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/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.


Introduction
Developing the herpesviruses vaccine is challenging because of the immunologically silent nature of its latency, and the virus mediates immune evasion [1,2]. Pseudorabies virus (PRV) is a swine herpesvirus, also known as Aujeszky virus, which belongs to the genus Varicellovirus in the subfamily Alphaherpesvirinae of the family Herpesviridae [3]. PRV is lethal to many domestic and wild animals, and pigs are the natural host [4]. Since 2011, PRV variants have emerged in China, and commercial vaccines fail to provide complete protection against PRV [5,6]. More seriously, PRV variants can spill over into humans and cause severely nerve-related diseases [7,8]. Developing a safe and effective PRV vaccine is one of the best choices for PRV control in related animals and humans.
Inactivated vaccines play a vital role in eradicating PRV in swine farms. However, the inactivated PRV vaccine mainly induces a humoral immune response, lacks effective T cell response, and inactivated PRV vaccines fail to stop viral shedding post-virus challenge [9,10]. Live-attenuated vaccines have shown the best efficacy against PRV; however, T cell response, and inactivated PRV vaccines fail to stop viral shedding post-virus challenge [9,10]. Live-attenuated vaccines have shown the best efficacy against PRV; however, this raises safety concerns, e.g., the attenuated PRV strains are lethal to dogs and can spread horizontally [11]. Therefore, developing a safe and effective PRV vaccine faces a dilemma.
Genetic code expansion (GCE) technology is an orthogonal translation system derived from the Methanosarcina barkeri. In this microbe, amber (TAG) stop codon can be read-through with the cooperation of Mb pyrrolysyl tRNA synthetase/tRNA Pyl pair (MbpylRS/tRNA Pyl ) and unnatural amino acids (UAA) [12][13][14]. The application of GCE technology in PTC harboring PRV is illustrated in Figure 1. The GCE technology provides a novel strategy to generate a live but replication-defective candidate vaccine. This technology has been successfully applied in the influenza A virus vaccine [14]. This study used PRV as a herpesvirus model and attempted to engineer a PTC site in an essential gene of PRV, gB, with amber codons (TAG). PTC harboring PRV could be successfully rescued in the orthogonal translation machinery system MbpylRS/tRNA Pyl pair and UAA. However, UAA incorporating efficacy seemed extremely low in our engineered PRV PTC virus. Furthermore, all our attempts to construct cell lines containing orthogonal translation machinery system MbpylRS/tRNA Pyl pair failed. These results suggested that several key issues should be resolved in PTC harboring herpesvirus production in the future. This study used PRV as a herpesvirus model and attempted to engineer a PTC site in an essential gene of PRV, gB, with amber codons (TAG). PTC harboring PRV could be successfully rescued in the orthogonal translation machinery system MbpylRS/tRNA Pyl pair and UAA. However, UAA incorporating efficacy seemed extremely low in our engineered PRV PTC virus. Furthermore, all our attempts to construct cell lines containing orthogonal translation machinery system MbpylRS/tRNA Pyl pair failed. These results suggested that several key issues should be resolved in PTC harboring herpesvirus production in the future.

Read-Through Efficacy for PTC Harboring gB by GCE
HEK-293T cells in good growth condition were plated in 24 well plates (4 × 10 5 cells/well). 0.5 µg of MbpylRS/tRNA Pyl plasmids were co-transfected with 0.5 µg of pCAGGS gB PTC plasmid using the jetPRIME transfection reagent (Polyplus, Illkirch, France). Two parallel experiments were conducted. pCAGGS gB was used as a positive control, and non-transfected cells were used as mock. The supernatant was replaced by fresh medium supplemented with 2% FBS in the presence of 1 mM NAEK (TRC, Ottawa, ON, Canada) or not, 6 h post-transfection. At 48 h post-transfection, a Western blot was performed to analyze the read-through efficacy of PTC harboring gB mutants. The cells were lysed by 70 µL lysis buffer (50 mM KCl, 100 mM NaCl, 0.25% NP-40, 1 mM DTT, and 50 mM herpes-NaOH) containing 1% protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA) for 30 min on ice and then centrifuged at 12,000× g for 10 min at 4 • C. Cell lysates were mixed with 5× loading buffer and boiled at 100 • C for 10 min. As previously described, the samples were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes [16].

