Engineering Herpesviruses using Synthetic Genomics

使用合成基因组学改造疱疹病毒

• 批准号: 8893391
• 负责人: PRASHANT J DESAI
• 金额: $ 27.21万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-15 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8893391
• 关键词: B-Lymphocytes; Bacterial Genome; Basic Science; Biological; Biological Assay; Biology; Cells; Chemicals; Code; Complex; DNA; DNA Viruses; Engineering; Episome; Escherichia coli; Future; Genetic; Genome; Genome engineering; Genomic approach; Genomics; Goals; Herpesviridae; Herpesvirus 1; Human Herpesvirus 4; Immunoglobulin G; Individual; Institutes; Laboratories; Lytic; Lytic Virus; Mammalian Cell; Measures; Methods; Microbe; Modeling; Molecular Biology; Molecular Genetics; One-Step dentin bonding system; Outcome; Pharmaceutical Preparations; Plasmids; Process; Production; Property; Proteins; Reagent; Scientist; Technology; Testing; Time; Transgenes; Viral Genome; Virion; Virus; Yeasts; base; crosslink; established cell line; gammaherpesvirus; genetic manipulation; genome sequencing; high risk; member; mutant; pathogen; prototype; public health relevance; reactivation from latency; recombinase; reconstitution; synthetic biology; time use; tool

 DESCRIPTION (provided by applicant): Herpesviruses are large DNA viruses whose genomes have the coding potential of in excess of 100 gene products. Historically, genetic manipulation of these large genomes was feasible for only a subset of these viruses. Subsequently, many of the herpesvirus genomes were cloned into BAC plasmids, which significantly advanced the technologies of genome engineering in an E.coli host and the successful reconstitution of infectious virus in the appropriate host cell. In this application, we propose to use synthetic biology to first build a wild-type clone of the Epstein-Barr Virus (EBV) genome and then reconstitute the infectious virus. Herpes simplex virus type 1 (HSV-1) will be used as a model to first test and optimize the assembly method. The successful outcome of this synthetic biology approach will have a transformational impact on the ability to synthetically clone and manipulate any herpesvirus genome. In addition, this approach offers a paradigm to help understand the molecular genetics and biology of emerging pathogens. Specific Aim 1. Use synthetic genomics methods to assemble an infectious genome of EBV. In this aim, we will clone the EBV genome, strain Akata, using synthetic genomics. Our approach will be based on recent advances made in this field by members of the J. Craig Venter Institute (JCVI) team that created the first synthetic microbe. The JCVI lab has developed methods that enable the assembly of large DNA fragments, ranging from hundred kilobases to megabase size genomes. This team will use these methods to assemble an infectious clone of EBV and at the same time, use the more "tractable" HSV-1 genome assembly to optimize and refine synthetic genomics methods. Specific Aim 2. Establish a cloned EBV genome in mammalian cells with biological activity and stability. The goal in this aim will be to recover infectious virus after introductionof assembled herpesvirus genomes into mammalian cells. EBV assembled genomes can be transfected into HEK-293 or EBV negative Akata cells. Cells that harbor the EBV episome, following drug selection, will be induced for lytic virus production. Biological activity will be measured using quantitative PCR for viral genomes, Raji GFP titers that measure establishment of latency and reactivation and finally by the ability to immortalize B cells. Our singular goal isto use the combined and complementary expertise of the JHU and JCVI laboratories to demonstrate we can assemble whole genome infectious clones of herpesviruses from the individual parts in an efficient process with high fidelity and stability. If successful, this woul provide a new powerful platform to clone and manipulate these viruses.