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

Abstract

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/tRNAPyl 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 tRNAPyl 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.

A novel viral vaccine platform based on engineered transfer RNA

“…Next, we used PRV as a model DNA virus and attempted to generate PTC-harbouring PRV. gB is an essential gene for PRV replication, and it was selected to engineer potential PTC sites and evaluate the PTC read-through efficacy of Arg-tRNA UGA and Gly-tRNA UGA [ 17 ]. In the current study, the PTC of the opal codon was introduced in the gB coding region, including 15 arginine sites and 14 glycine sites.…”

“…The pLVX-IRES-ZSGreen (Addgene, USA) or HIV-1-based luciferase (HIV-Luc) reporter plasmid [ 20 , 22 ] was cotransfected with the helper plasmids psPAX2 and pMD2.G (Addgene, USA). The MbPylRS/tRNA CUA plasmid was described in our recent study [ 17 ].…”

“…The PRV-PTC BAC infectious clone was constructed as previously described [ 17 , 19 ]. The obtained BAC clone with the TGA PTC in gB was termed PTC-pPRV-Bac-JS2012.…”

“…Genetic code expansion technology involves an orthogonal translation system that precisely controls the replication of viruses harbouring premature termination codons (PTCs) through the cooperation of the Methanosarcina barkeri Mb pyrrolysyl tRNA synthetase/tRNA CUA pair (MbpylRS/tRNA CUA ) with an orthogonal unnatural amino acid (UAA) [ 12 ]. Hepatitis D virus [ 13 ], HIV-1 [ 14 , 15 ], Zika virus [ 16 ] and pseudorabies virus[ 17 ] replication have been reported to be precisely controlled by GCE technology. A potent vaccine against influenza virus has been successfully developed using GCE, and it elicited robust immunity against both parental and antigenically distinct strains [ 12 ].…”

“…A potent vaccine against influenza virus has been successfully developed using GCE, and it elicited robust immunity against both parental and antigenically distinct strains [ 12 ]. The decisive, key step of GCE technology is the efficacy of UAA incorporation into the desired PTC engineering sites; however, it seemed extremely low in some tested viruses, which may limit the broad application of this technology [ 16 , 17 ].…”