Duck Plague Virus pUL48 Protein Activates the Immediate-Early Gene to Initiate the Transcription of the Virus Gene

Abstract

Duck plague caused by the duck plague virus (DPV) is an infectious disease that seriously harms the waterfowl breeding industry. The VP16 protein of α herpesvirus can bind to specific cis-acting elements upstream of the promoter of the immediate-early (IE, α) gene to promote the transcription of the IE gene, so it is also called the trans-inducer of IE gene (α-TIF). However, no studies on DPV α-TIF have been reported. This study investigated the DPV pUL48, a homolog of HSV-1 VP16, transcriptional activation region, target sequence, and viral protein affecting its transcriptional activation using a dual-luciferase reporter gene detection system, and pUL48 was identified as the α-TIF of DPV. (1) The regulation of pUL48 on DPV different gene promoters showed that pUL48 could activate all the promoters of IE genes (ICP4, ICP22, and ICP27) but not the promoters of early and late genes. (2) The activity of pUL48 to ICP4 and ICP22 promoters with different upstream lengths showed that pUL48 activated ICP4 and ICP22 promoters by acting on TAATGA (T) TAT element upstream of ICP4 promoter and TAATTATAT element upstream of ICP22 promoter, respectively. (3) Transcriptional activation of IE gene by truncated proteins of different lengths at the N-terminal of pUL48 was detected. The results showed that the transcriptional activation domain of pUL48 was amino acids 1-60 at the N-terminal, and amino acids 1-20 was its core region. In addition, it was found that pUL14, pUL46, and pUL47 significantly promoted the transcriptional activation of pUL48. The effects of loss of pUL47 and its nuclear localization signal on the nuclear entry and transcriptional activation function of pUL48 were further examined. The results showed that pUL47 could promote the nuclear entry of pUL48 through its nuclear localization signal at positions 40-50 and 768-777 amino acids, thus, enhancing the transcriptional activation function of pUL48 and synergistic promotion of viral gene transcription.

Keywords: duck plague virus; immediate early genes; pUL48; transcriptional activation domain; transcriptional activation function.

FIGURE 1

Regulation of pUL48 on duck plague virus (DPV) gene promoters of different types. (A) Expression of pCAGGS-pUL48-HA in DEF. pCAGGS and pCAGGS-pUL48-HA were transfected into DEF, respectively, and cell protein samples were collected at 12, 24, and 36 h for Western blot. (B) The regulation of pUL48 on the IE gene promoter of DPV. (C) The regulation of pUL48 on DPV early gene promoters. (D) The regulation of pUL48 on DPV late gene promoter. (E) Dose-dependent activation of pUL48 on ICP4-Pro (1,235 bp). pGL4-basic, ICP4-Pro-Luc, ICP22-Pro-Luc, ICP27-Pro-Luc, UL23-Pro-Luc, UL28-Pro-Luc, UL15-Pro-Luc, UL44-Pro-Luc, and UL48-Pro-Luc were cotransfected into DEF with pCAGGS, pCAGGS-pUL48-HA, and pRL-TK/0.05, 0.1, 0.2, 0.3, and 0.4 μg pCAGGS-pUL48-HA were cotransfected into DEF with pCAGGS, ICP4-Pro (1,235 bp)-Luc and pRL-TK. Except for the reference plasmid pRL-TK, the transfection ratio of other plasmids was 1:1:1, and the transfection quantity of pRL-TK was 1/20 of the double luciferase plasmids; pCAGGS was a blank group. Cell samples were collected 24 h after transfection. The activity of each promoter was detected by a dual-luciferase reporting system. The corresponding concentration of pCAGGS-pUL48-HA was transfected into DEF. At 24 h after transfection, cell protein samples were collected for Western blot analysis. Student’s t-test was used to analyze the differences of the two groups. The significance was as follows: NS showed no difference, *p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