Read-Through Efficacy for gB PTC Identified by Cell-to-Cell Fusion Assay
Cell-to-cell fusion assay was performed as previously described [18]. Briefly RK13 cells were placed in 24 well plates (4 × 10 5 cells/well), and 0.15 µg pCAGGS gB or indicated gB PTCs, 0.15 µg pCAGGS gD, 0.15 µg pCAGGS gH, 0.15 µg pCAGGS gL, 0.15 µg pDC315-GFP, and 0.4 µg MbpylRS/tRNA Pyl were co-transfected. The non-transfected cells were used as control. The supernatant medium was replaced by a fresh medium supplemented with 2% FBS in the presence of 1 mM UAA (NAEK) or not, 6 h post-transfection. 48 h after transfection, fluorescence microscopy was used to analyze the read-through efficacy by indicated gB mediated cell fusion analysis.

PTC Harboring PRV Construction
PRV-PTC construction was performed as previously described [15]. Briefly, the procedure is as follows. A DNA fragment with a galK expression cassette flanked by 50 bp homologous of gB gene was amplified by PCR using the primers gB-galKF/gB-galKR (gB-galKF: 5-GGGACCGCTTCTACGTCTGCCCGCCGCCGTCCGGCTCCACGGTGGT cctgttgacaattaatcatcggca-3; gB-galKR: 5-AGGCGGTCACCTTGTGGTTGTTGCGCACGTAC TCGGCCTTGGAGACGCACTTGCCtcagcactgtcctgctcctt-3) and KOD DNA polymerase (Toyobo, Osaka, Japan). The obtained PCR product was digested with DpnI (Thermo Fisher, Waltham, MA, USA) at 37 • C for 1 h to remove the original template plasmid, followed by agarose gel purification. To generate SW102-JS-galK, 25 µL SW102-JS electrocompetent cells were prepared and electro-transformed with 100 ng galK DNA fragment under the condition of 1.5 kV, 25 µF, 200 Ω. Then, 800 µL SOC medium was added immediately after electro-transformation and incubation at 32 • C, 200 rpm for 1.5 h. The recovered bacteria were washed twice with 1 mL M9 solution and took 150 µL M9 solution to plate the bacteria cells onto M63 plates containing galactose and chloramphenicol. PCR was used to confirm galK positive colonies with primers LgalKup/LgalKdown (LgalKup: 5-TGCTGCGCCTCGACCCCAA-3; LgalKdown: 5-AAGAACTTAACCCGGCACCCT-3). The galK positive colonies were further screened on a MacConkey plate containing chloramphenicol to realize the nucleotide substitution of galK at positions 141 to 187 of gB in pBac-JS2012. Finally, gB fragments harboring PTC points were used to remove the galK gene from pBac-JS2012-galK. A DNA fragment with PTC points in gB ORF was amplified from the template gB mutant plasmids with the primers gB-LF/gB-RR (gB-LF: 5-cgacggtatcgataagcttgatCGCTGGTGGCGGTCTTTG-3; gB-RR: 5-ccgggctgcaggaattcgatGAG TCCAGGTCGATGGGGTAG-3). The obtained PCR product was also digested with DpnI (Thermo Fisher, Waltham, MA, USA) at 37 • C for 1 h. The indicated PTC harboring gB fragment was electro-transformed into SW102-JS-galK as described above. After 3 h at 32 • C, the transformed cells were washed and suspended in an M9 medium. The positive clone with mutant gB fragment replacing galK was screened on M63 minimal medium plates containing chloramphenicol, 2-deoxy-galactose, and glycerol. The obtained PRV Bac clone with TAG PTC in gB was termed PRV-PTC.

Rescue of PRV-PTC Virus
HEK-293T cells and Vero cells (2 × 10 6 ) were plated in 6-well plates in DMEM supplemented with 10% FBS. Then, 2 µg MbpylRS/tRNA Pyl plasmid were co-transfected with the 2 µg pPRV-Bac or the indicated pPRV-PTC-Bac using the transfection reagent jetPRIME (Polyplus, Illkirch, France) according to the manufacturer's instructions. At 6 h post-transfection, the supernatant was replaced with DMEM containing 2% FBS and 1 mM UAAs, Nε-2-azidoethyloxycarbonyl-L-lysine (NAEK). To identify the UAA-dependence of PRV-PTC virus, a parallel packaging experiment was conducted in which the medium was not supplemented with UAA. The cells were further incubated at 37 • C in 5% CO 2 until cytopathic effect (CPE) or syncytium was observed.

