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.

在过去的一年里，这项研究继续关注 水泡性口炎病毒 (VSV) 的病毒基质蛋白 M 细胞病变发生和病毒组装。我们之前曾报道过 VSV M 蛋白负责细胞病变效应，其中涉及 VSV 感染后细胞骨架的紊乱。其他 研究人员还发现，基质蛋白仍然是唯一的 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蛋白的这个特性 分离编码以下任一者的 HIV-1 易感细胞克隆后 野生型或温度敏感的M蛋白在控制下 诱导型 HIV-1 LTR 启动子。编码该蛋白质的细胞克隆是 感染 HIV-1 和 HIV-2。基因表达和 两种病毒的复制均受到野生型 M 的显着抑制 与 HIV Tat1 和 Tat2 蛋白反式激活后的蛋白， 分别。尽管 HIV-1 能够在这些细胞中建立原病毒 细胞的效率与没有 M 蛋白的细胞相同，随后 病毒基因表达和传播在 M 中被显着抑制 表达细胞系。由于野生型 VSV M 蛋白的基础水平为 它对细胞无毒，似乎具有诱导型抗病毒药物的作用。 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 VSV 感染后将关闭。 一种新的核DNA结合 VSV 感染后通过凝胶迁移测定检测到蛋白质。装订 用特定的干扰素刺激反应元件检测到 顺序。通过与 Ozato 博士的小组合作，我们发现 该蛋白结合的诱导依赖于 VSV 基因表达 和复制。利用我们的一种温度 M 蛋白突变体，这 结合对温度敏感，这表明 VSV M 蛋白 可能在这种转变中发挥作用。 Supershift实验未发现 M 蛋白作为 DNA/蛋白质复合物本身的一部分。进一步的实验 正在进行中以检查 M 蛋白在诱导中的潜在作用 这种新的 DNA 结合活性及其在关闭 细胞转录。这些研究将提供重要的见解 VSV 诱导细胞病变的机制以及具体的 基因调控机制，可能涉及新的负细胞 转录因子。