{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,9]],"date-time":"2026-10-09T11:12:52Z","timestamp":1791544372445,"version":"4.3.4"},"reference-count":31,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2022,3,10]],"date-time":"2022-03-10T00:00:00Z","timestamp":1646870400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Heilongjiang Excellent Youth Fund Project","award":["YQ2019C032 and YQ2019C028"],"award-info":[{"award-number":["YQ2019C032 and YQ2019C028"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/v14030572","type":"journal-article","created":{"date-parts":[[2022,3,10]],"date-time":"2022-03-10T20:19:10Z","timestamp":1646943550000},"page":"572","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["Generation of Premature Termination Codon (PTC)-Harboring Pseudorabies Virus (PRV) via Genetic Code Expansion Technology"],"prefix":"10.3390","volume":"14","author":[{"given":"Tong-Yun","family":"Wang","sequence":"first","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guo-Ju","family":"Sang","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qian","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chao-Liang","family":"Leng","sequence":"additional","affiliation":[{"name":"Henan Provincial Engineering and Technology Center of Animal Disease Diagnosis and Integrated Control, Nanyang Normal University, Nanyang 473061, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhi-Jun","family":"Tian","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jin-Mei","family":"Peng","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7020-5410","authenticated-orcid":false,"given":"Shu-Jie","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ming-Xia","family":"Sun","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fan-Dan","family":"Meng","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"Zheng","sequence":"additional","affiliation":[{"name":"Shanghai Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Shanghai 200241, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xue-Hui","family":"Cai","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5690-2690","authenticated-orcid":false,"given":"Yan-Dong","family":"Tang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,3,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"482","DOI":"10.1016\/j.coviro.2012.06.005","article-title":"Vaccine prospect of Kaposi sarcoma-associated herpesvirus","volume":"2","author":"Wu","year":"2012","journal-title":"Curr. Opin. Virol."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Wang, T.Y., Yang, Y.L., Feng, C., Sun, M.X., Peng, J.M., Tian, Z.J., Tang, Y.D., and Cai, X.H. (2020). Pseudorabies Virus UL24 Abrogates Tumor Necrosis Factor Alpha-Induced NF-kappaB Activation by Degrading P65. Viruses, 12.","DOI":"10.3390\/v12010051"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"462","DOI":"10.1128\/MMBR.69.3.462-500.2005","article-title":"Molecular biology of pseudorabies virus: Impact on neurovirology and veterinary medicine","volume":"69","author":"Pomeranz","year":"2005","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/0147-9571(91)90128-Z","article-title":"Molecular biology of pseudorabies (Aujeszky\u2019s disease) virus","volume":"14","author":"Mettenleiter","year":"1991","journal-title":"Comp. Immunol. Microbiol. Infect. Dis."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Zhang, F., Wang, M., Michael, T., and Drabier, R. (2013). Novel alternative splicing isoform biomarkers identification from high-throughput plasma proteomics profiling of breast cancer. BMC Syst. Biol., 7.","DOI":"10.1186\/1752-0509-7-S5-S8"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1016\/j.vetmic.2014.09.003","article-title":"Pathogenicity and genomic characterization of a pseudorabies virus variant isolated from Bartha-K61-vaccinated swine population in China","volume":"174","author":"Luo","year":"2014","journal-title":"Vet. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1087","DOI":"10.3201\/eid2406.171612","article-title":"Human Endophthalmitis Caused By Pseudorabies Virus Infection, China, 2017","volume":"24","author":"Ai","year":"2018","journal-title":"Emerg. Infect. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e3690","DOI":"10.1093\/cid\/ciaa987","article-title":"A Novel Human Acute Encephalitis Caused by Pseudorabies Virus Variant Strain","volume":"73","author":"Liu","year":"2021","journal-title":"Clin. Infect. Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/j.virusres.2014.09.003","article-title":"A novel inactivated gE\/gI deleted pseudorabies virus (PRV) vaccine completely protects pigs from an emerged variant PRV challenge","volume":"195","author":"Gu","year":"2015","journal-title":"Virus Res."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Wang, J., Guo, R., Qiao, Y., Xu, M., Wang, Z., Liu, Y., Gu, Y., Liu, C., and Hou, J. (2016). An inactivated gE-deleted pseudorabies vaccine provides complete clinical protection and reduces virus shedding against challenge by a Chinese pseudorabies variant. BMC Vet. Res., 12.","DOI":"10.1186\/s12917-016-0897-z"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1016\/j.vetmic.2019.04.031","article-title":"Commercial vaccine against pseudorabies virus: A hidden health risk for dogs","volume":"233","author":"Lin","year":"2019","journal-title":"Vet. