VARICELLA ZOSTER--RECEPTORS AND INFECTIVE MECHANISMS

水痘带状疱疹——受体和感染机制

• 批准号: 6170198
• 负责人: Anne A. Gershon
• 金额: $ 35.07万
• 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/6170198
• 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)在感染细胞中成熟时被包裹两次。 核衣壳首先从内核获得一个临时的包膜 膜，因为他们芽成核周池。 这个信封使 未成熟的颗粒，缺乏皮层，与细胞膜融合， 粗面内质网（RER），将核衣壳运送到 胞质液 病毒糖蛋白（gps）和皮层在 trans-Golgi网络（TGN），核衣壳在其中接收其最终的 信封. 我们已经发现VZV gpI从质膜上回收 由于其信号序列（AYRV）和补丁， 胞质结构域（gpItail）。 皮层，在细胞质中合成， 粘附于富含gpi的TGN衍生膜的胞质表面， 核衣壳变成病毒包膜。 我们现在建议测试 假设GPS包含它们自己TGN目标信号（我们 将识别），或作为乘客在具有 包含导航器gp的信号，例如gpI。 选择目标和形成 将在用cDNA转染或共转染的细胞中研究复合物 编码GPS。 我们还将检验皮层蛋白质 粘附在TGN衍生的膜上，该膜包裹VZV，因为它们结合到 一个或多个GPS的胞质结构域。 目标的存在 gpI尾信号意味着细胞必须含有相互作用的蛋白质 这个信号。 这些很可能是内源性蛋白质， 胞质间囊泡的运输。 两种方法将 用于鉴定结合细胞溶质结构域的细胞蛋白质， gpI：使用重组gpI尾和基于酵母的2 检测两种蛋白质相互结合能力的杂交试验 通过使转录激活结构域与转录激活结构域紧密接近， 调节下游转录因子基因表达的DNA结合位点。 将用合成肽从gpItail洗脱细胞蛋白质 含有gpI TGN靶向序列AYRV。 对照肽将是 用于去除非特异性结合gpItail的蛋白质。 为 酵母试验中，我们构建了含有编码 gpItail与GAL 4结合结构域融合。 获得了cDNA文库 在编码与GAL 4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 6-磷酸（Man 6-P）残基，存在于病毒gps上， 与Man 6-P受体（MPR），存在于膜中， TGN衍生的运输囊泡，并可能影响其后TGN行程。 最后，我们将研究使VZV感染的信号 有丝分裂后的人类神经元（hNT细胞），临床上重要的，但不是很好 了解VZV的目标。 具体而言，我们将测试：（i）MPR的作用 在轴突终端的病毒进入和（ii）参与的一个 新发现的逆行转运/核输入途径， 含有核的立即早期皮层蛋白质的易位 定位信号（如IE62）从轴突末端的胞质溶胶， 神经细胞核。