FLOCK HOUSE VIRUS INFECTION OF DROSOPHILA LINE 1 CELLS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们试图将在体外获得的大量诺达病毒的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联系起来。鸡舍病毒(FHV)将被用来研究这些过程，因为它们发生在感染果蝇1系(DL1)细胞中。诺达病毒的进入需要形成T=3二十面体衣壳的单一基因产物。另外两种基因产物随后参与感染，蛋白A包括RNA导向的RNA聚合酶(RdRp)活性以及其他功能，蛋白B干扰细胞的RNAi活性。我们的研究集中在衣壳蛋白a(407aa)及其翻译后切割产物b(363aa)和g(44aa)的作用上。最初的研究(如下所述)通过用衣壳蛋白抗体标记的整个固定细胞的荧光显微镜和薄片细胞的电子显微镜来调查真正的FHV附着和进入DL1细胞。用杆状病毒表达系统制备的非传染性FHV病毒样颗粒(VLP)也对这一过程进行了研究。表达的A亚基自发组装形成与真正的病毒粒子难以区分的颗粒，但蛋白A和B的基因并不存在，从而停止了感染过程，而不产生后代病毒。这些粒子中的α亚基会自发分裂，就像真正的粒子一样。VLP的第二个缺陷是通过突变切割位点而导致只存在蛋白a，从而在未来的实验中确定g在感染过程中的作用。众所周知，g在体外会极大地改变人工膜，当它与绿色荧光蛋白的N端融合时，它会将GFP导向HeLa细胞的线粒体。相反，如果它与GFP的C末端融合，则不存在蛋白质定位。我们的工作假设是g参与了RNA的膜转位，并可能参与了额外的靶向。除了所描述的试剂外，FHV正在进行新的突变，纳入特定荧光配体Flash-EDT2结合的四半胱氨酸基序。已经证明，在形成b-结构域和C-末端g结构域的外壳中都可以进行类似大小的突变，而不会停止传染性。这些突变将在正宗病毒和VLP中进行，以便我们能够区分g肽在RNA复制之前的定位和它们在后代病毒中的定位。在用Flash-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，以表征后代病毒的晶体阵列以及它们与空泡或细胞细胞器的可能联系。

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.