INTERPLAY BETWEEN THE HA AND NA OF INFLUENZA VIRUSES

就发病率和死亡率而言，流感病毒感染仍然是最重要的呼吸道疾病。两种主要的表面糖蛋白血凝素（HA）和神经氨酸酶（NA）在流感病毒的组织和器官趋向性、传播和致病性中起重要作用。我们的长期目标是了解HA和NA的受体结合和受体破坏功能之间平衡的分子机制，以及这种平衡的变化如何影响人类流感病毒的致病性。一种新的NA抑制剂扎那米韦的抗病毒作用已在临床试验中得到证实。然而，当人类流感病毒在人类宿主中复制时，NA抑制剂对HA和NA特性的影响，在我们的知识方面存在重大差距。先前在体外细胞培养系统中研究NA抑制剂对流感病毒特性的影响时，没有考虑细胞受体的特异性，忽略了HA和NA功能的效率在很大程度上取决于这些受体（Siaalpha2,3Gal或Siaalpha2,6Gal）的特异性。因此，在特异性目标1中，我们将确定允许人类流感病毒在含有siaalpha2,6gal受体的人呼吸道上皮中存在NA抑制剂的情况下复制的机制。在特异性目标2中，我们将验证NA活性降低促进对HA受体亲和力降低的扎那米韦耐药病毒复制的假设，而在特异性目标3中，我们将利用具有类似于人类呼吸上皮受体特异性的雪貂模型，确定HA和NA突变对体内扎那米韦耐药性发展的影响。最后，在特异性目标4中，我们将确定扎那米韦耐药突变体的HA和/或NA的变化如何影响这些病毒在雪貂中的毒力。该应用的主要优势是：(1)从人呼吸道分离出具有良好特征的扎那米韦耐药突变体；(2)具有足够受体特异性的实验系统，人呼吸道上皮的原代组织培养（体外）和雪貂模型（体内）。总的来说，拟议的研究将提供流感病毒两种主要表面糖蛋白相互依赖的基本信息，重点关注在NA抑制剂压力下允许复制的适应性机制。对HA-NA相互作用的进一步了解无疑将有助于对抗特异性靶向NA糖蛋白的抗病毒药物的耐药性，并有助于阐明这种适应策略对人流感病毒毒力的影响。

Influenza virus infection continues to be the most important respiratory disease both in terms of morbidity and mortality. The two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), play important roles in tissue and organ tropism, spread, and pathogenicity of influenza viruses. Our long term goals are to understand the molecular mechanisms underlying the balance between the receptor-binding and receptor-destroying functions of the HA and NA and how changes in this balance affect the pathogenicity of influenza viruses in humans. The antiviral effect of a new NA inhibitor, zanamivir, has been demonstrated in clinical trials. However, there is a major gap in our knowledge of the effects of NA inhibitors on properties of the HA and NA of human influenza viruses when replication occurs in a human host. Previous studies on the effect of NA inhibitors on the properties of influenza viruses utilized in vitro cell culture systems without regard to the specificity of the cellular receptors, neglecting the fact that the efficiency of the HA and NA functions strongly depends on the specificity of these receptors (Siaalpha2,3Gal or Siaalpha2,6Gal). Therefore, in Specific Aim 1 we will identify the mechanism(s) that allow human influenza viruses to replicate in the presence of an NA inhibitor in human respiratory tract epithelium containing Siaalpha2,6Gal-receptors. In specific Aim 2 we will test the hypothesis that a decrease in NA activity promotes the replication of zanamivir-resistant viruses with reduced affinity for HA receptors, while in Specific Aim 3 we will determine the effect of HA and NA mutations on the development of zanamivir resistance in vivo, utilizing a ferret model that possesses receptor specificity similar to that of human respiratory epithelium. Finally, in Specific Aim 4, we will determine how changes in the HA and/or NA of zanamivir-resistant mutants affect the virulence of such viruses in ferrets. The major strength of this application is (i) availability of well-characterized zanamivir-resistant mutants isolated from human respiratory tract and (ii) experimental systems with adequate receptor specificity, primary tissue culture of the human respiratory tract epithelium (in vitro) and a ferret model (in vivo). Overall, the proposed studies will provide fundamental information on the interdependence of the two major surface glycoproteins of influenza viruses, focussing on adaptive mechanisms allowing replication under pressure from an NA inhibitor. Improved understanding of HA-NA interactions will undoubtedly aid in counteracting resistance to antiviral agents that specifically target the NA glycoprotein and in elucidating the impact on virulence of such adaptive strategies in human influenza viruses.