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.
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