Genetic Analysis of Papillomavirus Virion Morphogenesis

描述(申请人提供)：乳头瘤病毒(PV)的衣壳蛋白已经进化出功能结构域，用于它们之间、与PV病毒非结构蛋白、PV基因组DNA以及与宿主细胞因子的适当相互作用，从而允许感染性病毒颗粒的形态发生。使用病毒样颗粒(VLP)和伪病毒作为病毒形态发生的终点进行的分析可能等同于或可能不等同于在分化宿主组织中传播的真实病毒。我们的器官型病毒培养系统能够支持整个病毒生命周期，再加上我们在使用单抗研究衣壳构象方面的专业知识，使我们处于独特的地位，可以启动第一项研究，直接比较VLP自组装和真正的PV合成。这些研究将直接调查L2蛋白在真正的病毒形态发生中的功能作用，并包括调查L2在VLP自组装过程中的作用。我们的研究将集中在两种主要的遗传途径上。其中一个将使用嵌合遗传方法，通过寻找两种病毒类型相互作用蛋白的大结构域之间的特定类型差异来开始研究。另一种是对其他实验室提到的与病毒粒子形态发生有关的功能区域进行特异性突变。为了实现我们的研究目标，我们制定了四个具体的目标：(1)研究乳头瘤病毒类型特异性结构基因的相互作用；(2)比较病毒样颗粒(VLP)组装和真正的病毒粒子形态发生的遗传要求；(3)从基因上分析衣壳蛋白半胱氨酸残基在病毒粒子形态发生中的作用；以及(4)通过突变分析研究L1衣壳蛋白的N端在病毒粒子形态发生中的作用。完成后，这些研究将首次为病毒粒子形态发生的分子机制提供新的见解，该系统能够在分化的环境中再现光伏病毒形态发生和感染的自然复杂过程。

DESCRIPTION (provided by applicant): Capsid proteins of papillomaviruses (PV) have evolved functional domains for the proper interaction between themselves, with PV viral nonstructural proteins, with PV genomic DNA, and with host cell factors, allowing for morphogenesis of infectious viral particles. Analyses performed using virus-like particles (VLPs) and pseudoviruses as an endpoint for viral morphogenesis may or may not equate to the authentic virus propagated in differentiating host tissue. Our organotypic virus culture system, capable of supporting the complete viral life cycle, coupled with our expertise in using MAbs to study capsid conformation, place us in the unique position to initiate the first studies to directly compare VLP self-assembly with authentic PV synthesis. These studies will directly investigate a functional role for L2 protein in authentic virus morphogenesis and include investigating the role of L2 in the process of VLP self-assembly. Our studies will focus on 2 main genetic approaches. One will use a chimeric genetic approach allowing investigations to begin by looking for type-specific differences between large domains of the interacting proteins of 2 viral types. The other is to specifically mutate domains that have been alluded to by other laboratories as having a function related to virion morphogenesis. To accomplish our research goals we have developed four specific aims: (1) Investigate papillomavirus type-specific structural gene interactions; (2) Compare the genetic requirements for the assembly of virus-like particles (VLPs) to authentic virion morphogenesis; (3) Genetically analyze the role of capsid protein cysteine residues in virion morphogenesis; and (4) Investigate the role of the N-terminus of the L1 capsid protein in virion morphogenesis by mutational analyses. When completed the studies proposed will for the first time provide new insight into the molecular mechanisms of virion morphogenesis in a system capable of reproducing the natural complex processes of PV morphogenesis and infection in a differentiating environment.