REPLICATION AND PATHOGENESIS OF ENVELOPED VIRUSES

有包膜病毒的复制和发病机制

• 批准号: 3760290
• 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/3760290
• 关键词: DNA binding protein; Vesiculovirus; gel mobility shift assay; gene expression; gene induction /repression; genetic transcription; host organism interaction; human immunodeficiency virus 1; human immunodeficiency virus 2; interfering virus; temperature sensitive mutant; transcription factor; virus assembly; virus cytopathogenic effect; virus genetics; virus protein; virus replication; virus virus interaction

During the past year this study continued to focus on the role of the viral matrix protein M of vesicular stomatitis virus (VSV) in cytopathogenesis and in viral assembly. We had previously reported that the VSV M protein is responsible for cytopathic effects which involve the disorganization of the cytoskeleton after VSV infections. Other investigators also found that the matrix protein, which remains the only protein of this cytoplasmic virus found in cell nuclei is able to inhibit cellular transcription. We have studied this property of the M protein after isolation of HIV-1 susceptible cell clones which encode either a wild type or a temperature sensitive M protein under the control of the inducible HIV-1 LTR promoter. Cell clones which encode this protein were infected with HIV-1 as well as with HIV-2. Gene expression and replication of both viruses were drastically inhibited by wild type M protein after transactivation with the HIV Tat1 and Tat2 proteins, respectively. Although HIV-1 was able to establish a provirus in these cells at the same efficiency as in cells without M protein, subsequent viral gene expression and spread were drastically inhibited in M expressing cell lines. Since basal levels of wild type VSV M protein were not toxic to the cell, it appeared to function as an inducible antiviral. The mechanism for the inhibition of cellular transcription by the VSV M protein was further studied in the context of interferon inducible genes which are turned off after VSV infections. A novel nuclear DNA binding protein was detected in a gel shift assay after VSV infections. Binding was detected with a specific interferon stimulated response element sequence. In collaboration with Dr. Ozato's group, we found that the induction of this protein binding was dependent on VSV gene expression and replication. With one of our temperature M protein mutants, this binding was temperature sensitive, which indicated that the VSV M protein may play a role in that shift. Supershift experiments did not identify M protein as part of the DNA/protein complex itself. Further experiments are in progress to examine the potential role of M protein in inducing this new DNA binding activity as well as its role in the shut off of cellular transcription. These studies will allow important insights into the mechanism of VSV induced cytopathogenesis as well as into specific mechanisms of gene regulation, possibly involving new negative cellular transcription factors.

在过去的一年中，这项研究继续关注 囊泡口腔炎病毒（VSV）的病毒基质蛋白M 细胞病变和病毒组装中。我们以前曾报道过 VSV M蛋白负责涉及的细胞病毒作用 VSV感染后细胞骨架的混乱。其他 调查人员还发现，基质蛋白，它仍然是唯一的 在细胞核中发现的这种细胞质病毒的蛋白质能够抑制 细胞转录。我们研究了M蛋白的这种特性 分离出HIV-1易感细胞克隆后，该克隆编码A 野生型或温度敏感的M蛋白在控制下 诱导HIV-1 LTR启动子。编码该蛋白的细胞克隆是 感染HIV-1以及HIV-2。基因表达和 两种病毒的复制受到野生型M的巨大抑制 与HIV Tat1和Tat2蛋白反式激活后的蛋白质， 分别。尽管HIV-1能够在这些 细胞的效率与没有M蛋白的细胞相同，随后 病毒基因表达和扩散在M中得到了巨大抑制 表达细胞系。由于野生型VSV M蛋白的基础水平为 对细胞无毒，它似乎是诱导型抗病毒。 VSV M抑制细胞转录的机制 在干扰素诱导基因的背景下进一步研究了蛋白质 在VSV感染后将关闭。 一种新型的核DNA结合 VSV感染后，在凝胶转移测定中检测到蛋白质。结合 用特定的干扰素刺激响应元件检测到 顺序。与Ozato博士的小组合作，我们发现 该蛋白结合的诱导取决于VSV基因表达 和复制。使用我们的温度M蛋白突变体之一， 结合对温度敏感，这表明VSV M蛋白 可能在这一转变中发挥作用。 Supershift实验未识别 M蛋白作为DNA/蛋白质复合物本身的一部分。进一步的实验 正在进行研究M蛋白在诱导中的潜在作用 这种新的DNA结合活性以及其在关闭中的作用 细胞转录。这些研究将允许对 VSV的机制诱导了细胞病生成以及特定的 基因调节的机制，可能涉及新的阴性细胞 转录因子。