Cellular Entry of Influenza by Single-particle Imaging

该项目的长期目标是阐明细胞内的分子机制和细胞途径， 流感感染。为了实现这一目标，我们将开发最先进的仪器， 单个病毒和单个病毒基因的荧光成像。这些单粒子方法将使我们 直接可视化流感病毒进入细胞的过程，在真实的时间，解剖的各个阶段， 可能难以（如果不是不可能的话）通过集合方法检测的进入途径，并获得 更好地了解流感的细胞进入机制。 流感是许多医学上重要的病毒的代表，是理解病毒的范例。 进入过程。流感病毒通过几个步骤进入细胞，包括（1）受体介导的内吞作用， (2)病毒内吞运输到晚期内体，在晚期内体中病毒与内体融合导致 将病毒基因释放到细胞质中，和（3）这些病毒基因的核输入。三套 设计实验以研究这些病毒进入步骤。具体目标1：单粒子跟踪将 用于研究流感病毒的内吞作用机制。我们将讨论重要的 问题包括：（i）流感病毒利用哪些细胞内吞途径进入细胞;（ii） 是这些途径的分子特征;（iii）病毒如何靶向内吞机制; 和/或（iv）内吞机制如何在病毒周围组装？具体目标2：单粒子跟踪将 用于阐明流感病毒在细胞内的内吞运输。我们将讨论：（一）如何 病毒在细胞内运输;（ii）病毒如何在不同的内吞区室之间运输; 以及（iii）病毒是如何分类的。具体目标3：单粒子跟踪将用于表征 以病毒核糖核蛋白（vRNP）的形式调节流感基因的核输入。我们将 地址：（i）vRNP在细胞中的运输机制和（ii）调节细胞内的分子机制。 vRNP的核进口 上述实验有望为流感进入细胞过程提供新的见解 病毒这里开发的新方法可以扩展到研究其他病毒家族， 在未来导致抗病毒药物和治疗的新设计。这些技术也可以应用于 其他分子和细胞生物系统。

The long-term goal of this project is to elucidate the molecular mechanisms and cellular pathways of influenza infection. To accomplish this goal, we will develop state-of-the-art instrumentation for real-time fluorescence imaging of single viruses and single viral genes. These single-particle approaches will allow us to directly visualize the cellular entry process of influenza viruses in real time, to dissect individual stages of the entry pathway(s) that may be difficult if not impossible to detect by ensemble methods, and to obtain a better understanding of the cellular entry mechanisms of influenza. Influenza, representative of many medically important viruses, is a paradigm for understanding viral entry processes. Influenza viruses enter cells via several steps, including (1) receptor-mediated endocytosis, (2) endocytic trafficking of the viruses to late endosomes where viral fusion with endosomes leads to the release viral genes into the cytoplasm, and (3) nuclear import of these viral genes. Three sets of experiments are designed to investigate these viral entry steps. Specific Aim 1: Single-particle tracking will be used to investigate the endocytosis mechanisms used by the influenza viruses. We will address important questions including: (i) which cellular endocytic pathways are exploited by influenza to enter cells; (ii) what are the molecular characteristics of these pathways; (iii) How viruses are targeted to endocytic machinery; and/or (iv) how endocytic machinery assembles around viruses? Specific Aim 2: Single-particle tracking will be used to elucidate the endocytic trafficking of influenza viruses inside cells. We will address: (i) how viruses are transported inside cells; (ii) how viruses are trafficked between different endocytic compartments; and (iii) how viruses are sorted. Specific Aim 3: Single-particle tracking will be used to characterize the regulated nuclear import of influenzal genes, in the form of viral ribonucleoproteins (vRNPs). We will address: (i) the transport mechanisms of vRNPs in cells and (ii) the molecular mechanisms that regulate the nuclear import of vRNPs? The above experiments promise to provide new insights into the cellular entry process of influenza viruses. The new methodology developed here can be extended to study other families of viruses and may in the future lead to new designs of anti-viral drugs and therapeutics. These techniques can also be applied to other molecular and cellular biological systems.