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。血凝素(HA)和神经氨酸酶(NA)这两种主要的表面糖蛋白在流感病毒的组织器官趋向性、传播和致病性方面发挥着重要作用。我们的长期目标是了解HA和NA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，当人类宿主发生复制时，我们对NA抑制剂对人类流感病毒HA和NA特性的影响的了解存在重大差距。以前关于NA抑制剂对体外细胞培养系统中流感病毒特性的影响的研究没有考虑细胞受体的特异性，忽视了HA和NA功能的有效性强烈依赖于这些受体(Siaalpha2，3Gal或Siaalpha2，6Gal)的事实。因此，在特定目的1中，我们将确定允许人类流感病毒在安娜抑制剂存在下在含有Siaalpha2，6GalR的人呼吸道上皮细胞中复制的机制(S)。在特定目标2中，我们将检验假设，即NA活性的降低促进了与HA受体亲和力降低的扎那米韦耐药病毒的复制，而在特定目标3中，我们将利用具有与人类呼吸道上皮相似的受体特异性的雪貂模型，在体内确定HA和NA突变对扎那米韦耐药发展的影响。最后，在特定的目标4中，我们将确定扎那米韦耐药突变株的HA和/或NA的变化如何影响这种病毒在雪貂中的毒力。这一应用的主要优点是(I)从人类呼吸道中分离出具有良好特性的扎那米韦耐药突变株，(Ii)具有足够受体特异性的实验系统，人类呼吸道上皮的原代组织培养(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两种主要表面糖蛋白之间相互依赖的基本信息，重点是允许在安娜抑制剂压力下复制的适应性机制。提高对HA-NA相互作用的了解无疑将有助于对抗针对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类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.