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

描述（由申请人提供）：单纯疱疹病毒-1（HSV-1）在人类外周神经系统中建立潜伏期，并提供了隐形病原体的教科书示例。病毒蛋白质不会表达到任何显着水平，病毒转录仅限于潜伏期相关转录物（LAT），这些转录物加工成稳定的2.0 kb内含子和少数病毒微小RNA。缺乏潜伏抗原和缺乏可辨别的表型变化使得在没有需要固定的侵入性方法的情况下识别潜伏感染的神经元具有挑战性。异源性标记蛋白是没有信息的，因为随着潜伏期的建立，这些蛋白逐渐沉默。因此，关于神经系统内潜伏期、再激活和传播的时间和空间性质的关键问题仍然难以回答。这项为期2年的提案概述了构建重组HSV-1病毒的创新策略，这些病毒可以在病毒生命周期的潜伏和活跃复制（裂解）阶段在活神经元中容易地检测到。我们的新方法使用了一种基于短合成RNA（适体）的新活细胞成像技术，该技术结合细胞可渗透的无毒小分子，使其在可见光谱中发光。为了确保在神经元细胞核中的持续和高水平表达，将这种“绿色荧光RNA”（GFR）引入稳定的2.0-kb LAT内含子中。将使用我们实验室开发的培养的原代神经元潜伏期/再激活模型评价工程病毒的信号强度和完整性，随后在小鼠角膜感染模型中进行验证。GFR-LAT病毒潜伏感染神经元将在潜伏感染的神经元中产生独特的荧光特征，可以实时跟踪。然后通过在裂解基因启动子的控制下掺入红色荧光蛋白来构建另外的双色病毒。这些新病毒将用于探索LAT表达水平与潜伏病毒再活化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.