Generation of Premature Termination Codon (PTC)-Harboring Pseudorabies Virus (PRV) via Genetic Code Expansion Technology

Despite many efforts and diverse approaches, developing an effective herpesvirus vaccine remains a great challenge. Traditional inactivated and live-attenuated vaccines always raise efficacy or safety concerns. This study used Pseudorabies virus (PRV), a swine herpes virus, as a model. We attempted to develop a live but replication-incompetent PRV by genetic code expansion (GCE) technology. Premature termination codon (PTC) harboring PRV was successfully rescued in the presence of orthogonal system MbpylRS/tRNA^Pyl pair and unnatural amino acids (UAA). However, UAA incorporating efficacy seemed extremely low in our engineered PRV PTC virus. Furthermore, we failed to establish a stable transgenic cell line containing orthogonal translation machinery for PTC virus replication, and we demonstrated that orthogonal tRNA^Pyl is a key limiting factor. This study is the first to demonstrate that orthogonal translation system-mediated amber codon suppression strategy could precisely control PRV-PTC engineered virus replication. To our knowledge, this is the first reported PTC herpesvirus generated by GCE technology. Our work provides a proof-of-concept for generating UAAs-controlled PRV-PTC virus, which can be used as a safe and effective vaccine.