Kobayashi Mariko, Kim Ju-Youn, Camarena Vladimir, Roehm Pamela C, Chao Moses V, Wilson Angus C, Mohr Ian
Department of Microbiology, New York University School of Medicine, NY, USA.
J Vis Exp. 2012 Apr 2(62):3823. doi: 10.3791/3823.
Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility. Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA(+) neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor(1). A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.
1型单纯疱疹病毒（HSV-1）在外周神经元中建立终身潜伏感染。这个潜伏库是复发性激活事件的来源，这些事件确保了病毒传播并导致临床疾病。目前的抗病毒药物对潜伏库没有影响，且尚无疫苗。虽然裂解复制的分子细节已得到充分表征，但控制神经元潜伏的机制仍然难以捉摸。我们目前对潜伏的理解来自于使用小动物模型的体内研究，这些研究对于确定病毒基因需求和免疫反应的作用不可或缺。然而，在活体动物中，无法区分对病毒与神经元关系的特定影响与由免疫或非神经元支持细胞介导的感染的更普遍后果。此外，动物实验成本高、耗时，并且在操纵宿主过程的可用选项方面受到限制。为了克服这些限制，迫切需要一个仅包含神经元的系统，该系统能够重现潜伏和激活的体内特征，但在同质性和可及性方面具有组织培养的优势。在这里，我们展示了一种体外模型，该模型利用来自大鼠颈上神经节（SCG）的原代培养交感神经元（图1）来研究HSV-1的潜伏和激活，该模型符合大部分（如果不是全部）所需标准。在去除非神经元细胞后，在阿昔洛韦（ACV）存在的情况下，用HSV-1感染接近同质的TrkA(+)神经元培养物以抑制裂解复制。去除ACV后，能够忠实地展现公认潜伏特征的非生产性HSV-1感染得以有效建立。值得注意的是，即使在没有选择的情况下，裂解性mRNA、蛋白质和传染性病毒也变得无法检测到，但潜伏相关转录本（LAT）的表达在神经元细胞核中持续存在。病毒基因组在每个神经元中平均维持25个拷贝数，并且可以通过干扰PI3激酶/Akt信号通路或简单地去除神经生长因子来诱导其进行生产性复制(1)。一种编码与病毒裂解蛋白Us11融合的EGFP的重组HSV-1为激活导致的复制提供了一个功能性的实时标记，该标记易于定量。除了化学处理外，诸如RNA干扰或通过慢病毒载体进行基因传递等遗传方法也可以成功应用于该系统，从而进行在动物中非常困难（如果不是不可能）的机制研究。总之，基于SCG的HSV-1潜伏/激活系统提供了一个强大且必要的工具，用于揭示控制神经元中HSV1潜伏和激活的分子机制，这是病毒学中一个长期存在的难题，其解决方案可能为开发针对潜伏疱疹病毒库的新疗法提供新的见解。
