Department of Microbiology and Cell Biology, Montana State University, Bozeman, Montana, USA.
Department of Clinical Microbiology and Immunology, School of Medicine, Tel Aviv University, Tel Aviv-Yafo, Israel.
J Virol. 2024 May 14;98(5):e0003224. doi: 10.1128/jvi.00032-24. Epub 2024 Apr 23.
Critical stages of lytic herpes simplex virus type 1 (HSV-1) replication are marked by the sequential expression of immediate early (IE) to early (E), then late (L) viral genes. HSV-1 can also persist in neuronal cells via a non-replicative, transcriptionally repressed infection called latency. The regulation of lytic and latent transcriptional profiles is critical to HSV-1 pathogenesis and persistence. We sought a fluorescence-based approach to observe the outcome of neuronal HSV-1 infection at the single-cell level. To achieve this goal, we constructed and characterized a novel HSV-1 recombinant that enables discrimination between lytic and latent infection. The dual reporter HSV-1 encodes a human cytomegalovirus-immediate early (hCMV-IE) promoter-driven enhanced yellow fluorescent protein (eYFP) to visualize the establishment of infection and an endogenous mCherry-VP26 fusion to report lytic replication. We confirmed that viral gene expression, replication, and spread of infection are not altered by the incorporation of the fluorescent reporters, and fluorescent protein (FP) detection virtuously reports the progression of lytic replication. We demonstrate that the outcome of HSV-1 infection of compartmentalized primary neurons is determined by viral inoculating dose: high-dose axonal inoculation proceeds to lytic replication, whereas low-dose axonal inoculation establishes a latent HSV-1 infection. Interfering with low-dose axonal inoculation via small molecule drugs reports divergent phenotypes of eYFP and mCherry reporter detection, correlating with altered states of viral gene expression. We report that the transcriptional state of neuronal HSV-1 infection is variable in response to changes in the intracellular neuronal environment.IMPORTANCEHerpes simplex virus type 1 (HSV-1) is a prevalent human pathogen that infects approximately 67% of the global human population. HSV-1 invades the peripheral nervous system, where latent HSV-1 infection persists within the host for life. Immunological evasion, viral persistence, and herpetic pathologies are determined by the regulation of HSV-1 gene expression. Studying HSV-1 gene expression during neuronal infection is challenging but essential for the development of antiviral therapeutics and interventions. We used a recombinant HSV-1 to evaluate viral gene expression during infection of primary neurons. Manipulation of cell signaling pathways impacts the establishment and transcriptional state of HSV-1 latency in neurons. The work here provides critical insight into the cellular and viral factors contributing to the establishment of latent HSV-1 infection.
单纯疱疹病毒 1 型（HSV-1）的裂解复制的关键阶段通过立即早期（IE）到早期（E），然后晚期（L）病毒基因的顺序表达来标记。HSV-1 还可以通过称为潜伏的非复制、转录抑制的感染在神经元细胞中持续存在。裂解和潜伏转录谱的调节对 HSV-1 的发病机制和持续存在至关重要。我们寻求一种基于荧光的方法来观察单个细胞水平的神经元 HSV-1 感染的结果。为了实现这一目标，我们构建并表征了一种新型的 HSV-1 重组体，该重组体能够区分裂解和潜伏感染。双报告 HSV-1 编码人巨细胞病毒立即早期（hCMV-IE）启动子驱动的增强型黄色荧光蛋白（eYFP）以可视化感染的建立，并编码内源性 mCherry-VP26 融合以报告裂解复制。我们证实，荧光报告基因的掺入不会改变病毒基因表达、复制和感染的传播，并且荧光蛋白（FP）检测确实报告了裂解复制的进展。我们表明，分隔的原代神经元中 HSV-1 感染的结果取决于病毒接种剂量：高剂量轴突接种会进行裂解复制，而低剂量轴突接种会建立潜伏 HSV-1 感染。通过小分子药物干扰低剂量轴突接种会报告 eYFP 和 mCherry 报告基因检测的不同表型，这与病毒基因表达状态的改变相关。我们报告说，神经元 HSV-1 感染的转录状态会根据细胞内神经元环境的变化而变化。
重要性：单纯疱疹病毒 1 型（HSV-1）是一种流行的人类病原体，约 67%的全球人类人口感染了 HSV-1。HSV-1 入侵周围神经系统，潜伏的 HSV-1 感染在宿主中持续存在一生。HSV-1 的免疫逃避、病毒持续存在和疱疹病理由 HSV-1 基因表达的调节决定。研究神经元感染期间的 HSV-1 基因表达对于开发抗病毒治疗和干预措施至关重要。我们使用重组 HSV-1 来评估原代神经元感染期间的病毒基因表达。细胞信号通路的操纵会影响神经元中 HSV-1 潜伏期的建立和转录状态。这里的工作提供了对导致潜伏 HSV-1 感染建立的细胞和病毒因素的重要见解。
