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

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.