CAPSID STRUCTURE OF AN ENTRY-DEFECTIVE, MUTANT FLOCK HOUSE VIRUS BY CRYOTEM

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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. Abstract: : We are attempting to understand how non-enveloped viruses disrupt host cell membranes by using FHV (Flock House Virus), as a model system for this class of viruses. In several well-studied nonenveloped viruses, a small, membrane-active peptide has been identified as the agent responsible for host membrane disruption . FHV capsid contains an analogous, 44 amino acid peptide gamma, which has two distinct regions  the N-terminal 21 residues form an amphipathic helix, while the hydrophobic C-terminal region is involved in packaging of viral RNA during assembly. The N-terminal amphipathic helix, when synthetically produced, can disrupt membranes in vitro , and was previously thought to be sufficient for promoting membrane disruption during entry of FHV into its host drosophila cells. However, using in vitro and in vivo assays , we have found that the C-terminal region of gamma is specifically required for membrane disruption during entry of FHV into host cells. This surprising result indicates that in context of the virus capsid, the membrane active N-terminal helix of gamma is not sufficient for entry. A structure of FHV capsid determined using cyroelectron microscopy (4), has previously demonstrated that the N-terminal helices of gamma form pentameric helical bundles at the 5-fold axis of symmetry of the virus capsid. It is possible that the C-terminal region plays a significant structural role in promoting proper arrangement of these helices on the virus capsid. We propose to determine high-resolution structures of a mutated virus-like particle (VLP) of FHV, lacking the C-terminal region of gamma, by cryo-electron microscopy. This mutant, designated ¿384 FHV, can be made in milligram amounts by expressing the mutated coat protein of FHV in insect cells from a baculovirus vector. Since this mutant does not package viral genome, it is completely non-infectious. A comparison of this mutated capsid with wildtype FHV capsid will resolve whether the C-terminal region of the gamma peptide plays a role in positioning the amphipathic helices properly for entry.