Project 3

该项目将扩展Ahlquist小组与Loeb和Lambert小组的积极合作，以确定两种最重要的人类肿瘤病毒--乙肝病毒(HBV)和人乳头状瘤病毒(HPV)--的病毒粒子组装、基因组复制和病毒与宿主相互作用的关键方面。这些实验使用了对人类细胞和遗传易驯化的酿酒酵母的综合研究，Ahlquist小组率先将其作为模型宿主来研究病毒复制和病毒与宿主的相互作用。该项目有四个目标：对于乙肝病毒，我们将(I)使用共聚焦显微镜、电子显微镜和细胞分离 确定在人肝癌细胞和酵母中单独表达和选择组合表达的乙肝病毒结构蛋白[核心、逆转录酶和包膜蛋白(表面抗原)]在细胞内的定位、定位决定因素和相互作用；(Ii)使用靶向和全局酵母遗传筛选来确定酵母中乙肝病毒包膜蛋白驱动的出芽进入内质网相关的隔室是否依赖宿主细胞功能，包括逆转录病毒和其他病毒参与质膜萌发关键后期步骤的空泡蛋白分离和泛素化功能；(Iii)扩大正在进行的实验，以在酵母中复制前基因组RNA封装和随后的基因组复制步骤，以用于乙肝病毒及其主要实验室模型D乙肝(鸭乙肝病毒)，以便能够研究宿主的贡献 在基因组可以自由和系统地操纵的细胞中的这些复制步骤。对于HPV，我们将(IV)结合Ahlquist实验室在酵母中对HPV蛋白表达和衣壳组装的优化，以及Lambert实验室对酵母复制和稳定维护HPV基因组DNA的发现，以确定酵母中HPV基因组封装和传染性病毒粒子组装所需的蛋白质和DNA。除了确定组装决定因素外，这些研究还将克服目前器官型RAW培养的局限性，提供一个更容易获得可遗传操作的、具有感染性的HPV病毒粒子的来源，用于研究病毒进入哺乳动物细胞和早期感染步骤。

This project will extend active collaborations of the Ahlquist group with the Loeb and Lambert groups to define key aspects of virion assembly, genome replication, and virus-host interactions for two of the most important human tumor viruses, hepatitis B virus (HBV) and human papillomavirus (HPV). These experiments use integrated studies in human cells and the genetically tractable yeast Saccharomyces cerevisiae, which the Ahlquist group has pioneered as a model host to study virus replication and virus-host interactions. The project has four aims: For HBV, we will (I) Use confocal microscopy, electron microscopy and cell fractionation to define the intracellular localization, localization determinants, and interaction of the HBV structural proteins [core, reverse transcriptase, and envelope proteins (surface antigens)] when expressed singly and in selected combinations in human hepatoma cells and in yeast; (II)Use targeted and global yeast genetic screens to determine if HBV envelope protein-driven budding into endoplasmic reticulum-associated compartments in yeast depends on host cell functions, including vacuolar protein sorting and ubiquitination functions involved in crucial late steps of plasma membrane budding by retroviruses and other viruses; (III) Extend ongoing experiments to duplicate pregenomic RNA encapsidation and subsequent genome replication steps in yeast for HBV and its major laboratory model, DHBV (duck HBV), to allow study of host contributions to these replication steps in cells whose genome can be freely and systematically manipulated. For HPV, we will (IV) combine the Ahlquist lab's optimization in yeast of HPV protein expression and capsid assembly with the Lambert lab's finding that yeast replicate and stably maintain HPV genomic DNA, to determine the protein and DNA requirements for HPV genome encapsidation and infectious virion assembly in yeast. In addition to defining the assembly determinants, these studies will overcome present limitations of organotypic raft cultures to provide a more facile source of genetically manipulatable, infectious HPV virions with which to study virus entry and early infection steps in mammalian cells.