Cellular Entry of Influenza by Single-particle Imaging

通过单粒子成像观察流感病毒的细胞进入

• 批准号: 7492222
• 负责人: XIAOWEI ZHUANG
• 金额: $ 27.21万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-22 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7492222
• 关键词: Address; Arts; Behavior; Biological; Caveolae; Caveolins; Cell Nucleus; Cell physiology; Cells; Cellular Structures; Characteristics; Clathrin; Complex; Cytoplasm; Drug Design; Endocytosis; Endosomes; Family; Foundations; Future; Gene Expression; Genes; Goals; Hand; Health; Human; Image; Imaging Techniques; Individual; Infection; Influenza; Influenza A virus; Kinetics; Lead; Life; Measurement; Mediating; Membrane; Methodology; Methods; Molecular; Nuclear; Nuclear Import; Pathway interactions; Pharmaceutical Preparations; Process; Reaction; Recycling; Ribonucleoproteins; Screening procedure; Sorting - Cell Movement; Staging; System; Techniques; Terrorism; Therapeutic; Time; Viral; Viral Genes; Viral Proteins; Virion; Virus; Virus Diseases; caveolin 1; design; fluorescence imaging; influenzavirus; insight; instrumentation; late endosome; pandemic disease; particle; receptor mediated endocytosis; research study; single molecule; trafficking; viral RNA

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.

描述（由申请人提供）：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，我们将开发最新的仪器，用于单个病毒和单个病毒基因的实时荧光成像。 这些单粒子方法将使我们能够实时直接可视化流感病毒的细胞进入过程，从而剖析进入途径的各个阶段，如果不可能通过集合方法检测到可能很难检测到，并且可以更好地理解流感的细胞进入机制。 流感是许多医学重要病毒的代表，是理解病毒进入过程的范式。 流感病毒通过多个步骤进入细胞，包括（1）受体介导的内吞作用，（2）病毒的内吞输血到晚期内体内的内吞作用，其中病毒融合与内体的融合可导致细胞质中的病毒基因释放到细胞质中，以及（3）这些病毒基因的核导入。 设计了三组实验来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使用的内吞作用机制。 我们将解决重要的问题，包括：（i）流感被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）如何将病毒靶向内吞机械；和/或（iv）内吞机械如何围绕病毒组装？特定目标2：将使用单粒子跟踪来阐明细胞内流感病毒的内吞运输。我们将解决：（i）病毒如何在细胞内运输； （ii）如何在不同的内吞区室之间贩运病毒； （iii）病毒的分类方式。特定目标3：单粒子跟踪将用于表征 以病毒核糖核蛋白（VRNP）的形式进行了流感基因的核进口。 我们将解决：（i）细胞中VRNP的转运机制以及（ii）调节VRNP核进口的分子机制？ 上述实验有望为流感病毒的细胞进入过程提供新的见解。 这里开发的新方法可以扩展以研究其他病毒家族，并且将来可能会导致抗病毒药物和治疗剂的新设计。 这些技术也可以应用于其他分子和细胞生物系统。