Core C - Virus Core

核心C--项目摘要/摘要 核心C(病毒核心)的目的是为生成提供一种时间和成本高效的机制， 所有8项研究所需的野生型(WT)和突变病毒的验证、分布和档案存储 与本计划项目相关联的小组，用于他们建议的学习。具体目标是：(1)生产 WT、突变型和复变型EB病毒(EBV)和卡波西肉瘤疱疹病毒的有效库存 (KSHV)；(2)生产经过验证的WT和变异的乙肝病毒(HBV)；(3)生产经过验证的 存有传染性小鼠乳头瘤病毒(MmuPV1)和人乳头状瘤病毒(HPV)假病毒。 (1)。 突变的EBV(用于项目3、4和5)或KSHV(用于项目3)将在大肠杆菌中产生，以 除了标准方法外，我们还使用了无疤痕通过方法的适当的BAC 建立了生物信息管道来分析下一代疱疹病毒基因组测序数据，以 确保在独特的区域内没有意外的突变。只要有必要，表型就会 通过反互补或构建EBV/KSHV返回体基因组进行验证。病毒库 EBV或KSHV的WT、突变体和回复变种将使用Dr。 糖化、滴定和储存，以供所有四个EBV组使用。将生产乙肝病毒粒子(用于项目2)： 稳定转染四环素诱导的乙肝病毒表达的HepG2衍生物细胞 系统。乙肝病毒突变体将通过重组DNA方法在已经开发的载体中构建 勒布实验室。乳头瘤病毒病毒粒子和假病毒粒子将通过联合- 用(I)所需衣壳蛋白表达载体和(Ii)所需病毒或 荧光素酶/GFP编码的DNA，目标是使用先前由 兰伯特/阿尔奎斯特实验室。除了提高成本效率和质量控制外，还存在 这一核心有助于使用常见的病毒库，从而对补充数据进行解释 在不同的研究小组之间产生的，更可靠地合并，以实现 项目。与这5个项目有关的所有8个研究小组将根据需要由该核心设施提供服务。

CORE C – PROJECT SUMMARY/ABSTRACT The purpose of Core C (Virus Core) is to provide a time and cost efficient mechanism for the generation, validation, distribution, and archival storage of wild-type (WT) and mutant viruses needed by all 8 research groups associated with this Program Project for their proposed studies. The Specific Aims are: (1) to produce validated stocks of WT, mutant, and revertant Epstein-Barr virus (EBV) and Kaposi Sarcoma Herpesvirus (KSHV); (2) to produce validated stocks of WT and mutant hepatitis B viruses (HBVs); (3) to produce validated stocks of infectious mouse papillomavirus (MmuPV1) and human papillomavirus (HPV) pseudoviruses. (1). Mutant EBV (for Projects 3, 4, and 5) or KSHV (for Project 3) will be generated in E. coli starting with appropriate BACs using the scarless En Passant method In addition to standard methods, we have established a bioinformatic pipeline to analyze next generation sequencing data of herpesvirus genomes to ensure there are no unintended mutations within the unique regions. Whenever necessary, phenotypes will be validated using transcomplementation or the construction of revertant EBV/KSHV genomes. Virus stocks of WT, mutant, and revertant variants of EBV or KSHV will be generated using a protocol developed by Dr. Sugden, titered, and stored for use by all four EBV groups. HBV virions will be produced (for Project 2) from HepAD38 cells, a HepG2 derivative that is stably transfected with a tetracycline-inducible HBV expression system. HBV mutants will be constructed by recombinant DNA approaches in vectors already developed by the Loeb laboratory. Papillomavirus virions and pseudovirions will be generated (for Project 1) by co- transfection of 293T cells with (i) the desired capsid protein-expression plasmids, and (ii) the desired viral or luciferase/GFP-encoding DNAs targeted for encapsidation using protocols previously published by the Lambert/Ahlquist laboratories. In addition to increased cost efficiency and quality control, the existence of this Core facilitates the use of common virus stocks that will make the interpretation of complementary data generated among the various research groups more reliably merged toward achieving shared aims within the projects. All 8 research groups associated with the 5 projects will be served by this Core facility as needed.