FIGURE 2

Regulation of pUL48 on promoters of different lengths of intermediate-early (IE) gene. The regulation of pUL48 on ICP4/ICP22/ICP27 promoters of different lengths. ICP4-Pro (740 bp)-Luc, ICP4-Pro (1,235 bp)-Luc, ICP22-Pro (900 bp)-Luc, ICP4-Pro (1,200 bp)-Luc, ICP4-Pro (1,500 bp)-Luc, ICP27-Pro (100 bp)-Luc, and ICP27-Pro (1,500 bp)-Luc were cotransfected into DEF with pCAGGS and pCAGGS-pUL48-HA and pRL-TK, respectively. The transfection ratio of pCAGGS and pCAGGS-pUL48-HA with each double luciferase plasmid was 1:1, and the transfection quantity of pRL-TK was 1/20 of each double luciferase plasmid. Cell samples were collected 24 h after transfection. The activity of each promoter was detected by the dual-luciferase reporting system. pCAGGS-pUL48-HA was transfected into DEF. At 24 h after transfection, cellular protein samples were collected for Western blot analysis. Student’s t-test was used to analyze the differences of the two groups. The significance was as follows: *p < 0.05, ^***p < 0.001, ^****p < 0.0001.

FIGURE 3

The regulatory effects of pUL48 on ICP4 and ICP22 promoters containing different components. (A) Initial upstream cis-element sequence analysis of ICP4 and ICP22. Different colored squares represent different possible sequences of cis-elements. (B) The regulation of pUL48 on ICP4 promoter containing different elements. (C) The regulation of pUL48 on ICP22 promoter containing different elements. ICP4-Pro (740 bp)-Luc, ICP4-Pro (900 bp)-Luc, ICP4-Pro (1,226 bp)-Luc, ICP4-Pro (1,235 bp)-Luc, ICP22-Pro (300 bp)-Luc, ICP22-Pro (400 bp)-Luc, ICP22-Pro (900 bp)-Luc, pCAGGS, pCAGGS-pUL48-HA, and pRL-TK were cotransfected into DEF. At 24 h after transfection, cell samples were collected, and the activity of each promoter was detected by the dual-luciferase reporting system. pCAGGS-pUL48-HA was transfected into DEF. At 24 h after transfection, cell protein samples were collected for Western blot analysis. Student’s t-test was used to analyze the differences of the two groups. NS was not significant, ^****p < 0.0001.

FIGURE 4

Effect of pUL48-truncated protein on ICP4 promoter activity. (A) Structure diagram of N-terminal-truncated protein of pUL48. (B) Regulatory effects of pUL48 N-terminal-truncated protein on ICP4-Pro (1,235 bp). The pUL48 N-terminal-truncated plasmid was cotransfected with ICP4-Pro (1,235 bp) and pRL-TK into DEF. At 24 h after transfection, cell samples were collected, and the activity of each promoter was detected by the dual-luciferase reporting system. The pUL48 N-terminal-truncated plasmid was transfected into DEF. At 24 h after transfection, cell protein samples were collected for Western blot analysis. Student’s t-test was used to analyze the control and the differences of each experimental group. ***p < 0.001, ****p < 0.0001.

FIGURE 5

Identification of the core region of the DPV pUL48 transcriptional activation domain. The regulation of ICP4/ICP22/ICP27 promoter by a deletion in different regions of the pUL48 transcriptional activation domain. The N-terminal-truncated plasmids of pUL48 were cotransfected into DEF with ICP4-Pro (1,235 bp), ICP22-Pro (900 bp), ICP27-Pro (1,500 bp), and pRL-TK. pCAGGS was the negative control, and pCAGGS-pUL48-HA was the positive control. Cell samples were collected 24 h after transfection, and the activity of each promoter was detected by a dual-luciferase reporting system. The pUL48 N-terminal-truncated plasmid was transfected into DEF. At 24 h after transfection, cellular protein samples were collected for Western blot analysis. Student’s t-test was used to analyze the differences of the two groups, and the significance was as follows: ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

