Cellular Entry of Influenza by Single-particle Imaging

描述(申请人提供)：该项目的长期目标是阐明流感感染的分子机制和细胞途径。为了实现这一目标，我们将开发最先进的仪器，用于单病毒和单病毒基因的实时荧光成像。这些单颗粒方法将使我们能够实时直观地显示流感病毒进入细胞的过程，剖析即使不是不可能也很难用系综方法检测到的进入途径的各个阶段(S)，并更好地了解流感病毒的细胞进入机制。流感是许多医学上重要病毒的代表，是理解病毒进入过程的一个范例。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)病毒通过内吞运往晚期内体，其中病毒与内体融合导致病毒基因释放到细胞质中，以及(3)这些病毒基因的核输入。设计了三组实验来研究这些病毒进入步骤。具体目标1：将使用单粒子跟踪来研究流感病毒使用的内吞机制。我们将解决的重要问题包括：(I)流感利用哪些细胞内吞途径进入细胞；(Ii)这些途径的分子特征是什么；(Iii)病毒是如何针对内吞机制的；和/或(Iv)内吞机制是如何围绕病毒组装的？具体目标2：将使用单粒子跟踪来阐明流感病毒在细胞内的内吞运输。我们将讨论：(I)病毒是如何在细胞内传播的；(Ii)病毒是如何在不同的胞内室之间运输的；以及(Iii)病毒是如何分类的。具体目标3：将使用单粒子跟踪来表征 以病毒核糖核蛋白(VRNPs)的形式管制流感基因的核进口。我们将讨论：(I)vRNPs在细胞中的转运机制和(Ii)调控vRNPs核输入的分子机制？上述实验有望为流感病毒进入细胞的过程提供新的见解。这里开发的新方法可以扩展到研究其他病毒家族，并可能在未来导致新的抗病毒药物和疗法的设计。这些技术也可以应用于其他分子和细胞生物系统。

DESCRIPTION (provided by applicant): 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.