REPLICATION AND PATHOGENESIS OF ENVELOPED VIRUSES

有包膜病毒的复制和发病机制

• 批准号: 6163043
• 负责人: M SCHUBERT
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6163043
• 关键词: DNA binding protein; Vesiculovirus; antiviral agents; cytomegalovirus; cytotoxicity; gene expression; gene induction /repression; genetic transcription; interferon inducers; interferons; measles virus; molecular cloning; mutant; nervous system infection; subacute sclerosing panencephalitis; transcription factor; virus cytopathogenic effect; virus envelope; virus genetics; virus infection mechanism; virus protein; virus replication

A cytoplasmic and transient viral expression vector which is replication incompetent would be very useful to study cellular functions in vitro or in vivo. Vesicular stomatitus virus (VSV) has an extremely high infectious unit to particle ratio with up to 1 in every 5 virus particles leading to infection. With HIV-1 this ratio is only 1:10000. The goal of this project is to generate defective VSV particles that can be used as transient gene expression vectors. VSV is a nonsegmented negative strand RNA virus and does not have a DNA intermediate. This is the reason why it is very difficult to obtain viral nucleocapsids which can be replicated. To generate these vectors, our detailed knowledge of the basic virology and pathogenesis of VSV will be used together with the latest recombinant DNA technology that now allows for the first time to generate VSV particles from recombinant DNA. To be able to generate a defective, helper virus-free SVS particle, we have cloned the large polymerase gene of VSV into several eukaryotic expression plasmids with different promoters and expression levels. These plasmids will require integration or they will exist as high copy number episomes in the nucleus. HEK 293 cells have recently been transfected with these DNAs and cell clones have been selected for drug resistance. These cells are currently being tested for their ability to complement temperature sensitive polymerase mutants as the restrictive temperature. Studies on whether helper virus functions could be provided by conditional VSV polymerase mutants were also carried out. We evaluated the potential of some of these mutants with respect to encapsidating complete VSV genomic RNA when it was transcribed by T7 RNA polymerase from plasmid DNA in tissue culture. As an alternative the nucleocapsid and polymerse proteins necessary for replication are provided by cotransfection of plasmid DNAs.

一种可复制的细胞质瞬时病毒表达载体 无能对于体外研究细胞功能或 体内。 水泡口病毒（VSV）具有极高的 传染性单位与颗粒之比，每 5 个病毒颗粒中就有 1 个 导致感染。 对于 HIV-1，这一比例仅为 1:10000。 目标 该项目的目的是生成可使用的有缺陷的 VSV 颗粒 作为瞬时基因表达载体。 VSV 是不分段负数 链RNA病毒并且不具有DNA中间体。 这就是原因 为什么获得病毒核衣壳非常困难 复制。 为了生成这些向量，我们需要详细了解 VSV 的基本病毒学和发病机制将与 最新的重组 DNA 技术首次允许 从重组 DNA 生成 VSV 颗粒。 为了能够生成一个 有缺陷、无辅助病毒的 SVS 颗粒，我们已经克隆了大的 将 VSV 聚合酶基因转化为几种真核表达质粒 不同的启动子和表达水平。 这些质粒需要 整合，否则它们将以高拷贝数附加体的形式存在 核。 HEK 293 细胞最近被这些 DNA 转染并且 细胞克隆已被选择用于耐药性。 这些细胞是 目前正在测试它们补充温度的能力 敏感聚合酶突变体作为限制温度。 研究 条件VSV是否可以提供辅助病毒功能 还进行了聚合酶突变体。我们评估了潜力 其中一些突变体涉及包裹完整的 VSV 基因组 当 T7 RNA 聚合酶从质粒 DNA 转录 RNA 时 组织培养。 作为替代品，核衣壳和聚合酶蛋白 复制所需的DNA由质粒DNA的共转染提供。