Electron Microscopy for PRV-PTC Virus
HEK-293T cells were transfected with plasmids described above for conventional electron microscopy analysis. Then when the cytopathic effect (CPE) or syncytium was observed, cells were fixed with 2.5% (w/v) glutaraldehyde in 200 mM HEPES (pH 7.4) for 2 h at room temperature, followed by post-fixation with 1% OsO 4 and 1.5% K 3 Fe(CN) 6 in H 2 O at 4 • C for 30 min. According to standard procedures, samples were dehydrated with acetone and impregnated with epoxy at room temperature and further embedded overnight at 70 • C for polymerization. Then the samples were cut into 70 nm ultrathin sections by ultrathin slicer (Leica, Wetzlar, Germany), stained with 2% uranium acetate for 17 min, lead citrate for 12 min. Specimens were examined using a conventional transmission electron microscope (TEM, h7650, Hitachi, Tokyo, Japan).

Construction of GFP 39TAG Reporter Adenovirus
To generate adenovirus harboring GFP 39TAG reporter, HEK293 cells were transiently co-transfected with the pDC-315GFP 39TAG (plasmid with the TAG stop codon in GFP gene) with pBHGlox∆E1 and E3Cre helper plasmids as previously described [19]. 6 h after transfection, the medium was replaced with a fresh medium containing 2% FBS. The transfected cells were harvested after 7~9 d until plaque was observed.

Generation of Transgenic Cell Line Containing MbpylRS/tRNA Pyl Orthogonal System
Indicated cells were seeded in 6 well plates and were co-transfected with 3 µg of pB513B-puro-MbpylRS-12tRNA plasmid and 1 µg of pB220A-1 plasmid using the transfection reagent jetPRIME (Polyplus, Illkirch, France). Non-transfected cells were used as control. Then, 6 h later, the transfection medium was replaced by DMEM medium supplemented with 10% FBS and 1 mM UAAs (NAEK). 48 h after transfection, the cells were selected under the pressure of indicated concentrate puromycin (Gibco, Waltham, MA, USA). The medium was replaced every day until the cells in the control group completely died. The resultant cells were stably transfected and continued to cultivate in the presence of 4 µg/mL puromycin. Then these cells were infected with GFP 39TAG reporter adenovirus in the presence of UAA, and the single clones were further sorted by fluorescence-activated cell sorting (FACS) according to the GFP reporter.

Evaluation of UAA Site-Specific Incorporation for Potential PRV gB PTC Sites
gB was recognized as an essential gene for PRV replication. Therefore, we selected gB to engineer potential PTC sites. First, 149Q, 169K, 171K, 177K, 185W, 206Q, 217K, 221K, 267K, 285W, 319H, 331Q, 362W, 365W, 367W, 370K, 379K, 413Q of gB was separately engineered into amber codon (TAG). PTC containing gB constructs were confirmed by DNA sequencing (data not shown). Then the indicated gB-PTC constructs were co-transfected with the orthogonal MbpylRS/tRNA Pyl pair plasmid, respectively. The UAA in this study was Nε-2-azidoethyloxycarbonyl-L-lysine (NAEK) as illustrated in Figure 2A. Full-length gB protein can be cleavaged by furin, as demonstrated in Figure 2B. Western blot result showed that gB was successfully expressed in the presence of 1 mM NAEK for indicated gB PTC constructs ( Figure 2B,D). There was no gB expression without UAA ( Figure 2C,E). This result indicated that some gB PTC sites read-through by GCE technology as expected, although the expression level was lower than wild-type gB ( Figure 2B,D). As gB, gD, gH, and gL are the essential viral genes for virus-mediated cell-to-cell fusion, which is an important step for PRV spreading [20]. Next, a cell-to-cell fusion assay was used to test whether these NAEK incorporation sites influenced gB mediated cell-to-cell fusion. As previously described, a transient transfection-based cell-to-cell fusion assay was performed by co-transfection of PTC gB, gD, gH, gL and EGFP plasmids [18,21,22]. The result indicated that some gB PTC sites such as 149Q, 169K, 185W, 206Q, 379K successfully induced syncytia formation in the presence of NAEK, and no syncytia was formed without NAEK ( Figure 2F). These results indicated that the effect of substitution by NAEK on function varies with position, and the position of 149 and 185 was labeled in the gB crystal structure ( Figure 2G). and gL are the essential viral genes for virus-mediated cell-to-cell fusion, which is an important step for PRV spreading [20]. Next, a cell-to-cell fusion assay was used to test whether these NAEK incorporation sites influenced gB mediated cell-to-cell fusion. As previously described, a transient transfection-based cell-to-cell fusion assay was performed by co-transfection of PTC gB, gD, gH, gL and EGFP plasmids [18,21,22]. The result indicated that some gB PTC sites such as 149Q, 169K, 185W, 206Q, 379K successfully induced syncytia formation in the presence of NAEK, and no syncytia was formed without NAEK ( Figure 2F). These results indicated that the effect of substitution by NAEK on function varies with position, and the position of 149 and 185 was labeled in the gB crystal structure ( Figure 2G).

