Mechanisms of Influenza Hemagglutinin Membrane Fusion

描述（由申请人提供）：不同构象的透明质酸的高分辨率结构信息的可用性使其成为研究病毒和细胞膜融合蛋白的最佳模型系统。尽管如此，关于ha介导融合的分子力学的基本问题仍未得到解答。这些包括：(1)负责触发融合所需的酸诱导构象变化的分子决定因素；(2)发生核聚变所需的透明质酸结构重排的程度，以及采用由分子重排导致的最低自由能结构的重要性；(3)连接低pH HA的棒状结构与膜锚点和融合肽域的肽链的融合长度要求；(4)特定高度保守的融合肽残基对融合过程的意义。我们假设在中性pH HA中，融合肽附近的一个区域包含负责触发融合所需的酸诱导构象变化的残基，并旨在识别这些残基并评估每个残基的贡献。我们还假设，仅挤压融合肽不足以进行生物学相关的融合，而在低pH晶体结构中，完全转化为稳定形式的分子对于功能至关重要。我们的目标是产生突变体，以识别对低pH HA结构稳定性至关重要的残基，并将膜相关结构域置于彼此接近的位置，作为融合过程的一个组成部分。我们还旨在解决膜相关区域和低pH棒状结构末端之间融合的距离限制。此外，我们假设沿着23个残基融合肽长度的序列和结构特征将被证明对功能至关重要，我们将继续关注该区域，以努力理解这些考虑因素。我们将使用功能、生化、生物学和结构方法的组合来解决这些问题，使用表达的突变体HAs、分子片段和由反向遗传学产生的感染性病毒。我们提出的问题的阐明将有助于为未来病毒和细胞融合蛋白的研究提供一个平台，并为针对膜融合功能的抗病毒药物提供新的思路。

DESCRIPTION (provided by applicant): The availability of high-resolution structural information for HA in its different conformations has made it the best model system for studies on viral as well as cellular membrane fusion proteins. In spite of this, fundamental questions remain unanswered regarding the molecular mechanics of HA-mediated fusion. These include: (1) the molecular determinants responsible for triggering the acid-induced conformational changes required for fusion; (2) the extent to which the HA structural rearrangements must take place for fusion, and the significance of adopting the lowest free energy structure resulting from molecular rearrangement; (3) the length requirements for fusion of the peptide chains that link the rod-like structure of low pH HA to the membrane anchor and fusion peptide domains and; (4) the significance of specific highly conserved fusion peptide residues for the fusion process. We hypothesize that a region proximal to the fusion peptide in the neutral pH HA contains the residues responsible for triggering the acid-induced conformational changes required for fusion, and aim to identify these and assess the contributions of each. We also hypothesize that extrusion of the fusion peptide alone is not sufficient for biologically relevant fusion and that total conversion to the stable form of the molecule represented in the low pH crystal structures is critical for function. We aim to generate mutants to identify residues that are critical both for the structural stability of low pH HA, and for placing the membrane-associating domains in close proximity to one another as a component of the fusion process. We also aim to address distance constraints for fusion between the membrane-associating regions and the end of the low pH rod-like structure. Furthermore, we hypothesize that sequence and structural characteristics along the length of the 23-residue fusion peptide will prove critical for function, and we will continue to focus attention on this region in an effort to understand these considerations. We will address each of these issues using a combination of functional, biochemical, biological, and structural approaches using expressed mutant HAs, fragments of the molecule, and infectious viruses generated by reverse genetics. The elucidation of the questions that we pose will be instrumental in providing a platform for future studies on viral and cellular fusion proteins, and for instigating new ideas regarding antiviral drugs that target membrane fusion function.