VARICELLA ZOSTER--RECEPTORS AND INFECTIVE MECHANISMS

描述(摘自申请者摘要)：水痘带状疱疹病毒 (VZV)在感染细胞中成熟过程中被两次包膜。 核衣壳首先从内核获得一个临时包膜 当它们萌发到核周脑池中时，细胞膜。此信封启用 未成熟的颗粒，缺乏被膜，不能与膜融合 粗面内质网(RER)，将核衣壳运送到 胞浆。病毒糖蛋白(GPS)和被膜在 跨高尔基网络(TGN)，在那里核衣壳接受它们的最终 信封。我们发现VZV GPI是从质膜中回收的 并以TGN为目标，因为ITS中的信号序列(AYRV)和补丁 胞液结构域(GpIail)。被膜是在胞浆中合成的， 附着在包裹着富含GPI的TGN衍生膜的胞液表面 核衣壳并成为病毒的包膜。我们现在建议测试 假设GPS包含自己的TGN目标信号(我们 将被识别)，或作为乘客被送往TGN 包含信号的导航器GP，如GPI。靶向和目标的形成 复合体将在转染或与cdna共转染的细胞中进行研究。 对GPS进行编码。我们还将检验这样一种假设，即被膜蛋白 附着在包裹VZV的TGN衍生膜上，因为它们结合到 一个或多个GPS的胞浆结构域。目标的存在 GPI尾部的信号意味着细胞中必须含有相互作用的蛋白质 有了这个信号。这些很可能是内源性蛋白质参与了 胞质间小泡的运输。有两种方法将 可用于鉴定与胞浆结构域结合的细胞蛋白 GPI：重组gpItails与酵母基-2的亲和层析 一种检测两种蛋白质相互结合能力的杂交分析方法 通过使转录激活结构域与 调节下游报告基因表达的DNA结合位点。 细胞蛋白将用合成肽从gpI尾巴中洗脱出来 包含GPI TGN靶向序列AYRV。一个对照多肽将是 用于去除与gpIail非特异性结合的蛋白质。对于 酵母菌试验，我们已经构建了含有编码 GpIail与GAL4结合域融合。已获得一个cDNA文库 在相应的编码人脑蛋白的载体中融合到GAL4中 激活域。第三个目标是确定甘露糖是否 存在于病毒GPS上的6-磷酸(Man 6-P)残基相互作用 与存在于细胞膜上的Man 6-P受体(MPR)结合 TGN来源的运输囊泡，并可能影响它们在TGN后的行程。 最后，我们将研究使VZV感染的信号 人类有丝分裂后神经元(HNT细胞)，临床上很重要，但不是很好 了解VZV的目标。具体地说，我们将测试：(I)军事政策制定者的作用 在病毒进入的轴突终末和(Ii)一个 新发现的逆行转运/核进口途径 含有核的即刻-早期被膜蛋白的移位 定位信号(例如IE62)从轴突终末的胞浆到 神经元核团。

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.