Construction and Rescue of the PTC Site Harboring PRV
Next, we selected 149Q and 185W as potential PTC engineering sites in PRV, pPRV-149Q-TAG and pPRV-185W-TAG were subsequently constructed using pBac-JS2012 and the galK selection system as previously described [15]. Vero cells were co-transfected with

Construction and Rescue of the PTC Site Harboring PRV
Next, we selected 149Q and 185W as potential PTC engineering sites in PRV, pPRV-149Q-TAG and pPRV-185W-TAG were subsequently constructed using pBac-JS2012 and the galK selection system as previously described [15]. Vero cells were co-transfected with pPRV-149Q-TAG or pPRV-185W-TAG clones with plasmids containing orthogonal  Figure 3A). However, pPRV-185W-TAG failed induced syncytia in the presence of UAA ( Figure 3A). The same results were obtained in 293T cells ( Figure 3B). To test whether infectious PRV particles were produced in the presence of UAA, electron microscopic analysis was performed ( Figure 3C). Consistent with our above result, PRV particles were observed only in the pPRV-149Q-TAG transfected group in the presence of UAA. Furthermore, no PRV particles were observed in the control group and pPRV-185W-TAG transfected group ( Figure 3C). Taken together, UAA could be used as a precise switch for controlling PRV-PTC virus replication.
OR PEER REVIEW 7 of 12 pPRV-149Q-TAG or pPRV-185W-TAG clones with plasmids containing orthogonal translation systems to rescue the PRV-PTC virus. At 48 h post-transfection, typical PRV-induced syncytia were formed in cells transfected with pPRV-149Q-TAG in the presence of UAA. No syncytia were formed without UAA ( Figure 3A). However, pPRV-185W-TAG failed induced syncytia in the presence of UAA ( Figure 3A). The same results were obtained in 293T cells ( Figure 3B). To test whether infectious PRV particles were produced in the presence of UAA, electron microscopic analysis was performed ( Figure 3C). Consistent with our above result, PRV particles were observed only in the pPRV-149Q-TAG transfected group in the presence of UAA. Furthermore, no PRV particles were observed in the control group and pPRV-185W-TAG transfected group ( Figure 3C). Taken together, UAA could be used as a precise switch for controlling PRV-PTC virus replication.

Generation of MbpylRS/tRNA Pyl Pair Delivery Vector and Reporter Adenovirus
An efficient method to incorporate UAA into the viral PTC site is to generate a stable transgenic cell line harboring MbpylRS/tRNA Pyl pair in the host genome. Lentiviral vector and PiggyBac transposon system are powerful tools to generate stable cell lines [12,14]. However, the PiggyBac transposon system has the advantage of delivering large and complex DNA fragments into the genome of mammalian cells [23]. Therefore, in this study, we used the PiggyBac transposon system to deliver the MbpylRS/tRNA Pyl cassette. First, a PiggyBac transposon vector, pB513B-puro-MbpylRS-12tRNA, was constructed. It contained MbpylRS, which was promoted by chicken β-actin promoter, and 12 tandem tRNAexpression cassettes promoted by U6 or H1 promoters ( Figure 4A). To test whether this vector work normally, pB513B-puro-MbpylRS-12tRNA plasmid co-transfected with an amber codon-containing green fluorescent protein (GFP 39TAG ) reporter plasmid present, with or without UAA ( Figure 4B). The results showed that functional GFP was visualized in the presence of UAA ( Figure 4C), indicating pB513B-puro-MbpylRS-12tRNA was suc-

