BIOSYNTHESIS OF PARAMYXOVIRUSES

副粘病毒是导致呼吸道疾病的最主要原因 孩子们。长期目标是开发预防措施 由副流感病毒引起的呼吸道疾病。要做到这一点 目标，拟议的研究将集中在 血凝素-神经氨酸酶(HN)和融合(F)糖蛋白 使用两种副流感病毒1型感染：仙台病毒(SV)，a 小鼠的自然病原体和人副流感1型(hPIV-1)，以及 儿童的重要病原体。策略是应用于 每种病毒蛋白的结构/功能分析 在以下具体目标中。目标1：我们建议明确 确定HN在病毒融合中的作用，从结构上定义融合 促进领域与本质的融合促进功能。目标2： 确定结构相关性和生物学意义 SV HN神经氨酸酶活性。目的3：制备HN分子 和F形成的晶体适合于建立三维 结构。 目标1和目标2将使用表位映射、序列分析和功能 中和逃逸突变体、失败突变体的特征 HN的制备及其结合定点定位表达的研究 用突变来确定结构域和生物学意义 HN的融合促进和神经氨酸酶功能。这些方法 将用于定义抗原性结构、关键氨基酸和 每个领域的范围和复杂程度。使用ts突变体和 CDNAs的表达将检验融合促进的未解决的作用 病毒感染时HN的神经氨酸酶活性。SV HN突变体 神经氨酸酶活性增强并显著促进生长 将被用来检验这一假说 神经氨酸酶活性通常是病毒感染性的决定因素， 致病机理和致病力。目标3将用以下物质制备F分子 结晶和X射线分析的潜力。要求是(I) 通过以下方法去除疏水性膜锚定末端(尾部) 蛋白水解法，(Ii)离心法纯化无尾分子 和层析，以及(Iii)生物活性的保留。 抗原-单抗复合体将作为一种替代方法 结晶。

Paramyxoviruses are the most important cause of respiratory disease in children. The long-term objective is to develop means to prevent respiratory disease caused by parainfluenza viruses. To reach that objective, the proposed research will focus on the roles of the hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins during infection using two parainfluenza virus 1 subtypes: Sendai virus (SV), a natural pathogen of mice, and human parainfluenza type 1 (hPIV-1), an important pathogen of children. The strategy is to apply structure/function analysis to each of these viral proteins as outlined in the following specific aims. Aim 1: We propose to definitively establish a role of HN in virus fusion, structurally define the fusion promoting domain and the nature of the fusion promoting function. Aim 2: Determine the structural correlates and the biological significance of the neuraminidase activity of SV HN. Aim 3: To prepare molecules of HN and F that form crystals suitable for establishing the threedimensional structure. Aims 1 and 2 will use epitope mapping, sequence analysis and functional characterization of neutralization escape mutants, ts mutants that fail to make HN, and expression of cDNAs in conjunction with site-directed mutagenesis to define the structural domains and biological significance of the fusion promoting and neuraminidase functions of HN. These methods will be used to define the antigenic structure, critical amino acids, and the extent and complexity of each domain. Studies using ts mutants and cDNA expression will examine the unresolved roles of the fusion promoting and neuraminidase activities of HN in virus infection. SV HN mutants with enhanced neuraminidase activity and significantly increased growth in embryonated eggs will be used to test the hypothesis that neuraminidase activity in general is a determinant of virus infectivity, pathogenesis and virulence. Aim 3 will prepare F molecules with potential for crystallization and X-ray analysis. Requirements are (i) removal of the hydrophobic membrane anchoring termini (tails) by proteolysis, (ii) purification of tailless molecules by centrifugation and chromatography, and (iii) retention of biological activity. Antigen-MAb complexes will serve as an alternate method of crystallization.