The ILTV strain ILTV-LJS09 propagated in leghorn male hepatoma (LMH) cells was described in previous studies (Kong et al., 2013; Xu et al., 2022). Dulbecco’s modified Eagle’s medium (DMEM) was used to sustain LMH cells (ATCC CRL-2117), and 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 g/mL streptomycin, and 2 mM L-glutamine were supplemented. Cell cultures were maintained at 37°C with 5% CO₂. The final concentration of PFT-α (Sigma-Aldrich Corporation) utilized was 20 μM after being dissolved in DMSO (Sigma-Aldrich Corporation). No more than 0.1% of DMSO was added in cell culture in all groups, including the control group.

Three short-interfering RNAs (siRNAs) that precisely targeting chicken TP53 mRNA sequence (NM_205264; sip53-1, 5′-AGG AGG AGA ACU UCC GCA A-3′; sip53-2, 5′-GCG UGG CUA AGC GAG CCA U-3′; sip53-3, 5′-GCU GCU UCG AGG UGC GCG U-3′) were synthesized. A siRNA with no target site in any chicken mRNA (sicontrol, 5′-GCA CUU GAU ACA CGU GUA A-3′) was used as control. All siRNAs were ordered from Sigma-Aldrich Corporation. siRNA transfection was performed with a Lipofectamine^® 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA).

The recombinant plasmid Flag-chp53 was provided by Dr. Zhiyong Ma and Dr. Yafeng Qiu (Shanghai Veterinary Research Institute, CAAS). The transfection was carried out as described in our previous study (Chen et al., 2022), using Turbofect transfection reagent (Thermo Fisher Scientific, Rockford, IL).

Total RNA was extracted from LMH cells using RNAiso Plus (Takara Biotechnology, Dalian, China) at the given time points after infection, in accordance with the manufacturer’s procedure. Cells were infected with ILTV at a multiplicity of infection (MOI) of 1. A microvolume spectrophotometer was used to assess the concentration and quality of total RNA (Implen GmbH, Munich, Germany). The program Oligo 7 was used to create the primer sequences (version 7.6.0, Molecular Biology Insights, Inc., Colorado Springs, CO). The sequences of primers are presented in Supplementary Table 1. A One Step TB Green^® PrimeScript™ RT-PCR Kit II was used to for real-time quantitative PCR (RT-qPCR) (Takara Biotechnology, Dalian, China). Each sample was measured three times. By 2^–ΔΔCt approach, the relative quantification of target gene expression was computed using β-actin as the endogenous control, and the data are shown as the log₂ fold change. With three technical duplicates for every reaction, at least three separate experiments were carried out.

Plaque tests and ILTV-specific RT-qPCR assays were used to measure the amounts of viral replication, as previously described (Qiao et al., 2020). AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit was used for genomic DNA extraction (Axygen, Union City, CA, USA). Using Luna Universal qPCR Master Mix (NEB, Ipswich, MA, USA), absolute RT-qPCR was carried out in accordance with the manufacturer’s instructions. pMD18-T plasmid containing gC genes of ILTV was used as standards. Three or more independent experiments were performed for each experiment.

Western blot was performed strictly according to previously described procedures (Chen et al., 2022). Briefly, cells were washed with ice-cold PBS and soluble proteins were extracted with RIPA Lysis Buffer (Strong) (Beyotime Biotech, Shanghai, China) according to the manufacturer’s protocol. The protein concentration of each sample was determined by a BCA Kit (Beyotime Biotech, Shanghai, China). An equal amount of protein was separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Millipore, Billerica, MA). The membrane was then blocked with 5% non-fat milk for 2 h at room temperature and incubated with primary antibodies overnight at 4°C. Antibodies against P53 (Santa Cruz Biotechnology, Santa Cruz, USA) and β-actin (Proteintech, Wuhan, China) were used. Signals were visualized using infrared imaging systems (Odyssey CLX, LiCor Biosciences, Lincoln, NE).

