New tools to study the dynamics of HSV latency and reactivation in living neurons

描述(申请人提供)：单纯疱疹病毒-1(HSV-1)建立了人类周围神经系统的潜伏期，并提供了一个教科书上的隐形病原体的例子。病毒蛋白不表达到任何显著水平，病毒转录仅限于处理成稳定的2.0kb内含子和少数病毒microRNAs的潜伏期相关转录本(LAT)。潜伏抗原的缺乏和可识别的表型变化的缺乏使得识别潜伏感染的神经元变得具有挑战性，而不需要使用需要固定的侵入性方法。异源标记蛋白一直没有提供信息，因为随着潜伏期的确立，这些蛋白会逐渐沉默。因此，关于神经系统内潜伏期、重新激活和扩散的时间和空间性质的关键问题仍然很难回答。这项为期两年的重点计划概述了一种创新的战略，以构建重组HSV-1病毒，在病毒生命周期的潜伏期和活跃复制(裂解)阶段，可以在活的神经元中很容易地检测到这种病毒。我们的新方法使用了一种基于短合成RNA(适配子)的Ne活细胞成像技术，该技术结合了一个细胞渗透性的、无毒的小分子，使其在可见光光谱中发光。为了确保在神经元核中持续和高水平的表达，这种‘绿色荧光RNA’(GFR)将被引入到稳定的2.0kb LAT内含子中。工程病毒的信号强度和完整性将使用我们实验室开发的培养的初级神经元潜伏期/再激活模型进行评估，并随后在小鼠角膜感染模型中进行验证。潜伏感染GFR-LAT病毒的神经元将在潜伏感染的神经元中产生独特的荧光信号，并可实时跟踪。然后，在裂解基因启动子的控制下，通过掺入红色荧光蛋白来构建额外的双色病毒。这些新病毒将被用来探索LAT表达水平和潜伏病毒重新激活倾向之间的不确定关系。

DESCRIPTION (provided by applicant): Herpes simplex virus-1 (HSV-1) establishes latency in the human peripheral nervous system and provides a textbook example of a stealth pathogen. Viral proteins are not expressed to any significant level and viral transcription is limited to the latency-associated transcripts (LAT) that processed into a stable 2.0-kb intron and handful of viral microRNAs. The absence of latent antigens and lack of discernable phenotypic changes has made it challenging to recognize latently infected neurons without invasive methods that require fixation. Heterologous marker proteins have been uninformative because these are progressively silenced as latency is established. As a result, key questions about the temporal and spatial nature of latency, reactivation and spread within the nervous system remain difficult to answer. This focused 2-year proposal outlines an innovative strategy to construct recombinant HSV-1 viruses that can be readily detected in living neurons during the latent and actively replicating (lytic) stages of the viral lifecycle. Our novel approach uses a ne live cell imaging technology based on short synthetic RNAs (aptamers) that bind a cell-permeable, non-toxic small molecule causing it to emit light in the visible spectrum. To ensure persistent and high-level expression in the neuronal nucleus, this 'green fluorescent RNA' (GFR) will be introduced into the stable 2.0-kb LAT intron. Signal strength and integrity of the engineered viruses will be evaluated using a cultured primary neuron latency/reactivation model developed in our laboratories and subsequently validated in the mouse corneal infection model. Latent infection of neurons with GFR-LAT viruses will produce a distinctive fluorescent signature in latently infected neurons that can be followed in real-time. Additional dual-color viruses will then be constructed by incorporating a red fluorescent protein under the control of a lytic gene promoter. These new viruses will be used to explore the uncertain relationship between levels of LAT expression and propensity of a latent virus to reactivate.