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.

Automated summary

This study attempted to develop a live but replication-incompetent Pseudorabies virus (PRV) using genetic code expansion (GCE) technology. Although the premature termination codon (PTC) harboring PRV was successfully rescued, the incorporation efficacy of unnatural amino acids (UAA) was found to be extremely low. Moreover, the establishment of a stable transgenic cell line for PTC virus replication failed, with orthogonal tRNA(Pyl) identified as a limiting factor.