高效的无载体病毒及溶瘤性单纯疱疹病毒（HSV-1）合成策略

Efficient Strategy for Synthesizing Vector-Free and Oncolytic Herpes Simplex Type 1 Viruses

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ACS Synthetic Biology

影响因子:3.9

分区:生物学2区 / 生化研究方法1区

发表日期:2024 Oct 18

作者: Han Xiao, Hengrui Hu, Yijia Guo, Jiang Li, Wen-Bo Zeng, Min-Hua Luo, Manli Wang, Zhihong Hu

DOI: 10.1021/acssynbio.4c00360

摘要

病毒基因组的合成在基础病毒学研究及疫苗和抗病毒药物开发中起着重要作用。单纯疱疹病毒1型（HSV-1）是一种广泛用于溶瘤病毒治疗的大型DNA病毒。虽然已有报告提出HSV-1基因组的全新合成方法，但其合成效率仍有待提高，且合成的基因组中含有可能影响其复制和应用的载体序列。本研究开发了一种高效的无载体HSV-1合成与复苏策略。与传统的用完全合成的含载体基因组转染哺乳动物细胞的方法不同，采用酵母中通过转化相关重组（TAR）合成的重叠HSV-1片段线性化后，共转染入哺乳动物细胞以复苏合成病毒。利用该策略，成功合成了包含HSV-1 F株完整基因组的合成病毒F-Syn。电子显微镜和生长曲线分析显示，F-Syn的复制动力学和形态发生与野生病毒相似。此外，结合TAR和体外CRISPR/Cas9编辑技术，成功构建了缺失病毒基因ICP6、ICP34.5和ICP47的溶瘤病毒F-Syn-O。体外实验表明，F-Syn-O在多种人类肿瘤细胞株中实现了有效感染并表现出剂量依赖的细胞毒性。这些策略将有助于方便、系统性地操控HSV-1基因组，未来可用于设计和构建溶瘤性疱疹病毒。

Abstract

Synthesizing viral genomes plays an important role in fundamental virology research and in the development of vaccines and antiviral drugs. Herpes simplex virus type 1 (HSV-1) is a large DNA virus widely used in oncolytic virotherapy. Although de novo synthesis of the HSV-1 genome has been previously reported, the synthetic procedure is still far from efficient, and the synthesized genome contains a vector sequence that may affect its replication and application. In the present study, we developed an efficient vector-free strategy for synthesis and rescue of synthetic HSV-1. In contrast to the conventional method of transfecting mammalian cells with a completely synthesized genome containing a vector, overlapping HSV-1 fragments synthesized by transformation-associated recombination (TAR) in yeast were linearized and cotransfected into mammalian cells to rescue the synthetic virus. Using this strategy, a synthetic virus, F-Syn, comprising the complete genome of the HSV-1 F strain, was generated. The growth curve and electron microscopy of F-Syn confirmed that its replication dynamics and morphogenesis are similar to those of the parental virus. In addition, by combining TAR with in vitro CRISPR/Cas9 editing, an oncolytic virus, F-Syn-O, with deleted viral genes ICP6, ICP34.5, and ICP47 was generated. The antitumor effect of F-Syn-O was tested in vitro. F-Syn-O established a successful infection and induced dose-dependent cytotoxic effects in various human tumor cell lines. These strategies will facilitate convenient and systemic manipulation of HSV-1 genomes and could be further applied to the design and construction of oncolytic herpesviruses.