Assembly of human oncoviruses and bacteriophage

人类肿瘤病毒和噬菌体的组装

• 批准号: 6226080
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6226080
• 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.

与人类乳头瘤病毒（HPV）感染相关的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的组装和对其的免疫反应为预防疾病或改变其病程的药理学和免疫学干预提供了多种机会。这些干预措施的有效设计将有助于了解这些现象的结构基础。提出了阐明HPV衣壳结构及其抗原表位的调查。由于HPV不易在培养细胞中复制，因此很难通过传统的病毒学和病毒晶体学技术进行研究。为了规避这些限制，目前正在执行一种策略，该策略将HPV颗粒和HPV的EM图像重建：抗体复合物与单个蛋白质组分的晶体结构相结合。来自图像重建的信息将通过伪原子建模与来自晶体学的结构相结合。如PPG集团应用于其他系统。与co PI Baker一起，我们已经完成了HPV 6 b的高分辨率EM图像重建，并确定它至少在16埃是相当可靠的。扩展到更高的准确性和分辨率以及HPV：抗体复合物的类似研究将为我们的策略提供所需的数据。对于另一部分，我们已经以适合于结晶试验的可溶形式表达了HPV 6 b的主要和次要衣壳蛋白。虽然HPV的组装是完全“自我导向”的;但许多其他生物结构的组装仅在伴侣分子的帮助下发生。一种采用分子伴侣的系统是噬菌体λ的侧尾纤维的组装。侧尾纤维是在野生型λ上发现的非常长、细、有关节的纤维。这些长纤维，Stf基因产物的修剪物，类似于其他哺乳动物中的类似纤维，并且仅以pTfa分子伴侣为目的而形成。这些纤维增加了噬菌体吸收细胞的效率，可能是与第二个受体结合。一些研究表明，与任何其他分子伴侣不同，pTfa可以“主动”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目标。虽然所有这些都未能产生适合于X射线衍射的晶体，但我们最近生长了pTfa的胰蛋白酶片段的晶体，其衍射超过2 A。提出了确定这种结构和pTfa与非常长的、灵活的Stf蛋白的合适片段的复合物的结构。