CLONING AND EXPRESSION OF INFLUENZA VIRAL RNA SEGMENTS

描述：（改编自研究者摘要）：流感病毒，a 一组主要的人类病原体，造成10000 - 20000人死亡， 经济损失10 -20亿美元/年。流感病毒的萌芽来自 质膜，特别是从极化上皮细胞的顶侧 细胞本项目的长期目标是阐明 病毒蛋白质的极化转运与病毒的组装和出芽 粒子具体目标是：（i）确定HA的顶端决定因素 和NA，包膜病毒蛋白，（ii）定义HA和NA的相互作用 与M1，（iii）定义的包膜蛋白，HA和NA决定的作用， 顶侧与基底侧出芽。我们将使用嵌合结构， 位点特异性突变以及反向遗传学来定义 这些蛋白质参与病毒的组装和出芽。我们发现了一种新的 在顶端NA和HA蛋白的跨膜结构域（TMD）中的信号。我们将 分析并明确TMD中心尖信号的序列和要求 的HA和NA。用Triton X-100浮选梯度分析法 洗涤剂处理过的膜，我们将剖析TMD中的序列， 与M1特异性相互作用所需的HA和NA的胞质尾区。使用 反向遗传学，我们将确定TMD和HA的胞质尾区的作用， 病毒生物学中的NA。最后，使用基底外侧靶向HA和NA， 我们将检查HA和NA是否决定了出芽位点 （顶侧对基底侧）流感病毒在极化的MDCK细胞中的表达。 流感病毒的组装和出芽对于生长、复制和繁殖至关重要。 并因此参与流感病毒的发病机理。详细了解 这些过程将促进抗病毒药剂的合理开发 这可能干扰病毒装配中的一个或多个步骤。

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.