Structure and Mechanism of the Herpesvirus Fusogen Glycoprotein B

疱疹病毒融合糖蛋白B的结构和机制

• 批准号: 9122629
• 负责人: Rebecca Sisson Cooper
• 金额: $ 5.61万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9122629
• 关键词: Address; Architecture; Area; Blindness; C10; Cell membrane; Cells; Chimeric Proteins; Complement; Complex; Cryoelectron Microscopy; Crystallization; Cytoplasmic Tail; Data; Dependence; Detergents; Electron Microscopy; Electron Spin Resonance Spectroscopy; Electrons; Encephalitis; Energy Supply; Energy Transfer; Environment; Event; Glycoproteins; Goals; Herpes Labialis; Herpesviridae; Herpesvirus 1; Human Herpesvirus 2; Immunocompromised Host; Infection prevention; Investigation; Kinetics; Knowledge; Length; Life; Lipid Bilayers; Lipids; Location; Malignant Neoplasms; Measurement; Mediating; Melanoma Cell; Membrane; Membrane Fusion; Modeling; Modification; Molecular Conformation; Monitor; Movement; Mus; Mutation; Newborn Infant; Nucleic Acid Regulatory Sequences; Positioning Attribute; Process; Protein Dynamics; Proteins; Regulation; Resolution; Role; Shapes; Simplexvirus; Structure; System; Techniques; Testing; Therapeutic Agents; Transmembrane Domain; Vaccines; Vesicular stomatitis Indiana virus; Viral; Virion; Virus; Work; base; design; flexibility; latent infection; member; mimetics; novel; public health relevance; research study; response; therapeutic vaccine

 DESCRIPTION (provided by applicant): Viral fusogens are dynamic proteins that undergo large rearrangements to facilitate the merger of viral and target cell membranes. In Herpes simplex viruses (HSV-1 and HSV-2), this fusion event results from the sequential action of four viral glycoproteins, gD, gH/gL, and the fusogen gB. HSV causes lifelong, latent infections that reactivate to cause ailments ranging from cold sores to blindness and encephalitis, and the knowledge of how gB mediates membrane fusion during viral entry may suggest approaches for blocking entry, thereby preventing infections in the first place. The fusogenic mechanism of gB remains unclear, but it is likely to be unique from that of other fusogens due to the unusual reliance of gB upon other viral glycoproteins. A major barrier to elucidating the HSV-specific refolding process and its regulation is that while the structure of the inactive, postfusion form o the gB ectodomain is available, the structure of active, prefusion gB remains elusive. Furthermore, important membrane-interacting regulatory regions of gB, its membrane proximal region (MPR), transmembrane domain (TMD), and cytoplasmic domain (cytodomain), have not yet been resolved. The goals of this application are to determine the structure of full-length postfusion gB and characterize prefusion gB, requisite steps towards unraveling the HSV fusion mechanism. The prefusion form of gB has been the target of several prior investigations, but all of these studies focused on its soluble ectodomain, a typical approach to the study of fusogens. In contrast, this proposal is based upon the hypothesis that gB is maintained in its metastable prefusion conformation through the interaction of its MPR, TMD, and cytodomain with the viral membrane. In essence, the contacts that gB makes with the membrane are critical to its structure and function. Three specific aims underpinned by this hypothesis will be used to study the pre and postfusion gB structures. First, full-length HSV-1 gB will be crystallized in detergent or a detergent/lipid mixture to determine its postfusion structure and reveal the architecture of the membrane proximal region, transmembrane domain, and cytodomain. Information on the interaction of the cytodomain with the membrane will also be obtained by electron spin resonance (ESR) spectroscopy. Second, prefusion gB will be captured in lipid bilayer disks and characterized in this membrane environment by electron microscopy and ESR. Finally, Förster resonance energy transfer (FRET) will be used to assess large conformational changes that are predicted for the prefusion to postfusion transition. Together, these experiments are expected to reveal long- sought details about the prefusion structure and the conformational changes that gB undergoes, illuminating the complex herpesvirus entry process and advancing our general knowledge of how fusogens work.

 描述（由申请方提供）：病毒融合蛋白是动态蛋白，其经历大的重排以促进病毒和靶细胞膜的合并。在单纯疱疹病毒（HSV-1和HSV-2）中，这种融合事件由四种病毒糖蛋白gD、gH/gL和融合原gB的顺序作用引起。HSV会导致终身潜伏感染，这些感染会重新激活，导致从唇疱疹到失明和脑炎等疾病，而关于gB在病毒进入期间如何介导膜融合的知识可能会提示阻断病毒进入的方法，从而首先预防感染。gB的融合机制仍不清楚，但由于gB对其他病毒糖蛋白的异常依赖，它可能与其他融合原不同。阐明HSV特异性重折叠过程及其调控的主要障碍是，虽然gB胞外域的无活性融合后形式的结构是可用的，但活性融合前gB的结构仍然难以捉摸。此外，gB的重要的膜相互作用调节区域，其膜近端区域（MPR），跨膜结构域（TMD）和胞质结构域（cytodomain）尚未得到解决。本申请的目的是确定全长融合后gB的结构并表征融合前gB，这是解开HSV融合机制的必要步骤。gB的融合前形式已经是几个先前研究的目标，但是所有这些研究都集中在其可溶性胞外域，这是研究融合子的典型方法。相反，该建议是基于这样的假设，即gB通过其MPR、TMD和细胞结构域与病毒膜的相互作用而维持在其亚稳态融合前构象。从本质上讲，gB与膜的接触对其结构和功能至关重要。基于这一假设的三个具体目标将被用来研究融合前和融合后的gB结构。首先，全长HSV-1 gB将在洗涤剂中结晶 或去污剂/脂质混合物以确定其融合后结构并揭示膜近端区、跨膜结构域和细胞结构域的结构。还将通过电子自旋共振（ESR）光谱获得关于细胞结构域与膜相互作用的信息。第二，融合前gB将被捕获在脂质双层磁盘和其特征在于在这种膜环境中的电子显微镜和ESR。最后，Förster共振能量转移（FRET）将被用来评估预测的融合前到融合后过渡的大的构象变化。总之，这些实验有望揭示长期寻求的关于融合前结构和gB经历的构象变化的细节，阐明复杂的疱疹病毒进入过程，并推进我们对融合剂如何工作的一般知识。