Assembly of human oncoviruses and bacteriophage

人类肿瘤病毒和噬菌体的组装

• 批准号: 6340731
• 负责人: ALAN M FRIEDMAN
• 金额: $ 16.62万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6340731
• 关键词: X ray crystallography; antiviral antibody; bacteriophage lambda; chemical models; cryoelectron microscopy; crystallization; human papillomavirus; immune complex; molecular chaperones; structural biology; virus assembly

Cervical cancer associated with infection by human papillomavirus (HPV) is the second leading cause of cancer deaths among women worldwide, resulting in one in six deaths. Understanding the assembly of HPV and the immune response to it offers multiple opportunities for pharmacologic and immunologic intervention to prevent disease or alter its course. Effective design of these interventions would be aided by knowledge of the structural basis of these phenomena.. Investigations to elucidate the structure of the HPV capsid and its antigenic epitopes are proposed. Because HPV does not readily replicate in cultured cells, it has been extremely difficult to study by the traditional techniques of virology and virus crystallography. To circumvent these limitations, a strategy that combines EM image reconstruction of HPV particles and HPV: antibody complexes with crystal structures of individual protein components is currently being executed. Information from image reconstruction will be combined with structures from crystallography by pseudo-atomic modeling. as applied to other system by the PPG group. With co PI Baker, we have completed a high resolution EM image reconstruction of HPV6b, and determined that it is quite reliable to at least 16 angstroms. Extension to higher accuracy and resolution and similar studies on HPV: antibody complexes will provide the data required for part of our strategy. For the other part, we have expressed both major and minor capsid proteins of HPV6b in a soluble form suitable for crystallization trials. While assembly of HPV is completely "self-directed"; assembly of many other biological structures occurs only with the aid of chaperone molecules. One system that employs chaperones is the assembly of the side tail fibers of phage lambda. Side tail fibers are very long, thin, jointed fibers found on wild-type lambda. These long fibers, trimmers of the Stf gene product, are analogous to similar fibers in other phages, and are formed only with the aim of the pTfa chaperone. The fibers increase efficiency of phage absorption to cells, presumably engaging a second receptor. Some studies suggest that, unlike any other chaperone, pTfa can act "instructively" to alter the structure of the assembled fiber. To investigate this unusual behavior, pTfa has been the object of several attempts to crystallize the intact molecule. While all of these have failed to yield crystals suitable for x-ray diffraction, we have very recently grown crystals of a tryptic fragment of pTfa that diffract to beyond 2A. Determination of this structure and the structure of complexes of pTfa with suitable fragments of the very long, flexible Stf protein is proposed.

与人乳头瘤病毒（HPV）感染有关的子宫颈癌是全世界妇女癌症死亡的第二大原因，导致六分之一的死亡。了解HPV的组装及其免疫反应为药物和免疫干预提供了多种机会，以预防疾病或改变其进程。对这些现象的结构基础的了解将有助于这些干预措施的有效设计。研究阐明了HPV衣壳及其抗原表位的结构。由于人乳头瘤病毒不容易在培养细胞中复制，用传统的病毒学和病毒晶体学技术来研究它是极其困难的。为了规避这些限制，目前正在执行一种将HPV颗粒和HPV抗体复合物与单个蛋白质成分的晶体结构相结合的EM图像重建策略。图像重建的信息将通过伪原子建模与晶体学的结构相结合。适用于PPG集团的其他系统。我们与co PI Baker一起完成了HPV6b的高分辨率EM图像重建，并确定其至少在16埃范围内是相当可靠的。扩展到更高的准确性和分辨率，以及对HPV抗体复合物的类似研究将为我们的部分战略提供所需的数据。另一方面，我们以适合结晶试验的可溶性形式表达了HPV6b的主要和次要衣壳蛋白。而HPV的组装完全是“自我导向的”；许多其他生物结构的组装只有在伴侣分子的帮助下才能发生。使用伴侣蛋白的一个系统是噬菌体侧尾纤维的组装。侧尾纤维非常长，薄，在野生型lambda上发现的节理纤维。这些长纤维是Stf基因产物的修剪物，类似于其他噬菌体中的类似纤维，并且仅以pTfa伴侣蛋白为目的而形成。这些纤维增加了噬菌体对细胞的吸收效率，可能与第二受体结合。一些研究表明，与其他任何伴侣不同，pTfa可以“指导性地”改变组装纤维的结构。为了研究这种不寻常的行为，pTfa一直是几次尝试结晶完整分子的对象。虽然所有这些都没有产生适合x射线衍射的晶体，但我们最近已经培养出了pTfa色氨酸片段的晶体，其衍射范围超过2A。提出了这种结构和pTfa与非常长、灵活的Stf蛋白的合适片段的复合物的结构的测定。