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1038\/nmeth.3701","article-title":"Genetic code expansion in stable cell lines enables encoded chromatin modification","volume":"13","author":"Elsasser","year":"2016","journal-title":"Nat. Methods"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2330","DOI":"10.1080\/15476286.2021.1907055","article-title":"Amber codon is genetically unstable in generation of premature termination codon (PTC)-harbouring Foot-and-mouth disease virus (FMDV) via genetic code expansion","volume":"12","author":"Hao","year":"2021","journal-title":"RNA Biol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1170","DOI":"10.1126\/science.aah5869","article-title":"Generation of influenza A viruses as live but replication-incompetent virus vaccines","volume":"354","author":"Si","year":"2016","journal-title":"Science"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1016\/j.jviromet.2018.06.004","article-title":"Construction of an infectious bacterial artificial chromosome clone of a pseudorabies virus variant: Reconstituted virus exhibited wild-type properties in vitro and in vivo","volume":"259","author":"Wang","year":"2018","journal-title":"J. Virol. Methods"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"e0094421","DOI":"10.1128\/JVI.00944-21","article-title":"Aminopeptidase N Is an Entry Co-factor Triggering Porcine Deltacoronavirus Entry via an Endocytotic Pathway","volume":"95","author":"Yang","year":"2021","journal-title":"J. Virol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1080\/22221751.2020.1730245","article-title":"Trypsin promotes porcine deltacoronavirus mediating cell-to-cell fusion in a cell type-dependent manner","volume":"9","author":"Yang","year":"2020","journal-title":"Emerg. Microbes. Infect."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1186\/s12985-020-01312-2","article-title":"Isobavachalcone inhibits Pseudorabies virus by impairing virus-induced cell-to-cell fusion","volume":"17","author":"Wang","year":"2020","journal-title":"Virol. J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"12296","DOI":"10.1128\/JVI.01379-14","article-title":"Equine viperin restricts equine infectious anemia virus replication by inhibiting the production and\/or release of viral Gag, Env, and receptor via distortion of the endoplasmic reticulum","volume":"88","author":"Tang","year":"2014","journal-title":"J. Virol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"873","DOI":"10.1128\/JVI.72.1.873-875.1998","article-title":"Glycoproteins gB, gD, and gHgL of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a Cos cell transfection system","volume":"72","author":"Turner","year":"1998","journal-title":"J. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"6760","DOI":"10.1128\/JVI.74.15.6760-6768.2000","article-title":"Pseudorabies virus glycoprotein M inhibits membrane fusion","volume":"74","author":"Klupp","year":"2000","journal-title":"J. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e00061-17","DOI":"10.1128\/JVI.00061-17","article-title":"Functional Relevance of the N-Terminal Domain of Pseudorabies Virus Envelope Glycoprotein H and Its Interaction with Glycoprotein, L. J","volume":"91","author":"Vallbracht","year":"2017","journal-title":"Virol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1016\/j.cell.2005.07.013","article-title":"Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice","volume":"122","author":"Ding","year":"2005","journal-title":"Cell"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4867","DOI":"10.1002\/anie.201402092","article-title":"Construction of a live-attenuated HIV-1 vaccine through genetic code expansion","volume":"53","author":"Wang","year":"2014","journal-title":"Angew. Chem. Int. Ed. Engl."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"721","DOI":"10.1021\/acssynbio.6b00373","article-title":"Controlling Multicycle Replication of Live-Attenuated HIV-1 Using an Unnatural Genetic Switch","volume":"6","author":"Yuan","year":"2017","journal-title":"ACS Synth. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1007\/s11427-020-1737-3","article-title":"Construction and characterization of UAA-controlled recombinant Zika virus by genetic code expansion","volume":"64","author":"Zhang","year":"2021","journal-title":"Sci. China. Life Sci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"13970","DOI":"10.1002\/anie.201305787","article-title":"Site-specific engineering of chemical functionalities on the surface of live hepatitis D virus","volume":"52","author":"Lin","year":"2013","journal-title":"Angew. Chem. Int. Ed. Engl."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"5117","DOI":"10.1021\/acs.jcim.0c00725","article-title":"The Effects of p-Azidophenylalanine Incorporation on Protein Structure and Stability","volume":"60","author":"Wilkerson","year":"2020","journal-title":"J. Chem. Inf. Modeling"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"15577","DOI":"10.1021\/ja5069728","article-title":"Efficient multisite unnatural amino acid incorporation in mammalian cells via optimized pyrrolysyl tRNA synthetase\/tRNA expression and engineered eRF1","volume":"136","author":"Schmied","year":"2014","journal-title":"J. Am. Chem. Soc."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1231","DOI":"10.1002\/stem.679","article-title":"Genetically encoding unnatural amino acids in neural stem cells and optically reporting voltage-sensitive domain changes in differentiated neurons","volume":"29","author":"Shen","year":"2011","journal-title":"Stem Cells"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"383","DOI":"10.1016\/j.jviromet.2013.06.035","article-title":"PK-15 cells transfected with porcine CD163 by PiggyBac transposon system are susceptible to porcine reproductive and respiratory syndrome virus","volume":"193","author":"Wang","year":"2013","journal-title":"J Virol. 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