Mechanisms of Influenza Hemagglutinin Membrane Fusion

描述(由申请人提供)：HA不同构象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介导的融合的分子机制的基本问题仍然没有得到回答。这些问题包括：(1)引发融合所需的酸诱导构象变化的分子决定因素；(2)融合所需的HA结构重排的程度，以及采用分子重排产生的最低自由能结构的意义；(3)将低pH HA的棒状结构连接到膜锚定和融合多肽结构域的多肽链的融合长度要求；(4)特定的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，融合肽近端的一个区域包含负责触发酸诱导的融合所需构象变化的残基，并旨在识别这些残基并评估每个残基的贡献。我们还假设，仅有融合肽的挤出不足以进行生物相关的融合，并且完全转化为低pH晶体结构中所代表的分子的稳定形式对功能至关重要。我们的目标是产生突变体来识别残基，这些残基对低pH HA的结构稳定性以及作为融合过程的一个组成部分将膜结合结构域放置在一起是至关重要的。我们还旨在解决膜结合区域和低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个残基的融合肽长度的序列和结构特征将被证明对功能至关重要，我们将继续关注这一区域，以努力理解这些考虑。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，这些方法使用表达的突变HAS、分子片段和由反向遗传学产生的传染性病毒。我们提出的问题的阐明将有助于为未来对病毒和细胞融合蛋白的研究提供平台，并引发针对膜融合功能的抗病毒药物的新想法。

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.