The p53 inhibitor PFT-α (Sigma-Aldrich Corporation) were used at 20 μM before ILTV infection. Control groups were treated with DMSO at the same volumes. After the administration of PFT-α for 12 h, Cells were infected with ILTV at a multiplicity of infection (MOI) of 1 and cells were collected at 12 h post infection (hpi). Annoroad Gene Technology Co., Ltd. used RNA deep sequencing to undertake genome-wide gene expression profiling of LMH cells (Beijing, China). The Illumina platform (Illumina, Inc., San Diego, CA, USA) was used to build the library in accordance with the manufacturer’s guidelines. With the use of an Illumina HiSeq 2500 device, the samples were sequenced. There were four biological repetitions.

The web-based software Galaxy was used to analyze the results of RNA sequencing (Blankenberg et al., 2010). The DESeq R package was used to analyze DEGs and the read-count data obtained by analyzing gene expression levels were standardized. The hypothetical probability (p-value) was calculated according to the negative binomial distribution model. The screening criteria for DEGs were p-value < 0.05 and | log₂ (fold change) | > 1. KEGG pathway enrichment analysis was used to determine the primary functions of DEGs using DAVID (gene-enrichment analysis using EASE Score, a modified Fisher exact p-value, as the threshold) (Huang et al., 2009). Additionally, a corrected p-value < 0.05 was the threshold for a statistically significant correlation. RNA sequencing raw data were uploaded to the National Center for Biotechnology Information database (GSE193188).

ChIP assays were performed according to previous description (Chen et al., 2022). For each sample, 5 × 10⁶ cells were collected. A total of 5 μg of anti-flag (GenScript, Piscataway, NJ, USA) or isotype control IgG2b antibody (Cell Signaling Technology, Danvers, Massachusetts, USA) was used in each sample. Pull-down was performed using Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology, Santa Cruz, USA). A QIAquick PCR Purification Kit was used to purify the immunoprecipitated DNA (QIAGEN, Valencia, CA). Using Luna Universal qPCR Master Mix (NEB, Ipswich, MA, USA), ChIP followed by Quantitative PCR (ChIP-qPCR) was carried out according to previous description (Chen et al., 2022). The sequences of the primers are shown in Supplementary Table 2. Three biological repetitions were performed.

All statistical analyses were performed using the SPSS software suite (SPSS for Windows version13.0, SPSS Inc., Chicago, IL, USA). The mean ± standard deviation (SD) was used to present most data. The significance of differences between two groups was determined using two-tailed Student’s t-test.

P53 was knocked down in LMH cells using siRNAs before ILTV infection to clarify the impact of p53 on ILTV replication. Western blotting and RT-qPCR was performed to assess the knockdown efficacy (Figure 1A). P21, MDM2, and GADD45A have been identified as p53 target genes experimentally in chicken (Deng et al., 2010; Yang et al., 2013). Their transcription were detected by RT-qPCR to determine p53 transcriptional activity. β-actin was served as inner control. Upon p53 knockdown, the transcription of these p53 target genes were all repressed, suggesting successful inhibition of p53 transcriptional activity by knockdown (Figure 1A). The inhibition of p53 transcriptional activity by PFT-α was evidenced by RT-qPCR, which revealed significant downregulation of three well-known p53 target genes, p21, GADD45A, and MDM2, by the administration of PFT-α (Figure 1A). In LMH cells 6 h post infection (hpi) with ILTV, p53 knockdown significantly reduced the transcription of the immediate-early gene (IEG) ICP4, the early genes (EG) ICP27, VP16, and gC, the early/late gene (E/LG) gI, and the late gene (LG) gG (Mahmoudian et al., 2012), as determined by RT-qPCR, suggesting that p53 plays a regulatory role in the ILTV gene transcription (Figure 1B). Further, p53 knockdown significantly reduced subsequent viral genome replication and virion production, as determined by viral DNA detection using ILTV-specific RT-qPCR (Figure 2A) in LMH cells at 12 hpi and plaque assays in LMH cells at 3 dpi (Figure 2B), respectively. The titer of infectious virions in LMH cells was 7.77 × 10⁵ PFU/mL at 3 dpi but was 3.8 × 10⁵ PFU/mL, 5.27 × 10⁵ PFU/mL, and 3.00 × 10⁵ PFU/mL in cells upon p53 knockdown.

