Department of Cell Biology and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang Universitygrid.13402.34 School of Medicine, Hangzhou, China.
Liangzhu Laboratory, Zhejiang Universitygrid.13402.34 Medical Center, Hangzhou, China.
J Virol. 2022 Dec 21;96(24):e0092022. doi: 10.1128/jvi.00920-22. Epub 2022 Dec 1.
Real-time imaging tools for single-virus tracking provide spatially resolved, quantitative measurements of viral replication and virus-host interactions. However, efficiently labeling both parental and progeny viruses in living host cells remains challenging. Here, we developed a novel strategy using the CRISPR-Tag system to detect herpes simplex virus 1 (HSV-1) DNA in host cells. We created recombinant HSV-1 harboring an ~600-bp CRISPR-Tag sequence which can be sufficiently recognized by dCas9-fluorescent protein (FP) fusion proteins. RISPR-ssisted ingle ral genome racking (CASVIT) allows us to assess the temporal and spatial information of viral replication at the single-cell level. Combining the advantages of SunTag and tandem split green fluorescent protein (GFP) in amplifying fluorescent signals, dSaCas9-tdTomato and dSpCas9-GFP were constructed to enable efficient two-color CASVIT detection. Real-time two-color imaging indicates that replication compartments (RCs) frequently come into contact with each other but do not mix, suggesting that RC territory is highly stable. Last, two-color CASVIT enables simultaneous tracking of viral DNA and host chromatin, which reveals that a dramatic loss of telomeric and centromeric DNA occurs in host cells at the early stage of viral replication. Overall, our work has established a framework for developing CRISPR-Cas9-based imaging tools to study DNA viruses in living cells. Herpes simplex virus 1 (HSV-1), a representative of the family , is a ubiquitous pathogen that can establish lifelong infections and widely affects human health. Viral infection is a dynamic process that involves many steps and interactions with various cellular structures, including host chromatin. A common viral replication strategy is to form RCs that concentrate factors required for viral replication. Efficient strategies for imaging the dynamics of viral genomes, RC formation, and the interaction between the virus and host offer the opportunity to dissect the steps of the infection process and determine the mechanism underlying each step. We have developed an efficient two-color imaging system based on CRISPR-Cas9 technology to detect HSV-1 genomes quantitatively in living cells. Our results shed light on novel aspects of RC dynamics and virus-host interactions.
用于单病毒跟踪的实时成像工具提供了病毒复制和病毒-宿主相互作用的空间分辨、定量测量。然而，在活宿主细胞中有效地标记亲代和子代病毒仍然具有挑战性。在这里，我们使用 CRISPR-Tag 系统开发了一种新的策略来检测宿主细胞中的单纯疱疹病毒 1（HSV-1）DNA。我们创建了携带约 600bp CRISPR-Tag 序列的重组 HSV-1，该序列可以被 dCas9-荧光蛋白（FP）融合蛋白充分识别。RISPR 辅助的单基因组跟踪（CASVIT）允许我们在单细胞水平上评估病毒复制的时间和空间信息。结合 SunTag 和串联分裂绿色荧光蛋白（GFP）在放大荧光信号方面的优势，构建了 dSaCas9-tdTomato 和 dSpCas9-GFP，以实现高效的双色 CASVIT 检测。实时双色成像表明复制隔室（RCs）经常相互接触但不混合，表明 RC 区域高度稳定。最后，双色 CASVIT 能够同时跟踪病毒 DNA 和宿主染色质，这表明在病毒复制的早期阶段，宿主细胞中端粒和着丝粒 DNA 大量丢失。总的来说，我们的工作为开发基于 CRISPR-Cas9 的成像工具来研究活细胞中的 DNA 病毒奠定了框架。单纯疱疹病毒 1（HSV-1）是家族的代表，是一种普遍存在的病原体，可导致终生感染，并广泛影响人类健康。病毒感染是一个动态过程，涉及许多步骤，与各种细胞结构相互作用，包括宿主染色质。一种常见的病毒复制策略是形成浓缩病毒复制所需因子的 RC。高效的病毒基因组动力学成像、RC 形成以及病毒与宿主相互作用的策略为剖析感染过程的步骤和确定每个步骤的机制提供了机会。我们开发了一种基于 CRISPR-Cas9 技术的高效双色成像系统，用于在活细胞中定量检测 HSV-1 基因组。我们的结果揭示了 RC 动力学和病毒-宿主相互作用的新方面。