FIGURE 6

Effect of overexpression of pUL48 and its truncated protein on viral IE gene transcription. The relative transcription level of the IE gene at 4/8/12 h after infection with MOI = 1 DPV-CHv; pCAGGS, pCAGGS-pUL48-HA, pCAGGS-pUL48 (Δ1–20 aa)-HA, and pCAGGS-pUL48 (Δ1–60 aa)-HA were transfected into a 12-well plate DEF, respectively. At 12 h after transfection, DEF was infected with DPV CHv strain with MOI = 1. The virally infected cells were collected 4, 8, and 12 h after infection into the non-RNA enzyme EP tube using RNAiso Plus. According to the instructions of the RNA extraction kit, reverse transcription reagent and SYBR^®Premix Ex Taq™ II Kit, RNA extraction, reverse transcription, and quantitative fluorescence PCR were performed successively to detect the mRNA levels of IE gene ICP4, ICP22, and ICP27. The expression of each truncated protein of pUL48 was detected by WB. 1, pCAGGS; 2, pUL48; 3, pUL48 (Δ1–20 aa); 4: pUL48 (Δ1–60 aa). Relative quantitative test results were processed using 2^{– ΔΔ}Ct, and the results were normalized to the control group. Student’s t-test was used to analyze the experimental and control groups differences. *p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

FIGURE 7

Screening of viral proteins affecting the transcriptional activation of pUL48. (A) Various viral proteins affected the activation of pUL48 on ICP4-Pro (1,235 bp). The Mock: pCAGGS + ICP4-Pro (1,235 bp); Control: pUL48 + ICP4-Pro (1,235 bp); ICP22: ICP22 + pUL48 + ICP4-Pro (1,235 bp). The other groups were set up in the same way as ICP22. Will pGL4-ICP4-Pro(1235 bp)-Luc, reference plasmids pRL-TK, and pCAGGS-pUL48-HA were cotransfected with 26 eukaryotic expression plasmids of DPV protein, including pCAGGS-UL2-Flag and pCAGGS-UL47-Flag, respectively (the control group was transfected with the same amount of pCAGGS without loading), and the samples were collected at 24 h. The relative fluorescence intensity of the transfected virus protein group and the control group was detected and compared with screen out, the viral target protein that affected the transcriptional activation function of pUL48. (B) The activation of ICP4 and ICP22 promoters by pUL48 was regulated by pUL14/pUL46/pUL47/pUL49. It was verified that the target protein did not affect the IE gene promoter but regulated the transcriptional activation function of pUL48. Student’s t-test was used to analyze the differences of the two groups. *p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

FIGURE 8

Effects of pUL47 NLS on transcriptional activation of pUL48. (A) Cell localization of pUL48. (B) The effect of pUL47 on the nucleation of pUL48. (C) The influence of pUL47-Δ40–50 aa on pUL48 localization. (D) The influence of pUL47-Δ768–777 aa on pUL48 localization. (E) The influence of pUL47-Δ40–50 and 768–777 aa on pUL48 localization. First, pCAGGS-pUL48-HA was transfected separately. Then pCAGGS-pUL48-HA, pCAGGS-pUL47-Flag, pcDNA3.1-pUL47-Δ40–50 aa, pcDNA3.1-pUL47-Δ768–777 aa, and pcDNA3.1-pUL47-Δ40–50 and 768–777 aa with NLS deletion plasmids were cotransfected according to corresponding experimental groups, and indirect immunofluorescence assay was performed. (F) Effect of pUL47 NLS deletion on ICP4-Pro (1,235 bp) activation of pUL48. pCAGGS, pCAGGS-pUL48-HA, pCAGGS-UL47-Flag, pcDNA3.1-pUL47-Δ40–50 aa, pcDNA3.1-pUL47-Δ768–777 aa, and pcDNA3.1-pUL47-Δ40–50 and 768–777 aa were cotransfected into DEF with pGL4-ICP4-Pro (1,235 bp)-Luc and pRL-TK, except the reference plasmid pRL-TK. The transfection ratio of other plasmids with pGL4-ICP4-Pro (1,235 bp)-Luc was 1:1, and the transfection quantity of pRL-TK was 1/20 of pGL4-ICP4-Pro (1,235 bp)-Luc. Cell samples were collected 24 h after transfection, and ICP4-Pro (1,235 bp) promoter activity was detected by a dual-luciferase reporting system. Student’s t-test was used to analyze the differences of the two groups; *p < 0.05.