Plus- and minus-end directed microtubule motors bind simultaneously to herpes simplex virus capsids using different inner tegument structures.
Plus- and minus-end directed microtubule motors bind simultaneously to herpes simplex virus capsids using different inner tegument structures.
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DOI：
10.1371/journal.ppat.1000991
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发表时间：
2010-07-08
期刊：
影响因子：
6.7
通讯作者：
Sodeik B
中科院分区：
文献类型：
--
作者：
Radtke K;Kieneke D;Wolfstein A;Michael K;Steffen W;Scholz T;Karger A;Sodeik B
许多病毒依赖宿主微管马达到达其预定的细胞内位置。嗜神经性α疱疹病毒（例如单纯疱疹病毒1（HSV1））的病毒颗粒显示出向细胞中心和外围的双向运输，表明它们利用相反方向的微管马达。为了了解特定运动募集的机制，有必要表征这种运动病毒结构的分子组成。我们生成了具有不同表面特征的 HSV1 衣壳，而不损害其整体结构，并表明在哺乳动物无细胞系统中，微管马达动力蛋白和驱动蛋白-1 以及动力蛋白辅因子动力蛋白可以直接与衣壳相互作用，而不受其他宿主因素的影响。对病毒组装或细胞进入过程中可能暴露于胞质溶胶的 23 种结构蛋白的衣壳组成和表面进行了分析。其中许多蛋白质属于被膜，这是位于衣壳和病毒包膜之间的所有疱疹病毒的标志。使用免疫印迹、定量质谱和定量免疫电子显微镜，我们发现暴露内皮蛋白（例如 pUS3、pUL36、pUL37、ICP0、pUL14、pUL16 和 pUL21）的衣壳募集了动力蛋白、动力蛋白、驱动蛋白-1 和驱动蛋白-2。相反，暴露 VP5、VP26、pUL17 和 pUL25 的未覆盖衣壳和被外皮蛋白（例如 vhs、pUL11、ICP4、ICP34.5、VP11/12、VP13/14、VP16、VP22 或 pUS11）覆盖的衣壳均不结合微管马达。我们的数据表明，HSV1 利用内皮膜的不同结构特征来募集动力蛋白或驱动蛋白-1。各个衣壳同时容纳相反方向的马达以及同一马达的多个副本。因此，这些相关的马达要么参与拉锯战，要么协调调节它们的活动，以实现在细胞进入过程中向细胞核的净运输或在组装过程中向细胞质膜的包络实现净运输。许多病毒，特别是嗜神经性α疱疹病毒，例如单纯疱疹病毒（HSV），需要完整的微管网络才能有效复制和发病。在活细胞中，宿主和病毒货物在运输方向上表现出快速逆转，这表明它们可以同时招募相反方向的马达。为了阐明特定汽车货物识别的分子机制，有必要表征此类货物的表面。我们建立了一个无细胞系统，可以重建天然哺乳动物微管马达与完整被皮 HSV 衣壳的结合。我们的数据表明，入站运动动力蛋白和出站运动驱动蛋白-1 直接且独立于其他宿主因子与胞质运输过程中覆盖衣壳的内被膜结合。一方面，识别宿主转运机制的病毒受体将为我们提供抗病毒治疗的新的潜在靶标。另一方面，此类病毒蛋白结构域可以添加到病毒载体中，甚至添加到旨在将治疗基因或分子递送到细胞核或其他亚细胞目的地的人工纳米载体中。
Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1) show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during cell entry or to cytoplasmic membranes for envelopment during assembly. Many viruses, particularly neurotropic alphaherpesviruses such as herpes simplex virus (HSV), require an intact microtubule network for efficient replication and pathogenesis. In living cells, host and viral cargo show rapid reversals in transport direction, suggesting that they can recruit motors of opposing directionality simultaneously. To elucidate the molecular mechanisms for specific motor-cargo recognition, it is necessary to characterize the surface of such cargos. We established a cell-free system that reconstitutes the binding of native, mammalian microtubule motors to intact tegumented HSV capsids. Our data suggest that the inbound motor dynein and the outbound motor kinesin-1 bind directly and independently of other host factors to the inner tegument that coats the capsids during cytosolic transport. Identifying viral receptors for the hosts' transport machinery will provide us on the one hand with new potential targets for antiviral therapy. On the other hand, such viral protein domains could be added to viral vectors or even to artificial nano carriers designed to deliver therapeutic genes or molecules to the nucleus or other subcellular destinations.
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