Generation of MbpylRS/tRNA Pyl Pair Delivery Vector and Reporter Adenovirus
An efficient method to incorporate UAA into the viral PTC site is to generate a stable transgenic cell line harboring MbpylRS/tRNA Pyl pair in the host genome. Lentiviral vector and PiggyBac transposon system are powerful tools to generate stable cell lines [12,14]. However, the PiggyBac transposon system has the advantage of delivering large and complex DNA fragments into the genome of mammalian cells [23]. Therefore, in this study, we used the PiggyBac transposon system to deliver the MbpylRS/tRNA Pyl cassette. First, a PiggyBac transposon vector, pB513B-puro-MbpylRS-12tRNA, was constructed. It contained MbpylRS, which was promoted by chicken β-actin promoter, and 12 tandem tRNA-expression cassettes promoted by U6 or H1 promoters ( Figure 4A). To test whether this vector work normally, pB513B-puro-MbpylRS-12tRNA plasmid co-transfected with an amber codon-containing green fluorescent protein (GFP 39TAG ) reporter plasmid present, with or without UAA ( Figure 4B). The results showed that functional GFP was visualized in the presence of UAA ( Figure 4C), indicating pB513B-puro-MbpylRS-12tRNA was successfully constructed. To construct transgenic cells without reporter genes, we generate a recombinant adenovirus harboring GFP 39TAG ( Figure 4D). Recombinant adenovirus was confirmed in RK13 cells, which were first transfected with pB513B-puro-MbpylRS-12tRNA plasmid, then infected with recombinant adenovirus. The results showed that functional GFP was visualized in cells supplemented with UAAs, indicating that recombinant adenovirus was successfully generated ( Figure 4E).

FOR PEER REVIEW 8 of 12
recombinant adenovirus harboring GFP 39TAG ( Figure 4D). Recombinant adenovirus was confirmed in RK13 cells, which were first transfected with pB513B-puro-MbpylRS-12tRNA plasmid, then infected with recombinant adenovirus. The results showed that functional GFP was visualized in cells supplemented with UAAs, indicating that recombinant adenovirus was successfully generated ( Figure 4E).

Generation of Stable Cell Line Harboring GCE Machinery
To generate stable transgenic RK13, ST, and PK15 cell lines harboring GCE machinery, we co-transfected pB513B-puro-MbpylRS-12tRNA with pB220PA-1 (a vector expressing the PiggyBac transposase) together ( Figure 4A). 48 h post-transfection, puromycin (4 µg/mL) was added. Two weeks later, puromycin-resistant cells were infected with reporter adenovirus in the presence of UAA, and GFP expressing single cells were further sorted by fluorescence-activated cell sorting technology (FACS) ( Figure 5A). A single-cell of transgenic RK13, ST, PK15 cells was cultivated, to increase over approximately 2-3 weeks. Next, these cell lines were confirmed by infecting with GFP 39TAG adenovirus in the

Generation of Stable Cell Line Harboring GCE Machinery
To generate stable transgenic RK13, ST, and PK15 cell lines harboring GCE machinery, we co-transfected pB513B-puro-MbpylRS-12tRNA with pB220PA-1 (a vector expressing the PiggyBac transposase) together ( Figure 4A). 48 h post-transfection, puromycin (4 µg/mL) was added. Two weeks later, puromycin-resistant cells were infected with reporter adenovirus in the presence of UAA, and GFP expressing single cells were further sorted by fluorescence-activated cell sorting technology (FACS) ( Figure 5A). A single-cell of trans-genic RK13, ST, PK15 cells was cultivated, to increase over approximately 2-3 weeks. Next, these cell lines were confirmed by infecting with GFP 39TAG adenovirus in the presence of UAA, or not. The GFP 39TAG expressed well in these cells in the presence of UAA ( Figure 5B). The results demonstrated that MbpylRS/tRNA Pyl pair could be successfully delivered by the PiggyBac transposon system. To our surprise, unlike previous reports [12,14], all our transgenic cell lines are extremely unstable along with the increased passage. An overexpression assay was performed to test which element was lost in these cells. By transfecting MbpylRS, GFP 39TAG or tRNA Pyl individually or together, and by co-transfection together, the group was used as a positive control ( Figure 5C). Our result revealed that the tRNA Pyl and GFP 39TAG co-transfection group restored robust and efficient GFP 39TAG expression in the presence of UAA. However, the GFP 39TAG transfection group and MbpylRS and GFP 39TAG co-transfection group failed to restore efficient GFP 39TAG expression in the presence of UAA ( Figure 5C). Thus, we concluded that the expression of orthogonal tRNA is the key limiting factor in generating a stable cell line.

