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

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来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 摘要：:我们正试图了解无包膜病毒如何破坏宿主细胞膜，通过使用FHV（鸡舍病毒），作为这类病毒的模型系统。 在几种研究充分的无包膜病毒中，一种小的膜活性肽已被鉴定为负责宿主膜破坏的因子。 FHV衣壳含有类似的44个氨基酸的肽γ，其具有两个不同的区域  N-末端21个残基形成两亲性螺旋，而疏水性C-末端区域在装配期间参与病毒RNA的包装。 当合成产生时，N-末端两亲性螺旋可以在体外破坏膜，并且先前被认为足以在FHV进入其宿主果蝇细胞期间促进膜破坏。 然而，使用体外和体内测定，我们发现γ的C-末端区域是FHV进入宿主细胞期间膜破坏所特别需要的。 这一令人惊讶的结果表明，在病毒衣壳的情况下，γ的膜活性N-末端螺旋不足以进入。 使用冷冻电子显微镜测定的FHV衣壳结构（4）先前已证明γ的N-末端螺旋在病毒衣壳的5倍对称轴处形成五聚体螺旋束。 C-末端区域可能在促进病毒衣壳上这些螺旋的正确排列中起重要的结构作用。 我们建议确定高分辨率的结构突变的病毒样颗粒（VLP）的FHV，缺乏C-末端区域的γ，通过冷冻电子显微镜。 这种突变体，命名为384 FHV，可以通过在昆虫细胞中从杆状病毒载体表达FHV的突变外壳蛋白而以毫克量制备。 由于这种突变体不包装病毒基因组，因此它完全没有传染性。 这种突变的衣壳与野生型FHV衣壳的比较将解决γ肽的C-末端区域是否在适当定位两亲性螺旋以进入中起作用。

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.