REPLICATION AND PATHOGENESIS OF ENVELOPED VIRUSES

有包膜病毒的复制和发病机制

• 批准号: 3782381
• 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/3782381
• 关键词: DNA directed RNA polymerase; Vesiculovirus; gene induction /repression; gene therapy; genetic promoter element; human immunodeficiency virus 1; interfering virus; membrane fusion; molecular cloning; nucleic acid repetitive sequence; temperature sensitive mutant; transcription factor; transfection; transfection /expression vector; virus DNA; virus antigen; virus cytopathogenic effect; virus envelope; virus genetics; virus protein; virus replication

This project was initiated to study several aspects necessary for the design of viral expression vectors which can be targeted to specific cells for gene delivery. Understanding the mechanisms of viral assembly are crucial toward this goal. It is also essential to understand how the envelope of the virus, Whether it is a retrovirus or another envelope virus, could be altered so it could show a specific cell tropism, while maintaining membrane fusion activity. In viral assembly, the viral matrix protein M plays a central role. It brings the genome in contact with the plasma membrane at the site of virus budding. During the past fiscal year, we focused on the role of the matrix protein M of vesicular stomatitis virus (VSV), which is essential for viral assembly and, in part, for viral pathogenesis. Coexpression of the matrix protein with HIV~1 led to inhibition of HIV~1 replication, as indicated by a severe decrease in the level of p24 released and in the number of syncitia. We and others had earlier reported that the M protein can indiscriminately inhibit the expression of genes driven by several different RNA polymerase II promoters. If the cytopathic effect caused by M protein could be made inducible upon infection, for example, by the HIV~1 Tat protein, protection of the cell population may result. The infected cell itself may also be killed, thereby simultaneously clearing the virus from the cell population. To test for this possibility, a temperature~sensitive M protein was cloned under control of the HIV~1 LTR ,and individual cell clones harboring the construct were selected and challenged by transfection with HIV~1 DNA. HIV~1 replication, as evidenced by p24 antigen release and syncytia formation, was severely decreased at the permissive temperature of M (32oC ) as compared to the parental cell line which continued to support HIV~1 replication. In contrast, at the nonpermissive temperature for the M protein (40 0C), the cell clones efficiently supported HIV~1 replication, like the parental cells. The fact, that the cell clones were viable at these temperatures demonstrated that basal levels of the temperature ~ sensitive M expression from the HIV~1 LTR were not toxic to the cell. The expression of M protein at permissive temperature protected the cell population from virus spread. For the M protein to be potentially useful in a gene therapeutic approach, a lack of toxicity at the level of the uninduced wild type M protein expression needs to be demonstrated.

启动该项目是为了研究以下几个方面： 设计可针对特定目标的病毒表达载体 用于基因传递的细胞。 了解病毒组装的机制 对这一目标至关重要。 了解如何 病毒的包膜，无论是逆转录病毒还是其他包膜 病毒，可以被改变，因此它可以显示出特定的细胞向性，而 维持膜融合活性。 在病毒组装过程中，病毒 基质蛋白M起着核心作用。 它使基因组接触 质膜位于病毒出芽部位。 在过去的财年中，我们重点关注基质蛋白的作用 水疱性口炎病毒 (VSV) 的 M，这是病毒传播所必需的 组装，部分用于病毒发病机制。 共表达 HIV~1 的基质蛋白导致 HIV~1 复制的抑制，如 p24 释放水平严重下降表明 合胞体的数量。 我们和其他人早些时候曾报道过，M 蛋白质可以无差别地抑制由以下因素驱动的基因的表达 几种不同的 RNA 聚合酶 II 启动子。 如果细胞病变效应 由 M 蛋白引起的感染可以被诱导，例如， 通过 HIV~1 Tat 蛋白，可能会产生对细胞群的保护。 受感染的细胞本身也可能被杀死，从而同时 从细胞群中清除病毒。 为了测试这种可能性，温度敏感的 M 蛋白被 在 HIV~1 LTR 的控制下进行克隆，以及单个细胞克隆 选择含有该构建体的载体并通过转染进行攻击 HIV~1 DNA。 HIV~1 复制，p24 抗原释放和 合胞体形成，在允许的温度下严重减少 M (32oC) 与亲本细胞系相比，继续 支持HIV~1复制。 相反，在不允许的温度下 对于 M 蛋白 (40 0C)，细胞克隆有效支持 HIV~1 复制，就像亲本细胞一样。 事实上，细胞克隆 在这些温度下是可行的，证明了基础水平 HIV~1 LTR 的温度 ~ 敏感 M 表达没有毒性 到细胞。 M蛋白在允许温度下的表达 保护细胞群免受病毒传播。 对于M蛋白 在基因治疗方法中具有潜在用途，缺乏毒性 在未诱导的野生型 M 蛋白表达水平上需要 证明了。