VARICELLA-ZOSTER: RECEPTORS AND INFECTIVE MECHANISMS

水痘带状疱疹：受体和感染机制

• 批准号: 3141339
• 负责人: Anne A. Gershon
• 金额: $ 28.01万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 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 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3141339
• 关键词: Betaherpesvirinae; Golgi apparatus; Herpes simplex disease; Herpesviridae; Vesiculovirus; affinity chromatography; aniline dye; asparagine; autoradiography; beta glucuronidase; biological signal transduction; biomarker; cell membrane; chloroquine; conformation; enzyme linked immunosorbent assay; enzyme mechanism; exocytosis; fibroblasts; fluorescent dye /probe; glucosamine; glycoprotein biosynthesis; glycoproteins; glycosylation; host organism interaction; human tissue; hydrolase; immunocytochemistry; immunoelectron microscopy; intracellular transport; mannose 6 phosphate isomerase; membrane permeability; monoclonal antibody; monosaccharides; nuclear membrane; nucleic acid sequence; oligosaccharides; protein purification; radiotracer; receptor binding; secretory protein; skin; thymidine monophosphate; transfection; tritium; varicella zoster virus; virion; virus envelope; virus infection mechanism; virus protein; virus receptors; virus replication

Varicella-zoster virus (VZV), the highly contagious agent of chickenpox (varicella) and shingles (zoster), is spread by infectious virions that are abundant in the vesicular fluid of cutaneous lesions and which are shed from the skin. Virions secreted from cells grown in vitro, however, are not infectious. Electron microscopic (EM) observations have led to the postulate that in cultured cells nucleocapsids of VZV are enveloped while passing through the inner nuclear membrane and travel within the cisternal compartment to the trans Golgi network (TGN), where they are diverted from the secretory pathway to prelysosomes, in which the virions are degraded. Since VZV envelope glycoproteins (gps), like those of newly synthesized lysosomal enzymes, contain phosphorylated oligosaccharides, it is proposed that binding of VZV to Man 6-P receptors (MPRs) in the TGN is responsible for directing newly synthesized VZV to prelysosomes. Because MPRs also cycle to the cell surface, binding of VZV to MPRs of the plasma membrane might facilitate infection of target cells if VZV in vivo were to escape diversion to prelysosomes. Preliminary data indicate that: (i) the phosphorylated mannose residues of viral envelope gps are present on complex oligosaccharides; the enzymes responsible for phosphorylating these residues are different from those which phosphorylate acid hydrolases; (ii) purified cation independent 125I-MPR (MPRci) binds to immobilized VZV gp I; (iii) Man 6-P and other phosphorylated monosaccharides protect cells from infection by cell-free VZV in vitro with a rank order of efficacy that is comparable to that for affinity for MPRs; Man 6-P protection declines within 30 min of application of VZV, a time course compatible with an action directed against viral entry (iv) dephosphorylation of virions by exposure to alkaline phosphatase destroys viral infectivity; (v) LM and EM immunocytochemistry reveal that within infected cells in culture VZV is found in the TGN and co-distributes in intracellular vacuoles with the immunoreactivity of the MPRci. We now propose to determine where in infected cells VZV gps are assembled into the viral envelope, the identity and relative internal acidity of the organelles through which enveloped VZV virions pass during their maturation, and the identity of the intracellular compartments in which VZ virions bind to MPRci. We will also ascertain the structures of VZV-associated oligosaccharides, how they are synthesized in infected cells, and how comparable they are to oligosaccharides of gps of other viruses. In addition, we will characterize the binding of MPRci to VZV-associated Man 6-P bearing oligosaccharides, using as reagents iodinated or colloidal gold-labeled affinity purified ectodomain of the MPRci. The role of the MPRci in viral entry will be tested with human epidermoid KB cells (HEKB), which lack MPRci. If HEKB cells cannot be infected with VZV, we will determine whether transfection with cDNA encoding the MPRci confers infectability on them. Also to be studied are whether unoccupied MPRci at cell surfaces are necessary for infection of HELF by VZV and whether VZV infection disrupts recycling of MPRci to the plasma membrane. Finally, the mechanism by which infectious cell-free VZV particles escape intracellular degradation and reach vesicle fluid in vivo will be investigated.

水痘-带状疱疹病毒（VZV），水痘的高度传染性因子 （水痘）和带状疱疹（带状疱疹），是由传染性病毒粒子， 在皮肤病变的水泡液中丰富， 从皮肤上。 然而，从体外生长的细胞分泌的病毒体是 没有传染性 电子显微镜（EM）观察导致了 假设在培养的细胞中，VZV的核衣壳被包膜， 穿过内核膜进入脑池 隔室的transGolgi网络（TGN），在那里他们被转移， 前溶酶体的分泌途径，在其中病毒体被降解。 由于VZV包膜糖蛋白（GPS），像那些新合成的 溶酶体酶，含有磷酸化的寡糖，建议 VZV与TGN中Man 6-P受体（MPR）的结合是TGN中VZV与Man 6-P受体（MPR）结合的原因。 用于将新合成的VZV导向前溶酶体。 因为MPR还 VZV与细胞膜MPR的结合 如果VZV在体内逃逸， 转移到前溶酶体。 初步数据表明：（一） 病毒包膜GPS的磷酸化甘露糖残基存在于 复合寡糖;负责磷酸化这些寡糖的酶 残基不同于使酸水解酶磷酸化的那些残基;（ii） 纯化的阳离子非依赖性125 I-MPR（MPRci）与固定化的VZV gp I结合; (iii)Man 6-P和其他磷酸化单糖保护细胞免受 无细胞VZV体外感染的效力排序， 与MPRs亲和力相当; Man 6-P保护下降 在应用VZV的30分钟内，时间过程与 直接对抗病毒进入的作用（iv）通过 暴露于碱性磷酸酶破坏病毒感染性;（v）LM和EM 免疫细胞化学显示，在培养的感染细胞内， 在TGN中发现，并与 MPRci的免疫反应性。 我们现在建议确定 被感染的细胞VZV GPS被组装到病毒包膜中， 和相对内部酸性的细胞器，通过它包膜VZV 病毒粒子在其成熟过程中通过，并且细胞内的 VZ病毒体与MPRci结合的区室。 我们还将确定 VZV相关低聚糖的结构及其在体内的合成方式 感染的细胞，以及它们与 其他病毒。 此外，我们将表征MPRci与 VZV相关的Man 6-P携带寡糖，用作试剂 碘化或胶体金标记的亲和纯化的胞外域 MPRci。 MPRci在病毒进入中的作用将用人类进行测试。 表皮样KB细胞（HEKB），其缺乏MPRci。 如果HEKB细胞不能被 感染VZV，我们将确定是否转染cDNA 编码MPRci赋予它们传染性。 还需要研究的是 细胞表面未被占据的MPRci是否是感染 VZV的HELF以及VZV感染是否破坏了MPRci循环到 质膜 最后，感染性无细胞VZV 颗粒逃避细胞内降解并在体内到达囊泡液 将进行调查。