Core C - Virus Core

核心 C - 病毒核心

• 批准号: 10414882
• 负责人: ERIC C JOHANNSEN
• 金额: $ 7.85万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10414882
• 关键词: Archives; Bacterial Artificial Chromosomes; Capsid Proteins; Cells; Collaborations; Core Facility; Cost efficiency; DNA; Data; Ensure; Escherichia coli; Generations; Genome; HepG2; Hepatitis B Virus; Herpesviridae; Human Genetics; Human Herpesvirus 4; Human Herpesvirus 8; Human Papillomavirus; Infectious hepatitides; Laboratories; Luciferases; Methods; Molecular Biology; Molecular Genetics; Mus; Mutation; Oncogenic Viruses; Papillomavirus; Phenotype; Plasmids; Protocols documentation; Publishing; Quality Control; Recombinant DNA; Research; System; Tetracyclines; Time; Transfection; Validation; Variant; Viral; Virion; Virus; bioinformatics pipeline; cost efficient; mutant; next generation sequencing; programs; protein expression; vector; virus core

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.

核心C -项目总结/摘要 核心C（病毒核心）的目的是提供一个时间和成本效益的机制， 所有8项研究所需的野生型（WT）和突变病毒的验证、分发和存档 与本计划项目相关的团体，以供其拟议的研究。具体目标是：（1）生产 经验证的WT、突变型和回复突变型EB病毒（EBV）和卡波西肉瘤疱疹病毒贮备液 （KSHV）;（2）生产经验证的WT和突变型B型肝炎病毒（HBV）储备液;（3）生产经验证的 感染性小鼠乳头瘤病毒（MmuPV 1）和人乳头瘤病毒（HPV）假病毒的原种。 （一）. 突变EBV（项目3、4和5）或KSHV（项目3）将在E.大肠杆菌起始于 使用无疤痕的En Passant方法进行适当的BAC除了标准方法外，我们还 建立了一个生物信息学管道来分析疱疹病毒基因组的下一代测序数据， 确保在独特区域内没有非预期的突变。在必要时，表型将 使用反式互补或构建回复突变EBV/KSHV基因组进行验证。病毒原液 的WT、突变体和EBV或KSHV的回复突变体变体将使用Dr. Sugden、滴定并储存以供所有四个EBV组使用。HBV病毒体将由以下来源生产（用于项目2）： HepAD 38细胞，一种稳定转染四环素诱导的HBV表达的HepG 2衍生物 系统HBV突变体将通过重组DNA方法在已经开发的载体中构建， Loeb实验室乳头瘤病毒病毒粒子和假病毒粒子将通过以下方法生成（对于项目1）： 用（i）所需衣壳蛋白表达质粒，和（ii）所需病毒或载体转染293 T细胞， 荧光素酶/GFP编码的DNA靶向使用先前由 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， 该核心有助于使用共同的病毒库来解释补充数据 在各种研究小组之间产生的更可靠的合并，以实现共同的目标， 项目与5个项目相关的所有8个研究小组将根据需要由该核心设施提供服务。