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

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