VARICELLA ZOSTER--RECEPTORS AND INFECTIVE MECHANISMS

水痘带状疱疹——受体和感染机制

• 批准号: 2671933
• 负责人: Anne A. Gershon
• 金额: $ 33.07万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2671933
• 关键词: Golgi apparatus; biological signal transduction; chimeric proteins; glycoproteins; mannose 6 phosphate; molecular cloning; tissue /cell culture; transfection; varicella zoster virus; virus infection mechanism; virus protein; virus receptors

DESCRIPTION (Adapted from Applicant's Abstract): Varicella zoster virus (VZV) is enveloped twice during its maturation in infected cells. Nucleocapsids first acquire a temporary envelope from the inner nuclear membrane as they bud into the perinuclear cisterna. This envelope enables the immature particles, which lack tegument, to fuse with the membrane of the rough endoplasmic reticulum (RER), delivering nucleocapsids to the cytosol. Viral glycoproteins (gps) and tegument come together at the trans-Golgi network (TGN), where the nucleocapsids receive their final envelope. We have found that VZV gpI is retrieved from the plasma membrane and targeted to the TGN because of a signal sequence (AYRV) and patch in its cytosolic domain (gpItail). Tegument, which is synthesized in the cytosol, adheres to the cytosolic face of a gpI-rich TGN-derived membrane that wraps nucleocapsids and becomes the viral envelope. We now propose to test the hypotheses that gps contain a TGN targeting signal of their own (which we will identify), or are routed to the TGN as passengers in a complex with a signal-containing navigator gp, such as gpI. Targeting and the formation of complexes will be studied in cells transfected or co-transfected with cDNA encoding the gps. We will also test the hypothesis that tegument proteins adhere to the TGN-derived membrane that envelops VZV because they bind to the cytosolic domains of one or more gps. The presence of a targeting signal in gpI tail implies that cells must contain proteins that interact with this signal. These are likely to be endogenous proteins involved in the traffic of vesicles between cytoplasmic compartments. Two methods will be used to identify cellular proteins that bind to the cytosolic domain of gpI: affinity chromatography with recombinant gpItails and a yeast-based 2 hybrid assay that detects the ability of two proteins to bind to one another by bringing a transcription activation domain into close proximity with a DNA-binding site that regulates the expression of a downstream reportergene. Cellular proteins will be eluted from gpItail with a synthetic peptide containing the gpI TGN targeting sequence, AYRV. A control peptide will be used to remove proteins that bind non-specifically to gpItail. For the yeast assay, we have constructed vectors containing hybrid genes that encode gpItail fused to the GAL4 binding domain. A cDNA library has been obtained in a corresponding vector encoding human brain proteins fused to the GAL4 activation domain. A third aim will be to determine whether the mannose 6-phosphate (Man 6-P) residues, which are present on viral gps, interact with the Man 6-P receptors (MPRs) that are present in the membranes of TGN-derived transport vesicles and may influence their post-TGN itinerary. Finally, we will investigate the signals that enable VZV to infect post-mitotic human neurons (hNT cells), a clinically important, but not well understood target of VZV. Specifically, we will test: (i) the role of MPRs at axon terminals in viral entry and (ii) the participation of a newly-discovered retrograde transport/nuclear import pathway in the translocation of immediate-early tegument proteins that contain a nuclear localization signal (such as IE62) from the cytosol of an axon terminal to the neuronal nucleus.

描述（改编自申请人的摘要）：水痘带状疱疹病毒 (VZV) 在受感染细胞的成熟过程中被包裹两次。 核衣壳首先从内核获得一个临时的包膜 当它们芽进入核周池时形成膜。 该信封使 未成熟的颗粒，缺乏外皮，与细胞膜融合 粗面内质网 (RER)，将核衣壳输送至 细胞质。 病毒糖蛋白（gps）和外皮在 跨高尔基体网络 (TGN)，核衣壳在此接收最终的 信封。 我们发现 VZV gpI 是从质膜中回收的 并由于其信号序列 (AYRV) 和补丁而针对 TGN 胞质结构域（gpItail）。 外皮是在细胞质中合成的， 粘附在富含 gpI 的 TGN 衍生膜的胞质表面，该膜包裹 核衣壳并成为病毒包膜。 我们现在建议测试 假设 GPS 包含自己的 TGN 目标信号（我们 将识别），或者作为复合设施中的乘客被路由到 TGN 包含信号的导航器 gp，例如 gpI。 目标定位和形成 将在转染或共转染 cDNA 的细胞中研究复合物 对 GPS 进行编码。 我们还将检验皮层蛋白的假设 粘附在包裹 VZV 的 TGN 衍生膜上，因为它们结合 一个或多个 GPS 的胞质结构域。 目标的存在 gpI 尾部的信号意味着细胞必须含有相互作用的蛋白质 有了这个信号。 这些可能是参与的内源蛋白质 细胞质区室之间囊泡的运输。 两种方法将 用于鉴定与胞质结构域结合的细胞蛋白 gpI：使用重组 gpItails 和基于酵母的 2 亲和层析 检测两种蛋白质相互结合能力的杂交测定 通过将转录激活域与 调节下游报告基因表达的 DNA 结合位点。 使用合成肽从 gpItail 中洗脱细胞蛋白 含有 gpI TGN 靶向序列 AYRV。 对照肽将是 用于去除与 gpItail 非特异性结合的蛋白质。 对于 酵母分析中，我们构建了含有编码杂合基因的载体 gpItail 与 GAL4 结合域融合。 已获得cDNA文库 在编码与 GAL4 融合的人脑蛋白的相应载体中 激活域。 第三个目标是确定甘露糖是否 病毒 GPS 上存在的 6-磷酸 (Man 6-P) 残基相互作用 与存在于细胞膜中的 Man 6-P 受体 (MPR) TGN 衍生的转运囊泡可能会影响其 TGN 后的行程。 最后，我们将研究使 VZV 感染的信号 有丝分裂后人类神经元（hNT细胞），临床上很重要，但效果不佳 了解 VZV 的目标。 具体来说，我们将测试：(i) MPR 的作用 在病毒进入的轴突末端以及（ii）的参与 新发现的逆行运输/核输入途径 含有核的早期被膜蛋白的易位 定位信号（如 IE62）从轴突末端的胞质溶胶到 神经元核。