Buckingham Erin M, Jarosinski Keith W, Jackson Wallen, Carpenter John E, Grose Charles
Departments of Pediatrics and Microbiology, University of Iowa, Iowa City, Iowa, USA.
Departments of Pediatrics and Microbiology, University of Iowa, Iowa City, Iowa, USA
J Virol. 2016 Sep 12;90(19):8673-85. doi: 10.1128/JVI.00915-16. Print 2016 Oct 1.
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.
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.
水痘带状疱疹病毒（VZV）是一种与细胞高度相关的疱疹病毒，病毒颗粒的释放有限。在VZV感染的单层细胞中自噬的诱导很容易检测到；自噬的抑制会导致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感染周期产生病毒促进作用，我们推测二次包膜后的VZV胞吐途径可能与自噬途径汇合。这一假设依赖于VZV gE与自噬相关（Atg）Atg9/Atg16L1运输途径之间已知的相似性。使用高度纯化的VZV病毒粒子组分进行了研究。当测试病毒粒子组分中自噬和内体蛋白的存在时，检测到微管相关蛋白1轻链（MAP1LC3B）和类Ras GTP酶11（Rab11）。通过免疫标记后的二维（2D）和三维（3D）成像，这两种蛋白在一部分细胞质囊泡中也与VZV gE共定位。免疫电子显微镜后对纯化的VZV病毒粒子进行计数时，在用抗VZV gE（约100%）、Rab11（50%）和LC3B（30%）抗体孵育后的病毒上检测到金珠。对大量电子显微镜照片的检查表明，包膜病毒粒子存在于单膜囊泡中；在自噬体中未观察到病毒粒子。综上所述，我们的数据表明，二次包膜后的一些病毒粒子积聚在异质性的单膜囊泡区室中，这些区室装饰有来自内吞途径（Rab11）和自噬途径（LC3B）的成分。后者的细胞质病毒囊泡类似于两性体。
VZV感染导致自噬流增加，而自噬的抑制导致病毒传播显著减少。在这项关于自噬的病毒促进作用的研究中，我们发现了病毒胞吐途径和自噬途径相交的证据。具体而言，LC3-II和Rab11蛋白都与一些感染性VZV颗粒共纯化。结果表明，一部分VZV颗粒被携带到具有两性体特征的单壁囊泡中的细胞表面，两性体是由内体和自噬体融合形成的细胞器。我们的结果还涉及了对缺乏其ICP34.5神经毒力基因（HSVΔ34.5）的突变单纯疱疹病毒的自噬/异体吞噬结果的解释。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感染的细胞中未观察到异体吞噬，这是在感染HSVΔ34.5后记录的一种降解过程。
