VARICELLA-ZOSTER--RECEPTORS AND INFECTIVE MECHANISMS

水痘-带状疱疹病毒(VZV)，水痘的高传染性病原体 (水痘)和带状疱疹(带状疱疹)，是通过传染性病毒粒子传播的 在皮损的水泡液中含量丰富，并脱落 从皮肤上。然而，从体外培养的细胞中分泌的病毒粒子是 不会传染。电子显微镜(EM)观察导致了 假设在培养细胞中，VZV的核衣壳是被包被的，而 穿过内核膜，在脑池内行进 分隔到跨高尔基网络(TGN)，在那里他们被分流 进入前溶酶体的分泌途径，其中病毒粒子被降解。 由于VZV包膜糖蛋白(GPS)与新合成的糖蛋白一样 溶酶体酶，含有磷酸化低聚糖，它被建议 VZV与TGN中的Man 6-P受体(MPR)的结合起作用 用于将新合成的VZV导向前溶酶体。因为MPR也 循环到细胞表面，VZV与质膜MPR结合 如果VZV在体内逃逸，可能会促进靶细胞的感染 转移到溶酶体前。初步数据显示：(I) 病毒包膜GPS的磷酸化甘露糖残留物存在于 复杂的低聚糖；负责将其磷酸化的酶 残基不同于使酸性水解酶磷酸化的残基； 纯化的阳离子非依赖性125I-MPR(MPRci)与固定化VZV gp I结合； (Iii)Man 6-P和其他磷酸化单糖保护细胞免受 体外感染无细胞VZV的疗效排序为 与MPR的亲和力相当；Man 6-P保护功能下降 在应用VZV的30分钟内，与 针对病毒侵入的行动(Iv)病毒粒子去磷酸化 暴露于碱性磷酸酶可破坏病毒的传染性；(V)LM和EM 免疫细胞化学显示在培养的感染细胞内VZV是 发现于TGN中，并共同分布在细胞内的空泡中 MPRCI的免疫反应性。我们现在建议确定在 被感染的细胞VZV GPS被组装成病毒的包膜，身份 以及包被VZV的细胞器的相对内部酸度 病毒粒子在成熟过程中通过，细胞内的身份 VZ病毒粒子与MPRCI结合的隔间。我们还将确定 VZV相关低聚糖的结构及其合成方法 感染细胞，以及它们与GPS的寡糖有多大的可比性 其他病毒。此外，我们还将描述MPRci与 VZV相关的含低聚糖的Man 6-P用作试剂 碘化金或胶体金标记的亲和纯化胞外结构域 MPRCI。MPRCI在病毒进入中的作用将在人类身上进行测试 表皮样KB细胞(HEKB)，缺乏MPRCi。如果HEKB细胞不能 感染VZV后，我们将确定是否用cDNA转染法 对MPRCI进行编码使它们具有感染性。同样需要研究的还有 细胞表面空置的MPRCI是否为感染所必需的 VZV感染HELF以及VZV感染是否扰乱了MPRCI向 质膜。最后，感染性无细胞VZV的机制 体内微粒逃脱细胞内降解，进入囊泡液 将会被调查。

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.