FLOCK HOUSE VIRUS INFECTION OF DROSOPHILA LINE 1 CELLS

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们试图将体外获得的诺达病毒的大量结构和生物物理数据与体内病毒进入、分解、组装和细胞退出相关的事件关联起来。 羊群病毒 (FHV) 将用于研究果蝇 1 系 (DL1) 细胞感染过程中发生的这些过程。 诺达病毒的进入需要形成 T=3 二十面体衣壳的单基因产物。 另外两种基因产物后来参与感染，蛋白 A 包括 RNA 指导的 RNA 聚合酶 (RdRp) 活性以及其他功能，蛋白 B 干扰细胞 RNAi 活性。 我们的研究重点是衣壳蛋白 a (407aa) 及其翻译后裂解产物 b (363aa) 和 g (44aa) 的作用。 初步研究（如下所述）通过衣壳蛋白抗体标记的整个固定细胞的荧光显微镜和薄切片细胞的电子显微镜研究了真正的 FHV 附着和进入 DL1 细胞。 还利用杆状病毒表达系统中制备的 FHV 非感染性病毒样颗粒 (VLP) 研究了该过程。 表达的亚基自发组装形成与真实病毒体无法区分的颗粒，但蛋白 A 和 B 的基因不存在，从而停止感染过程而不产生子代病毒。 这些粒子中的 a 亚基会自发分裂，就像真正的粒子一样。 VLP 中的第二个缺陷是通过突变切割位点而引入的，导致仅存在蛋白 a，从而允许在未来的实验中确定 g 在感染过程中的作用。 众所周知，g 在体外会显着改变人工膜，当与绿色荧光蛋白的 N 末端融合时，它会将 GFP 引导至 HeLa 细胞的线粒体。 相反，如果它融合到 GFP 的 C 末端，则蛋白质不会定位。我们的工作假设是 g 参与 RNA 的膜易位以及可能的额外靶向。除了所描述的试剂之外，FHV 的新突变也正在开发中，其中掺入了与特定荧光配体 FlAsH-EDT2 结合的四半胱氨酸基序。 已经证明，可以在形成壳的 b 结构域和 C 末端 g 结构域中进​​行相当大小的突变，而不会停止感染性。 这些突变将在真实病毒和 VLP 中发生，以便我们能够区分 RNA 复制前 g 肽的定位和它们在子代病毒中的定位。 在用 FlaAsH-EDT2 标记所有传入病毒后，将 ReAsH-EDT2 作为第二个探针，应该可以识别后代 g 或 b 肽。 最后，我们正在进行三维全细胞断层扫描，以表征子代病毒的晶体阵列及其与液泡或细胞器的可能关联。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.