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嗜性、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，当在人类宿主中发生复制时，NA抑制剂对人流感病毒HA和NA性质的影响的知识存在重大差距。 先前关于NA抑制剂对流感病毒性质的影响的研究在体外细胞培养系统中使用，而不考虑细胞受体的特异性，忽略了HA和NA功能的效率强烈依赖于这些受体（Siaa 2，3Gal或Siaa 2，6 Gal）的特异性的事实。 因此，在具体目标1中，我们将确定允许人流感病毒在NA抑制剂存在下在含有Siaalpha 2，6 Gal受体的人呼吸道上皮中复制的机制。 在具体目标2中，我们将测试假设NA活性的降低促进扎那米韦耐药病毒的复制，降低HA受体的亲和力，而在具体目标3中，我们将确定HA和NA突变对体内扎那米韦耐药发展的影响，利用雪貂模型，具有类似于人呼吸道上皮的受体特异性。 最后，在具体目标4中，我们将确定扎那米韦耐药突变体的HA和/或NA的变化如何影响此类病毒在雪貂中的毒力。 本申请的主要优势在于：（i）从人呼吸道分离的充分表征的扎那米韦耐药突变体的可用性和（ii）具有足够受体特异性的实验系统，人呼吸道上皮细胞的原代组织培养（体外）和雪貂模型（体内）。 总体而言，拟议的研究将提供流感病毒的两个主要表面糖蛋白的相互依赖性的基本信息，重点是适应机制，允许复制的压力下，从NA抑制剂。 对HA-NA相互作用的进一步了解无疑将有助于抵消对特异性靶向NA糖蛋白的抗病毒剂的耐药性，并阐明这种适应性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.