Cryo electron tomography of herpes simplex virus during axonal transport and secondary envelopment in primary neurons.
Cryo electron tomography of herpes simplex virus during axonal transport and secondary envelopment in primary neurons.
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轴突运输过程中单纯疱疹病毒的冷冻电子断层扫描和原发性神经元中的次级包膜。
DOI：
10.1371/journal.ppat.1002406
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发表时间：
2011-12
期刊：
影响因子：
6.7
通讯作者：
Grünewald K
中科院分区：
文献类型：
--
作者：
Ibiricu I;Huiskonen JT;Döhner K;Bradke F;Sodeik B;Grünewald K
在单纯疱疹病毒1（HSV 1）在神经元中外出期间，病毒颗粒从神经元细胞体沿着轴突向突触行进。HSV 1粒子是否如“已婚”模型所提出的那样作为包膜病毒体或如“分离”模型所提出的那样作为无包膜衣壳运输是有争议的。特定的病毒蛋白质可以形成催化这种运输的微管马达的募集平台。然而，它们的亚病毒位置仍然难以捉摸。在这里，我们建立了一个系统，分析疱疹病毒的出口冷冻电子断层扫描。在感染后16小时，我们观察到轴突内运输子代HSV 1病毒颗粒在分离的海马神经元活细胞荧光显微镜。冷冻电子断层扫描冷冻水化的神经元显示，大多数外出衣壳运输独立的病毒包膜。出乎意料的是，我们发现不仅含有DNA的衣壳（胞质C-衣壳），但也缺乏DNA的衣壳（胞质A-/B-衣壳）中轴突区域。子体积平均显示细胞质A-/B-衣壳上的被膜量低于C-衣壳。然而，所有的衣壳类型进行积极的轴突运输。因此，即使是少数的壳顶被蛋白似乎足以运输。在轴突终末观察到衣壳的次级降解。在它们的腔面上，包膜囊泡布满了典型的糖蛋白样刺突。此外，我们注意到在接近衣壳的囊泡膜的凹胞质面处的皮层密度增加。三维分析表明，这些组装点缺乏细胞骨架元素，但丝状肌动蛋白包围它们，形成一个组装室。我们的数据支持“分离模型”的HSV 1出口，即后代疱疹病毒被运输沿着轴突作为载体，而不是作为完整的病毒体在运输囊泡。单纯疱疹病毒1（HSV 1）在外周神经系统中建立终身潜伏感染。再激活后，子代病毒颗粒在感觉神经元内向初始感染部位移动。有什么类型的病毒结构的运输相互矛盾的报告：一些研究观察到无包膜衣壳旅行，而其他报告的运输囊泡内的完全包膜病毒。在这里，我们使用冷冻电子断层扫描分析HSV 1在海马神经元轴突的三维结构。在中间轴突区域，我们发现主要是无包膜衣壳。有趣的是，我们观察到含有基因组的衣壳和空衣壳在结合蛋白的量上存在显著差异。因此，不同的胞质衣壳类型之间的病毒蛋白质招募不同，但有效的运输发生，尽管这些差异。此外，我们观察到轴突末端二级衣壳包裹的三维快照。总之，这项研究提供了有价值的结构细节轴突HSV 1颗粒支持的概念，即病毒载体被传送沿着轴突组装后，只有轴突运输。
During herpes simplex virus 1 (HSV1) egress in neurons, viral particles travel from the neuronal cell body along the axon towards the synapse. Whether HSV1 particles are transported as enveloped virions as proposed by the ‘married’ model or as non-enveloped capsids suggested by the ‘separate’ model is controversial. Specific viral proteins may form a recruitment platform for microtubule motors that catalyze such transport. However, their subviral location has remained elusive. Here we established a system to analyze herpesvirus egress by cryo electron tomography. At 16 h post infection, we observed intra-axonal transport of progeny HSV1 viral particles in dissociated hippocampal neurons by live-cell fluorescence microscopy. Cryo electron tomography of frozen-hydrated neurons revealed that most egressing capsids were transported independently of the viral envelope. Unexpectedly, we found not only DNA-containing capsids (cytosolic C-capsids), but also capsids lacking DNA (cytosolic A-/B-capsids) in mid-axon regions. Subvolume averaging revealed lower amounts of tegument on cytosolic A-/B-capsids than on C-capsids. Nevertheless, all capsid types underwent active axonal transport. Therefore, even few tegument proteins on the capsid vertices seemed to suffice for transport. Secondary envelopment of capsids was observed at axon terminals. On their luminal face, the enveloping vesicles were studded with typical glycoprotein-like spikes. Furthermore, we noted an accretion of tegument density at the concave cytosolic face of the vesicle membrane in close proximity to the capsids. Three-dimensional analysis revealed that these assembly sites lacked cytoskeletal elements, but that filamentous actin surrounded them and formed an assembly compartment. Our data support the ‘separate model’ for HSV1 egress, i.e. progeny herpes viruses being transported along axons as subassemblies and not as complete virions within transport vesicles. Herpes simplex virus 1 (HSV1) establishes lifelong latent infections in the peripheral nervous system. After reactivation, progeny viral particles travel within sensory neurons towards sites of initial infection. There are conflicting reports what type of viral structures are transported: some studies observed non-enveloped capsids traveling while others reported transport of fully enveloped viruses within vesicles. Here, we used cryo electron tomography to analyze the three-dimensional architecture of HSV1 in axons of hippocampal neurons. In mid-axonal regions we found predominantly non-enveloped capsids. Interestingly, we observed both genome-containing and empty capsids that differed significantly in the amount of bound proteins. Viral protein recruitment thus varied between the different cytosolic capsid types, but effective transport occurred despite these differences. Furthermore, we observed three-dimensional snapshots of secondary capsid envelopment in axon terminals. Altogether, this study provides valuable structural detail on axonal HSV1 particles supporting the notion that viral subassemblies are conveyed along the axons to be assembled only after axonal transport.
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