Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy Pathways.
Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy Pathways.
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DOI：
10.1128/jvi.00915-16
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发表时间：
2016-10-01
影响因子：
5.4
通讯作者：
Grose C
中科院分区：
文献类型：
--
作者：
Buckingham EM;Jarosinski KW;Jackson W;Carpenter JE;Grose C
水痘-带状疱疹病毒（VZV）是一种高度细胞相关的疱疹病毒，病毒颗粒的排出有限。VZV感染单层细胞中自噬的诱导很容易检测到;自噬的抑制导致VZV糖蛋白生物合成减少和病毒滴度降低。为了解释自噬流如何对VZV感染周期产生前病毒效应，我们假设VZV胞吐途径在继发性炎症后可能与自噬途径汇合。这一假设依赖于VZV gE和自噬相关（Atg）Atg 9/Atg 16 L1运输途径之间的已知相似性。用高度纯化的VZV病毒粒子级分进行研究。当检测病毒体组分中是否存在自噬和内体蛋白时，检测到微管相关蛋白1轻链（MAP 1 LC 3B）和Ras样GT3蛋白11（Rab 11）。通过二维（2D）和3D成像免疫标记后，这两种蛋白质也共定位与VZV gE的细胞质囊泡的比例。当纯化的VZV病毒粒子计数后，免疫电子显微镜，金珠检测病毒孵育后与抗体VZV gE（100%），Rab 11（50%），和LC 3B（30%）。大量的电子显微镜检查表明，包膜病毒粒子被安置在单膜囊泡中，在自噬体中未观察到病毒颗粒。两者合计，我们的数据表明，一些病毒颗粒后，二次沉淀积累在一个异质性人口的单膜囊泡室，这是装饰与组件从内吞途径（Rab 11）和自噬途径（LC 3B）。后者的细胞质病毒囊泡类似于一个两性体。重要性VZV感染会导致自噬通量增加，而自噬的抑制会导致病毒传播显着减少。在对自噬的前病毒作用的研究中，我们发现了病毒胞吐和自噬途径交叉的证据。具体而言，LC 3-II和Rab 11蛋白与一些感染性VZV颗粒共纯化。结果表明，一个亚群的VZV颗粒进行到细胞表面的单壁囊泡的属性，一个细胞器形成的内体和自噬体的融合。我们的研究结果还解释了缺乏ICP34.5神经毒力基因的突变型单纯疱疹病毒（HSVΔ34.5）的自噬/异噬结果。VZV基因组缺乏ICP34.5直系同源物，但我们没有发现VZV颗粒存在于双膜自噬体中的证据。换句话说，在VZV感染的细胞中没有观察到食异，即HSVΔ34.5感染后记录的降解过程。
Varicella-zoster virus (VZV) is an extremely cell-associated herpesvirus with limited egress of viral particles. The induction of autophagy in VZV-infected monolayers is easily detectable; inhibition of autophagy leads to decreased VZV glycoprotein biosynthesis and diminished viral titers. To explain how autophagic flux could exert a proviral effect on the VZV infectious cycle, we postulated that the VZV exocytosis pathway following secondary envelopment may converge with the autophagy pathway. This hypothesis depended on known similarities between VZV gE and autophagy-related (Atg) Atg9/Atg16L1 trafficking pathways. Investigations were carried out with highly purified fractions of VZV virions. When the virion fraction was tested for the presence of autophagy and endosomal proteins, microtubule-associated protein 1 light chain (MAP1LC3B) and Ras-like GTPase 11 (Rab11) were detected. By two-dimensional (2D) and 3D imaging after immunolabeling, both proteins also colocalized with VZV gE in a proportion of cytoplasmic vesicles. When purified VZV virions were enumerated after immunoelectron microscopy, gold beads were detected on viruses following incubation with antibodies to VZV gE (∼100%), Rab11 (50%), and LC3B (30%). Examination of numerous electron micrographs demonstrated that enveloped virions were housed in single-membraned vesicles; viral particles were not observed in autophagosomes. Taken together, our data suggested that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B). The latter cytoplasmic viral vesicles resembled an amphisome. IMPORTANCE VZV infection leads to increased autophagic flux, while inhibition of autophagy leads to a marked reduction in virus spread. In this investigation of the proviral role of autophagy, we found evidence for an intersection of viral exocytosis and autophagy pathways. Specifically, both LC3-II and Rab11 proteins copurified with some infectious VZV particles. The results suggested that a subpopulation of VZV particles were carried to the cell surface in single-walled vesicles with attributes of an amphisome, an organelle formed from the fusion of an endosome and an autophagosome. Our results also addressed the interpretation of autophagy/xenophagy results with mutated herpes simplex virus lacking its ICP34.5 neurovirulence gene (HSVΔ34.5). The VZV genome lacks an ICP34.5 ortholog, yet we found no evidence of VZV particles housed in a double-membraned autophagosome. In other words, xenophagy, a degradative process documented after infection with HSVΔ34.5, was not observed in VZV-infected cells.