Discussion
In recent decades, GCE technology has been widely used to engineer PTC sites in essential genes to control different kinds of virus replication, such as HDV, HIV, Zika, FMDV [13,[24][25][26][27]. Application of GCE technology in Influenza A virus makes it wellknown as a novel tool for vaccine development [14]. In this study, we generated PTC harboring PRV, and the results suggested that PRV-PTC virus was successfully rescued in the presence of orthogonal system MbpylRS/tRNA Pyl pair and UAA. This study was the To our surprise, unlike previous reports [12,14], all our transgenic cell lines are extremely unstable along with the increased passage. An overexpression assay was performed to test which element was lost in these cells. By transfecting MbpylRS, GFP 39TAG or tRNA Pyl individually or together, and by co-transfection together, the group was used as a positive control ( Figure 5C). Our result revealed that the tRNA Pyl and GFP 39TAG co-transfection group restored robust and efficient GFP 39TAG expression in the presence of UAA. However, the GFP 39TAG transfection group and MbpylRS and GFP 39TAG co-transfection group failed to restore efficient GFP 39TAG expression in the presence of UAA ( Figure 5C). Thus, we concluded that the expression of orthogonal tRNA is the key limiting factor in generating a stable cell line.

Discussion
In recent decades, GCE technology has been widely used to engineer PTC sites in essential genes to control different kinds of virus replication, such as HDV, HIV, Zika, FMDV [13,[24][25][26][27]. Application of GCE technology in Influenza A virus makes it wellknown as a novel tool for vaccine development [14]. In this study, we generated PTC harboring PRV, and the results suggested that PRV-PTC virus was successfully rescued in the presence of orthogonal system MbpylRS/tRNA Pyl pair and UAA. This study was the first to demonstrate that UAA was incorporated into PRV gB protein by GCE technology. However, efficiency was generally low. Western blot showed different degrees of weak protein expression in 149Q, 169K, 185W, 206Q, 379K PTC sites, indicating that the incorporation of UAA may have a site preference ( Figure 2B). However, only a few of these manifested the UAA-dependent gB mediated cell-to-cell fusion phenotype ( Figure 2F). So we speculated that UAA incorporation in target proteins might have deleterious effects on gB function for some PTC sites [28]. More PRV essential genes and potential PTC sites should be screened in the future to obtain ideal PTC sites with efficient, site-specific incorporation of UAAs into PRV.
According to the Western blot and cell-to-cell fusion assay, we chose 149Q and 185W as potential sites to engineer amber codon TAG based on the pPRV-Bac infectious clone and successfully generated pPRV-149Q-TAG or pPRV-185W-TAG PTC virus. pPRV-149Q-TAG PTC virus was successfully rescued in the presence of UAA, demonstrating that GCE technology could control PRV PTC replication in vitro. Unfortunately, in our study the viral titer is extremely low, making it difficult to perform animal experiments in order to evaluate its efficacy as a potential vaccine. pPRV-185W-TAG PTC virus failed to rescue in the presence of UAA. We envisaged that the surrogate of tryptophan at position 185 by UAA might destroy other functions besides a cell-to-cell fusion of gB protein. Therefore, we concluded that the structure of UAA and the incorporation sites might influence the protein function.
Previous reports have demonstrated that the lentivirus vector [13,14] or PiggyBac transposon system successfully constructed stable cell lines harboring the orthogonal translation system [12,13]. Efficient incorporation of UAA requires multiple copies of tRNA Pyl [29], which makes it difficult to package lentivirus efficiently [30]. The PiggyBac transposon system is characterized by rapid and efficient integration of large and complex sequences into mammalian cells' genomes [23,31]. Therefore, this study used the PiggyBac transposon system to generate cell lines containing orthogonal translation machinery. Unfortunately, unlike the previous reports [12][13][14], all our attempts failed to generate a stable orthogonal MbpylRS/tRNA Pyl system in the current work. The result indicated that insufficient orthogonal tRNA Pyl copies were the limitation steps. Wolfgang H. Schmied et al. developed an optimized pyrrolysyl-tRNA synthetase/tRNA CUA expression system and engineered eukaryotic release factor subunit 1 (eRF1) to efficient incorporate UAA in mammalian cells [29]. Future work will investigate the optimized approaches to efficiently incorporating UAAs at PTC sites in eukaryotic cells.

Conclusions
In conclusion, we demonstrated that an orthogonal translation system-mediated amber codon suppression strategy could precisely control PRV-PTC virus replication. To our knowledge, this is the first study reported for herpesvirus generated by GCE technology. However, there are still many challenges remaining to be addressed. Our further work will establish transgenic cell lines with high-efficiency expression of the orthogonal translation system.