FLOCK HOUSE VIRUS INFECTION OF DROSOPHILA LINE 1 CELLS

果蝇 1 系细胞的羊群病毒感染

• 批准号: 8361901
• 负责人: John Emil Johnson
• 金额: $ 2.47万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361901
• 关键词: Antibodies; Artificial Membranes; Baculovirus Expression System; Binding; C-terminal; Capsid; Capsid Proteins; Cells; Cleaved cell; Data; Defect; Drosophila genus; Electron Microscopy; Event; Fluorescence Microscopy; Funding; Future; Gene Proteins; Grant; Green Fluorescent Proteins; Hela Cells; Housing; Image Analysis; In Vitro; Infection; Label; Ligands; Membrane; Microtomy; Mitochondria; Mutate; Mutation; National Center for Research Resources; Organelles; Peptides; Principal Investigator; Process; Production; Proteins; RNA; RNA Interference; RNA replication; RNA-Directed RNA Polymerase; Reagent; Research; Research Infrastructure; Resources; Role; Site; Source; Translations; United States National Institutes of Health; Vacuole; Viral; Virion; Virus; Virus Diseases; Virus-like particle; Work; cell fixing; cost; in vivo; particle; protein B; research study; tomography

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We seek to correlate an extensive amount of structural and biophysical data obtained in vitro for nodaviruses with events associated with viral entry, disassembly, assembly and cellular exit in vivo. Flock house virus (FHV) will be used to study these processes as they occur in the infection of drosophila line 1 (DL1) cells. Nodavirus entry requires a single gene product that forms the T=3 icosahedral capsid. Two other gene products participate later in infection, protein A that includes the RNA directed RNA polymerase (RdRp) activity as well as other functions and protein B that interferes with the cellular RNAi activity. Our studies are focused on the role of the capsid protein a (407aa) and its post translation cleavage products b (363aa) and g (44aa). Initial studies (described below) investigated attachment and entry of authentic FHV into DL1 cells with fluorescence microscopy of whole fixed cells labeled with antibodies to the capsid protein and electron microscopy of thin-sectioned cells. The process was also studied with noninfectious virus-like particles (VLPs) of FHV made in a baculovirus expression system. Expressed a subunits spontaneously assemble to form particles that are indistinguishable from authentic virions, but the genes for protein A and B are not present, stopping the infection process without production of progeny virus. The a subunits in these particles spontaneously cleave, just like authentic particles. A second defect in the VLPs was introduced by mutating the cleavage site resulting in the presence of only protein a, thus allowing, in future experiments, the determination of the role of g in the infection process. It is known that g will dramatically alter artificial membranes in vitro and that, when fused to the N-terminus of green fluorescent protein, it directs GFP to the mitochondria of HeLa cells. In contrast, if it is fused to the C-terminus of GFP, there is no localization of the protein. Our working hypothesis is that g participates in membrane translocation of RNA and possibly additional targeting. In addition to the reagents described, new mutations of FHV are being made that incorporated the tetracysteine motif to which specific fluorescent ligand FlAsH-EDT2 binds. It has already been demonstrated that mutations of comparable size can be made in both the shell forming b-domain and the C-terminal g domain without stopping infectivity. These mutations will be made in both authentic virus and in the VLPs so that we can distinguish between localization of g peptides before RNA replication and their localization in progeny virus. Incorporating the ReAsH-EDT2 as a second probe after labeling all incoming virus with FlAsH-EDT2, it should be possible to identify the progeny g or b peptides. Finally, we are performing three-dimensional whole cell tomography to characterize the crystalline arrays of progeny virus and their possible association with vacuoles or cellular organelles.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 我们试图将大量的结构性和生物物理数据与疾病中获得的大量结构和生物物理数据与与病毒入口，拆卸，组装和细胞出口相关的事件相关联。 羊群病毒（FHV）将用于研究这些过程，因为它们发生在果蝇1（DL1）细胞的感染中。 Nodavirus进入需要一个形成T = 3二十面体帽的单个基因产物。 另外两个基因产物在后来参与感染，包括RNA定向RNA聚合酶（RDRP）活性以及其他功能和蛋白B的蛋白A，这些功能和蛋白B会干扰细胞RNAI活性。 我们的研究集中在capsid蛋白A（407AA）及其翻译后裂解产物B（363AA）和G（44AA）的作用上。 最初的研究（如下所述）研究了真实的FHV的附着和进入DL1细胞中的荧光显微镜，这些细胞用抗帽膜蛋白抗体标记的整个固定细胞和薄切片细胞的电子显微镜标记。 还研究了在杆状病毒表达系统中制造的非感染性病毒样颗粒（VLP）的过程。 表达的亚基自发地组装以形成与正宗病毒体无法区分的颗粒，但是蛋白A和B的基因不存在，从而在没有产生后代病毒的情况下停止了感染过程。 这些颗粒中的A亚基自发裂解，就像真实的颗粒一样。 通过突变裂解位点引入VLP中的第二个缺陷，导致仅存在蛋白A，从而允许在将来的实验中确定G在感染过程中的作用。 众所周知，G会在体外显着改变人造膜，并且当与绿色荧光蛋白的N末端融合时，它将GFP引导到HeLa细胞的线粒体。 相反，如果将其融合到GFP的C端，则没有蛋白质的定位。我们的工作假设是G参与RNA的膜易位以及可能的其他靶向。除了描述的试剂外，还制作了新的FHV突变，并结合了特异性荧光配体闪存闪存flash-eDT2结合的四环素状态基序。 已经证明，在形成B域和C末端G结构域的壳体中，可以进行相当大小的突变，而不会阻止感染性。 这些突变将在正宗病毒和VLP中进行，以便我们可以在RNA复制之前区分G肽的定位及其在后代病毒中的定位。 将REASH-EDT2纳入将所有传入病毒标记为Flash-Edt2后，将REASH-EDT2纳入第二探针，应该可以识别后代G或B肽。 最后，我们正在执行三维全细胞断层扫描，以表征后代病毒的结晶阵列及其与液泡或细胞细胞器的可能关联。