病毒的高度相似性，本研究中确定的技术、结果和机制可以 适用于其他黄病毒。将这些必要的实验研究与建议的培训技能相结合 通过写作、演讲和指导进行有效科学交流的合作机会 能让我为将来从事生物医学研究做好准备

Project Summary/Abstract Flaviviruses (a family of over 90 known viruses, including Zika, dengue and West Nile) are significant human pathogens affecting 3.1 billion people annually, and most of these enveloped viruses do not have any viable vaccines or antivirals capable of combating their spread. The primary objective of current flavivirus antiviral design is to disrupt specific mechanisms in the flavivirus life cycle that are key for virus survival, including virus attachment to the host cell, viral endocytosis, genome uncoating, genome replication, and virus maturation through the Golgi. These potentially druggable mechanisms have been explored in great detail both within our lab and elsewhere. However, little is known about the assembly mechanisms that drive infectious virus particle formation. Because of the high structural homology among all flaviviruses, the identification of assembly mechanisms will provide ubiquitous targets for therapeutic intervention in virus proliferation. In other enveloped virus systems, assembly depends on the interaction of virus core proteins with lipid membranes or membrane bound glycoproteins to produce infectious virus particles. Exploration via single particle Cryo-EM reconstruction has shown that the nucleocapsid core (NC) of immature Zika virus, is found in close proximity with the envelope glycoproteins on the inner side of the virus’s lipid bilayer. Due to this close proximity, I hypothesized that the NC interacts with the envelope glycoproteins during virus assembly. I further hypothesized and that these interactions occur between the capsid protein (CP) and transmembrane helices of the precursor membrane (prM) protein and the envelope (E) protein while the particle is in the immature state. Since no information currently exists on virus assembly relying on CP-prM/E interactions, these interactions have the potential to be exploited as new drug targets capable of inhibiting the proliferation of flaviviruses. To this end, I am investigating two independent strategies to validate the hypothesized NC-prM/E interactions using dengue virus serotype 2 (DENV2) as a model system. The Kuhn lab is particularly adept in mutagenesis studies using DENV2, which is why the decision was made to use this virus as our model system instead of Zika. Firstly, amino acids within the prM/E transmembrane helices that were posited to be key in the assembly process of DENV2 and other flaviviruses have been or will be mutated to investigate their role in promoting particle assembly. Secondly, the ability of the DENV2 prM and E transmembrane helices to interact with CP is being examined through the use of reconstituted prM and E proteins within SMA lipid nanoparticles. Due to the high similarity of all flaviviruses, the techniques, results and mechanisms identified in this study can be applied to other flaviviruses. Combining these essential experimental studies with the proposed training skills and collaborative opportunities for effective scientific communication through writing, speaking and mentoring will prepare me well for a future in biomedical research.