Next, we conducted the genome-wide transcription analysis to explore the molecular mechanism by which p53 inhibition decreased ILTV replication. Bioinformatics analyses using following criteria found 1,595 genes that were differently expressed between groups: (i) a p-value < 0.05, (ii) | log₂ fold change| > 1. Using these differentially expressed genes (DEGs), hierarchical clustering analysis showed effective clustering of biological replicates. While, the transcription profiles of uninfected cells were clustered separately from those of other cells (Figure 3A). To address the potential biological processes directly regulated by p53 during ILTV infection, further transcriptional analysis with the genes bound by chicken p53 (Chen et al., 2022) was conducted. Among 15,108 p53 bound genes, 438 DEGs were regulated in PFT-α-treated cells, while 983 DEGs were regulated in ILTV-infected cells pretreated with PFT-α; 138 DEGs were regulated in the ILTV treatment group (Figure 3B). With a p-value of 0.05, pathway analysis using DAVID tools identified 20 pathways enriched by these DEGs (Figure 3C). PFT-α repressed 13 of the 20 pathways, and majority of these repressed pathways (8/13) were metabolic pathways such as nucleotide metabolism, pyrimidine metabolism, purine metabolism, and glutathione metabolism, demonstrating that repression of metabolic pathways plays a key part in the antiviral function of p53 inhibition.

Our previous metabolome and transcriptome analysis identified nucleotide metabolism and ATP synthesis as targets of PFT-α during its repression of ILTV replication in LMH cells (Xu et al., 2022). Ten genes related to nucleotide metabolism and 62 genes related to ATP synthesis were screened. As shown in Figures 4A,B, eight of the 10 nucleotide metabolism genes were bound by p53. Comparing with DMSO control group, the transcription of one gene, namely RRM2, among these eight p53 bound genes were repressed by the administration of PFT-α (Figure 4A). Comparing with ILTV-infected cells, there were three p53 bound genes, including RRM2, repressed by the administration of PFT-α in ILTV-infected cells (Figure 4B). The three genes are considered as the putative chicken p53 direct target genes. As shown in Figures 4C,D, 37 of the 62 ATP synthesis related genes were bound by p53. Comparing with DMSO control group, the transcription of four genes, namely NDUFA9, NDUFA4, COX4I1, and ATP5C1, among these 37 p53 bound genes were repressed by the administration of PFT-α (Figure 4C). Comparing with ILTV-infected cells, there were fifteen p53 bound genes, including NDUFA4, COX4I1, and ATP5C1, repressed by the administration of PFT-α in ILTV-infected cells (Figure 4D). The 15 genes are considered as the putative chicken p53 direct target genes. Among these putative chicken p53 direct target genes, three genes involved to nucleotide metabolism and five genes involved to ATP synthesis were selected randomly for validation. After p53 was expressed ectopically in LMH cells, the level of p53 bound to the putative binding sites was determined using chromatin immunoprecipitation followed by qPCR (ChIP-qPCR). The amount of non-specific antibody binding to DNA was measured using ChIP with isotype IgG2b antibody and input DNA was used as the negative control. In cells with ectopic overexpression of chicken p53, ChIP-qPCR revealed increased bindings of the overexpressed protein to the transcriptional regulatory areas of these eight genes (Figure 5A). While, p53 knockdown decreased the transcription of these eight genes as measured by RT-qPCR, suggesting the transcriptional control of these genes by p53 (Figure 5B).