P53 maintains gallid alpha herpesvirus 1 replication by direct regulation of nucleotide metabolism and ATP synthesis through its target genes

Abstract

P53, a well-known tumor suppressor, has been confirmed to regulate the infection of various viruses, including chicken viruses. Our previous study observed antiviral effect of p53 inhibitor Pifithrin-α (PFT-α) on the infection of avian infectious laryngotracheitis virus (ILTV), one of the major avian viruses economically significant to the poultry industry globally. However, the potential link between this antiviral effect of PFT-α and p53 remains unclear. Using chicken LMH cell line which is permissive for ILTV infection as model, we explore the effects of p53 on ILTV replication and its underlying molecular mechanism based on genome-wide transcriptome analysis of genes with p53 binding sites. The putative p53 target genes were validated by ChIP-qPCR and RT-qPCR. Results demonstrated that, consistent with the effects of PFT-α on ILTV replication we previously reported, knockdown of p53 repressed viral gene transcription and the genome replication of ILTV effectively. The production of infectious virions was also suppressed significantly by p53 knockdown. Further bioinformatic analysis of genes with p53 binding sites revealed extensive repression of these putative p53 target genes enriched in the metabolic processes, especially nucleotide metabolism and ATP synthesis, upon p53 repression by PFT-α in ILTV infected LMH cells. Among these genes, eighteen were involved in nucleotide metabolism and ATP synthesis. Then eight of the 18 genes were selected randomly for validations, all of which were successfully identified as p53 target genes. Our findings shed light on the mechanisms through which p53 controls ILTV infection, meanwhile expand our knowledge of chicken p53 target genes.

Keywords: P53; PFT-α; alphaherpesviruses; virus replication; virus-host interactions.

FIGURE 1

P53 is a host determinant of ILTV gene transcription in LMH cells. LMH cells were transiently transfected with p53 siRNA (sip53) or negative control siRNA (sicontrol) for 24 h. (A) The knockdown efficiencies of p53 siRNA were analyzed by immunoblotting and RT-qPCR and the effect of PFT-α on p53 target genes were analyzed by RT-qPCR. (B) The transcription levels of six ILTV genes covering all stages of ILTV transcription, namely ICP4, ICP27, VP16, gC, gI, and gG, in LMH cells were quantitated 6 h after ILTV infection (MOI = 1) using RT-qPCR. The results are presented as the mean ± SD, n = 3. *p < 0.05 and **p < 0.01 indicated the levels of significance.

FIGURE 2

P53 is a host determinant of ILTV replication in LMH cells. (A) At 12 hpi, viral genome copy numbers were determined by ILTV-specific RT-qPCR assays. (B) The number of infectious virions was quantified by plaque assays at 3 dpi. The results are presented as the mean ± SD, n = 3. **p < 0.01 indicates the levels of significance.

FIGURE 3

Genome-wide transcriptome analyses. (A) Hierarchical clustering analysis of 1,595 genes differentially expressed in LMH cells at p-value < 0.05, | log₂fold change | > 1. Columns indicate arrays, and rows indicate genes. Values are normalized by row. Blue indicates repression, and red indicates promotion. (B) Bioinformatic analysis of the genes bound by p53 and differentially expressed in mock cells or ILTV-infected cells upon PFT-α or DMSO treatment. These DEGs of PFT-α treatment group, ILTV treatment group and ILTV-infected cells pretreated with PFT-α intersect with p53 bound genes, respectively. (C) Combined pathway analysis of significantly expressed genes (p-value < 0.05). Red indicates upregulated gene enrichment pathways, blue indicates downregulated gene enrichment pathways, and white represents unchanged pathways.

FIGURE 4

Relationship between genes bound by p53 and key nucleotide metabolizing or ATP synthesis related enzyme genes. (A) Venn diagram showing the intersections of these DEGs (p-value < 0.05) of the administration of PFT-α, nucleotide metabolism related genes and genes bound by p53. (B) Venn diagram showing the intersections of these DEGs (p-value < 0.05) of the administration of PFT-α in ILTV-infected cells, nucleotide metabolism related genes and genes bound by p53. (C) Venn diagram showing the intersections of these DEGs (p-value < 0.05) of the administration of PFT-α, ATP synthesis related genes and genes bound by p53. (D) Venn diagram showing the intersections of these DEGs (p-value < 0.05) of the administration of PFT-α in ILTV-infected cells, ATP synthesis related genes and genes bound by p53.

FIGURE 5

Identification bona fide direct target genes of p53. (A) LMH cells were transiently transfected with Flag-chp53 or empty vector p3xFLAG-CMV-7.1 plasmid (vector) and harvested at 24 h. ChIP-qPCR analysis of the relative p53 occupancy in the promoters of eight putative chicken p53 direct target genes including RRM2, NME2, NME3, ATP5C1, NDUFA4, COX5A, NDUFC2, and NDUFB4 in LMH cells. (B) LMH cells were transiently transfected with p53 siRNA (sip53) or negative control siRNA (sicontrol) for 24 h and harvested. The transcriptional levels of eight putative chicken p53 direct target genes including three nucleotide metabolism related genes and five ATP synthesis related genes were detected by RT-qPCR in LMH cells. The results are presented as the mean ± SD, n = 3. *p < 0.05 and **p < 0.01 indicated the levels of significance.