Structural insights into pandemic and emerging influenza viruses

描述(申请人提供)：甲型流感病毒通过血凝素(HA 1-16)和神经氨酸酶(NA 1-9)表面抗原的多种血清型表现出极大的多样性。到目前为止，在鸟类和动物宿主中发现的144种可能的组合中，只有3种与人类大流行有关(H1N1、H2N2、H3N2)。最近，在蝙蝠身上发现了一种独特的甲型流感病毒谱系，进一步增加了可能感染人类的人畜共患病毒的谱系。这一建议试图在结构水平上阐明流感病毒的关键脆弱部位，以发展可持续的跨血清型免疫反应，并了解作为大流行和季节性流感病毒致病性和传播性基础的表面糖蛋白(HA，NA)和核糖核蛋白(RNP)复合体的活性关系，包括聚合酶(PA，PB1，PB2)。抗体介导的流感病毒中和对于人类免疫系统来说是一个复杂的组合问题，因为它呈现出多样化、高度可变和不断进化的病毒。虽然针对人类流感的中和抗体传统上被认为是毒株特异性的，但最近的研究表明，在特定亚型(例如H3N2型)数十年的进化过程中，可以跨组1或组2，甚至跨两个主要的系统发育组(I和2)进行更广泛的反应。虽然这些例子提供了令人信服的证据，表明免疫系统能够对流感产生持续的、跨血清型的反应，但如何通过接种疫苗来诱导广泛的中和抗体却知之甚少。因此，我们建议确定广泛中和的结构基础，并划定HA上的脆弱部位，以开发新的疫苗支架，甚至小分子抑制剂，以改善或防止疾病进展。此外，由于我们不明白为什么某些病毒，如最近的H1N1 2009年猪流感，能够进入人类群体并导致大流行，我们也将研究致病性的分子基础。通过使用新设计的糖链微阵列研究HA和NA底物的特异性和活性的新策略，我们将评估NA和HA活性之间的功能关系。通过这种方式，我们将检验这一假设，即流感病毒有效感染人类需要在HA的结合和特异性与NA与宿主多糖受体的酶活性之间取得功能平衡。宿主特异性致病的另一个关键因素是由RNP和相关聚合酶组成的复制机制，其中突变可以改变聚合酶活性和与宿主细胞因子的相互作用。对RNP结构和功能的理解 将使抗击流感感染的其他方法成为可能。三个实验室使用最先进的X射线结晶学、电子显微镜和葡聚糖阵列技术的生物物理和生物化学相结合的方法将被用来提供关于流感病毒中和、取向和发病机制的关键见解，以揭示控制和抗击未来大流行的新战略。

DESCRIPTION (provided by applicant): Influenza A viruses exhibit extreme diversity via multiple serotypes of the hemagglutinin (HA 1-16) and neuraminidase (NA 1-9) surface antigens. To date, only three of the possible 144 combinations found in bird and animal reservoirs have been associated with human pandemics (H1N1, H2N2, H3N2). Recently, a distinct lineage of influenza A viruses has been identified in bats, further increasing the spectrum of possible zoonotic viruses that could infect humans. This proposal seeks to elucidate at the structural level, key sites of vulnerability on influenza virus for development ofa sustainable cross-serotype immune response, and understand activity relationships of the surface glycoproteins (HA, NA) and ribonucleoprotein (RNP) complex, including the polymerase (PA, PB1, PB2), that underlie the pathogenicity and transmissibility of pandemic and seasonal influenza viruses. Antibody-mediated neutralization of influenza virus is a complex combinatorial problem for the human immune system as it is presented with diverse, highly variable and constantly evolving viruses. While neutralizing antibodies against human flu are traditionally regarded as being strain specific, recent studies have shown that a much broader response can be mounted over decades of evolution of a particular subtype (e.g. H3N2), across group 1 or group 2, and even across two major phylogenetic groups (I and 2). While these examples provide compelling evidence that the immune system is capable of mounting a sustained, cross-serotype response against influenza, how to elicit broadly neutralizing antibodies by vaccination is poorly understood. Therefore, we propose to determine the structural basis of broad neutralization and delineate the sites of vulnerability on the HA to enable development of novel vaccine scaffolds and even small molecule inhibitors that ameliorate or prevent disease progression. Furthermore, because we do not understand why certain viruses, such as the recent H1N1 2009 swine flu, are able to enter the human population and cause pandemics, we will also study the molecular basis of pathogenicity. Using a novel strategy to investigate both HA and NA substrate specificity and activity using newly designed glycan microarrays, we will assess the functional relationships between NA and HA activity. In this way, we will test the hypothesis that efficient infection of humans by influenza viruses requires a functional balance between the binding and specificity of HA and enzymatic activity of the NA with host glycan receptors. Another key factor in host-specific pathogenicity is the replication machinery composed of the RNP with associated polymerase, where mutations can alter polymerase activity and interaction with host cell factors. Structural and functional understanding of the RNP will enable other approaches to combat influenza infection. A combined biophysical and biochemical approach from three laboratories employing state of the art x-ray crystallography, electron microscopy and glycan array technologies will be used to provide key insights into influenza virus neutralization, tropism and pathogenesis, to reveal novel strategies to control and combat future pandemics.