An in vitro model of latency and reactivation of varicella zoster virus in human stem cell-derived neurons.
An in vitro model of latency and reactivation of varicella zoster virus in human stem cell-derived neurons.
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DOI：
10.1371/journal.ppat.1004885
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发表时间：
2015-06
期刊：
影响因子：
6.7
通讯作者：
Goldstein RS
中科院分区：
文献类型：
--
作者：
Markus A;Lebenthal-Loinger I;Yang IH;Kinchington PR;Goldstein RS
水痘带状疱疹病毒(VZV)在感觉神经元和自主神经中的潜伏时间一直是个谜，很难研究，而且还没有实现实验性的再激活。我们之前已经证明，人类胚胎干细胞(HESC)来源的神经元允许产生和传播VZV感染。我们现在证明，hESC来源的神经元也可以持续数周的持续非生产性感染，随后可以通过多种实验刺激重新激活。静止性感染是通过将神经元暴露于低滴度的无细胞VZV来建立的，要么使用阿昔洛韦，要么通过感染分隔的微流体室中的轴突而不使用阿昔洛韦。在长达七周的时间里，qPCR可以检测到VZV DNA和低水平的病毒转录。通过去除生长因子或抑制PI3-Kinase活性，静止期感染的人类神经元培养物可重新表达病毒基因和蛋白。引人注目的是，在较低温度(34摄氏度)下诱导重新激活的培养物可增强VZV的重新激活，从而导致传播和生产性感染。用RNAseq比较静止期感染和产生期感染的神经元中VZV基因组转录，发现特定基因组区域，特别是重复区域的优先转录。这些实验建立了一个强大的新系统来模拟VZV的潜伏期，并揭示了温度在VZV重新激活和疾病中的潜在作用。全世界大多数成年人在他们的神经节中藏匿着潜伏的VZV，由此重新激活会导致带状疱疹。这种痛苦的疾病经常并发长期疼痛、神经后遗症或视力丧失，需要改进预防和治疗策略。由于不能在体外或体内复制可通过实验重新激活的持久状态，对VZV潜伏期和重新激活的研究一直受到严重阻碍。我们的研究建立了一个使用来自胚胎干细胞的人类神经元的系统，在这个系统中，多种刺激可以从长期的实验潜伏期诱导重新激活。该系统揭示了温度在VZV复活中的潜在作用，可首次用于研究VZV的潜伏/溶解开关。
Varicella zoster virus (VZV) latency in sensory and autonomic neurons has remained enigmatic and difficult to study, and experimental reactivation has not yet been achieved. We have previously shown that human embryonic stem cell (hESC)-derived neurons are permissive to a productive and spreading VZV infection. We now demonstrate that hESC-derived neurons can also host a persistent non-productive infection lasting for weeks which can subsequently be reactivated by multiple experimental stimuli. Quiescent infections were established by exposing neurons to low titer cell-free VZV either by using acyclovir or by infection of axons in compartmented microfluidic chambers without acyclovir. VZV DNA and low levels of viral transcription were detectable by qPCR for up to seven weeks. Quiescently-infected human neuronal cultures were induced to undergo renewed viral gene and protein expression by growth factor removal or by inhibition of PI3-Kinase activity. Strikingly, incubation of cultures induced to reactivate at a lower temperature (34°C) resulted in enhanced VZV reactivation, resulting in spreading, productive infections. Comparison of VZV genome transcription in quiescently-infected to productively-infected neurons using RNASeq revealed preferential transcription from specific genome regions, especially the duplicated regions. These experiments establish a powerful new system for modeling the VZV latent state, and reveal a potential role for temperature in VZV reactivation and disease. Most adults worldwide harbor latent VZV in their ganglia, and reactivation from it causes herpes zoster. This painful disease is frequently complicated by long-term pain, neurological sequelae, or vision loss that require improved prevention and treatment strategies. Study of VZV latency and reactivation has been severely hampered by the inability to reproduce a persistent state in vitro or in vivo that can be experimentally reactivated. Our study establishes a system using human neurons derived from embryonic stem cells where multiple stimuli can induce reactivation from long term experimental latency. A potential role for temperature in VZV reactivation has been revealed with this system, which can now be used to study the latent/lytic switch of VZV for the first time.
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