FLOCK HOUSE VIRUS INFECTION OF DROSOPHILA LINE 1 CELLS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 我们试图将大量的体外获得的野田病毒的结构和生物物理数据与体内病毒进入，拆卸，组装和细胞退出相关的事件相关联。 将使用鸡舍病毒（FHV）研究这些过程，因为它们发生在果蝇1号系（DL 1）细胞的感染中。 野田病毒进入需要形成T=3二十面体衣壳的单个基因产物。 另外两种基因产物稍后参与感染，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。 我们的研究集中在衣壳蛋白a（407 aa）及其翻译后切割产物B（363 aa）和g（44 aa）的作用。 最初的研究（如下所述）研究了真实的FHV附着和进入DL 1细胞，用荧光显微镜观察了用衣壳蛋白抗体标记的整个固定细胞，并用电子显微镜观察了薄切片细胞。 该过程还研究了非感染性病毒样颗粒（VLP）的FHV在杆状病毒表达系统。 表达的a亚基自发组装形成与真实病毒体难以区分的颗粒，但不存在蛋白A和B的基因，从而停止感染过程而不产生子代病毒。 这些粒子中的a亚基自发地分裂，就像真实的粒子一样。 VLP中的第二个缺陷是通过突变切割位点引入的，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。 已知的是，g将在体外显著改变人工膜，并且当与绿色荧光蛋白的N-末端融合时，其将GFP引导至HeLa细胞的线粒体。 相反，如果它与GFP的C末端融合，则蛋白质没有定位。我们的工作假设是，g参与RNA的膜易位和可能的额外靶向。除了所描述的试剂之外，正在进行FHV的新突变，其并入特异性荧光配体FlAsH-EDT 2结合的四半胱氨酸基序。 已经证明，可以在形成外壳的b结构域和C末端g结构域中进行相当大小的突变，而不停止感染性。 这些突变将在真实病毒和VLP中进行，以便我们可以区分RNA复制前g肽的定位和它们在子代病毒中的定位。 在用FlAsH-EDT 2标记所有进入的病毒后，将ReAsH-EDT 2作为第二探针，应该可以鉴定子代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空泡或细胞器的可能联系。

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.