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

通过低温冷冻技术观察入口缺陷型突变羊群病毒的衣壳结构

• 批准号: 7723581
• 负责人: John Emil Johnson
• 金额: $ 0.63万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723581
• 关键词: Amino Acids; Baculoviruses; Biological Assay; Biological Models; C-terminal; Capsid; Capsid Proteins; Cell membrane; Cells; Class; Computer Retrieval of Information on Scientific Projects Database; Cryoelectron Microscopy; Disruption; Drosophila genus; Funding; Grant; Helix (Snails); Housing; In Vitro; Insecta; Institution; Membrane; Microscopy; Mutate; N-terminal; Peptides; Play; Positioning Attribute; Research; Research Personnel; Resolution; Resources; Role; Source; Structure; Thinking; United States National Institutes of Health; Viral Genome; Viral Packaging; Virus; Virus-like particle; abstracting; ear helix; in vivo; milligram; mutant; vector; viral RNA

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.

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。 摘要：我们试图通过使用FHV(Flock House Virus)作为这类病毒的模型系统来了解非包膜病毒是如何破坏宿主细胞膜的。在几种经过充分研究的无包膜病毒中，一种小的膜活性多肽已被确定为导致宿主膜破坏的因素。FHV衣壳含有一个类似的、由44个氨基酸组成的多肽伽马，它有两个截然不同的区域，N-端21个残基形成两亲性螺旋，而疏水的C-端区参与病毒RNA的组装。合成的N-末端两亲性螺旋在体外可以破坏细胞膜，以前被认为足以促进FHV进入寄主果蝇细胞的膜破坏。然而，通过体外和体内实验，我们发现在FHV进入宿主细胞的过程中，伽马的C-末端区域是膜破坏所特需的。这一令人惊讶的结果表明，在病毒衣壳的背景下，伽马的膜活性N端螺旋不足以进入。用循环电子显微镜(4)测定的FHV衣壳结构(4)先前证明，伽马的N端螺旋在病毒衣壳的5倍对称轴上形成五聚体螺旋束。可能的是，C末端区域在促进这些螺旋在病毒衣壳上的正确排列方面起着重要的结构作用。我们建议用冷冻电子显微镜来确定FHV突变的病毒样颗粒(VLP)的高分辨结构，该颗粒缺乏伽马的C-末端区域。这种突变体命名为384 FHV，可以通过在昆虫细胞中表达来自杆状病毒载体的突变的FHV外壳蛋白，以毫克量制造。由于该突变体不包装病毒基因组，因此它完全不具传染性。将这一突变衣壳与野生型FHV衣壳进行比较，将确定伽马肽的C-末端区域是否在正确定位两亲性螺旋进入的过程中发挥作用。