CLONING AND EXPRESSION OF INFLUENZA VIRAL RNA SEGMENTS

描述：(改编自调查人员摘要)：流感病毒，a 一组主要的人类病原体，造成10,000-20,000人死亡和 每年100-200亿美元的经济损失。流感病毒从一开始就发芽了 质膜，特别是来自极化上皮顶侧的质膜 细胞。该项目的长期目标是阐明 病毒蛋白的极化运输与病毒的组装和萌发 粒子。具体目标是：(1)界定HA的根尖决定因素 和NA，被膜病毒蛋白，(Ii)定义HA和NA的相互作用 与M1，(Iii)定义包膜蛋白的作用，HA和NA决定 顶端对基侧萌芽。我们将使用嵌合式结构， 定点突变以及反向遗传学来定义基因的功能 这些蛋白质参与病毒的组装和萌发。我们发现了一种新颖的根尖 顶端NA和HA蛋白跨膜区(TMD)的信号。我们会 剖析和定义TMD心尖信号的顺序和要求 HA和NA。Triton X-100浮选梯度分析的应用 ，我们将剖析TMD和TMD中的序列 与M1特异性相互作用所需的HA和NA的细胞质尾巴。vbl.使用 反向遗传学，我们将确定TMD和细胞质尾部的HA和 病毒生物学方面的NA。最后，使用在基础上定向的HA和NA 如果HA和NA决定了出芽位置，我们将检查转基因病毒 在极化的MDCK细胞中流感病毒的(顶端与基底端)。 流感病毒的组装和萌芽对生长、复制至关重要 并因此在流感病毒的发病机制中发挥作用。详细了解 这些过程将促进抗病毒药物的合理开发 这可能会干扰病毒组装的一个或多个步骤。

DESCRIPTION: (Adapted from the Investigator's abstract): Influenza viruses, a group of major human pathogens, are responsible for 10,000-20,000 deaths and economic loss of $10-20 billions/yr. Influenza viruses asemble and bud from the plasma membrane, specifically from the apical side of polarized epithelial cells. Long term goal of this project is to elucidate the processes involved in polarized transport of viral proteins and assembly and budding of virus particles. Specific objectives are to: (i) define the apical determinants of HA and NA, the envelope viral proteins, (ii) define the interactions of HA and NA with M1, (iii) define the role of envelope proteins, HA and NA determining the apical vs. basolateral budding. We shall use chimeric constructions, site-specific mutations as well as reverse genetics to define the function of these proteins in virus assembly and budding. We have discovered a novel apical signal in the transmembrane domain (TMD) of apical NA and HA proteins. We will dissect and define the sequences and requirements of apical signal in the TMD of HA and NA. Using the floatation gradient analysis of Triton X-100 detergent-treated membranes, we will dissect the sequences in the TMD and cytoplasmic tail of HA and NA required for specific interaction with M1. Using reverse genetics, we will determine the role TMD and cytoplasmic tail of HA and NA in virus biology. Finally, using basolaterally targeted HA and NA in transfectant viruses we will examine if HA and NA determine the budding site (apical vs. basolatral) of influenza viruses in polarized MDCK cells. Assembly and budding of influenza viruses are critical for growth, replication and consequently in pathogenesis of influenza viruses. A detailed understanding of these processes will facilitate the rational development of antiviral agents which could interfere with one or more steps in virus assembly.