Novel Function of Avian p53 in Binding to ALV-J LTR Contributes to Its Antiviral Roles

Abstract

Accumulating evidence suggests that p53 is involved in viral infection. However, it remains elusive whether avian p53 orchestrates avian leukosis virus (ALV) replication. We showed that p53 recruits the histone deacetylase 1 and 2 (HDAC1/2) complex to the ALV promoter to shut off ALV's promoter activity and viral replication. HDAC1/2 binding to the ALV promoter was abolished in the absence of p53. Moreover, we collected samples in ALV-infected chickens and found that the acetylation status of ALV-bound H3 and H4 histones correlated with ALV viremia. HDAC inhibitors (HDACi) potently increase ALV replication, but HDACi-promoted viral replication is dramatically reduced in cells with p53 depletion. These data demonstrate that p53 is critical for inhibition ALV replication and suggest that future studies of ALV replication need to account for the potential effects of p53 activity. IMPORTANCE Rous sarcoma virus (RSV)/ALV was the first retrovirus to be discovered, which was really the first hint that cancer, or a tumor, could be transmitted by a virus. The specific mechanisms that regulate ALV replication during infection remain poorly understood. Here, we show that avian p53 and HDAC complex inhibit ALV promoter activity and replication, and p53 inhibits ALV replication through binding to the ALV promoter. We demonstrated that the acetylation status of ALV-bound H3 and H4 histones correlates with ALV viremia level using clinical samples collected from commercial poultry. These findings identify both p53-mediated inhibition on ALV replication and a potential role for virus-induced tumorigenesis.

Keywords: histone deacetylase; p53; viral replication.

FIG 1

p53 inhibits ALV-J promoter activity. (A) p53 overexpression inhibits ALV-J LTR promoter activity in a dose-dependent manner. (B) ALV-J LTR promoter activity was increased in DF-1 cells with depletion of endogenous p53. (C) Depletion of endogenous p53 prevents recruitment of HDAC1 and HDAC2 to the ALV-J promoter. DF-1 cells were transfected with ALV-J LTR-mediated reporter followed by ChIP assays. Each ChIP DNA fraction's threshold cycle (C_T) value was normalized to the IgG DNA fraction's C_T value (ΔΔCT) at the same time point. IgG is defined as 1. Error bars stem from three technical replicates; n = 3. (D) Re-ChIP PCR assay of p53-HDAC1/2 interaction at ALV-J LTR promoter in DF-1 cells after ALV-J LTR-mediated reporter transfection. First-round ChIP (1st antibody [Ab]) against p53 (P3), HDAC1 (H1), HDAC2 (H2), or IgG (Ig). Eluted samples were subject to re-ChIP (2nd antibody). Lane 2 was input (in). (E, Left) siRNA-mediated knockdown of HDAC1 in DF-1 cells was performed and confirmed by Western blot analysis. (E, Middle) DF-1 cells were then transfected with an ALV-J LTR-mediated reporter. (E, Right) ALV-J infection and replication in control and siRNA-mediated knockdown of HDAC1 in DF-1 cells. ALV-J replication was measured on day 6 postinfection by p27 ELISA. Error bars represent ± SD for triplicate experiments. (F, Left) siRNA-mediated knockdown of HDAC2 in DF-1 cells was performed and confirmed by Western blot analysis. (F, Middle) DF-1 cells were then transfected with an ALV-J LTR-mediated reporter. (F, Right) ALV-J infection and replication in control and siRNA-mediated knockdown of HDAC2 in DF-1 cells. ALV-J replication was measured on day 6 postinfection by p27 ELISA. Error bars represent ± SD for triplicate experiments. (G) p53 and ALV-J LTR-mediated reporter overexpression in siRNA-mediated knockdown of HDAC1 or HDAC2 in DF-1 cells. Thirty-six hours after transfection, cells were harvested and lysed for assessment of luciferase activity.

FIG 2

p53 binds to ALV-J LTR. (A, Top) Diagram of ALV-J long terminal repeat (LTR) and DNA fragments used in electrophoretic mobility shift assay (EMSA). The shaded boxes indicate the EMSA probe-corresponding fragment. The size and location of DNA fragments used in the EMSA are indicated. (A, Bottom) EMSA of in vitro binding of p53 to probes A and C but not probe B corresponding to the LTR region. Nuclear extract from Hep3B cells transfected with a plasmid expressing avian p53 protein (NEP53) was used for EMSA and band shift of protein-DNA was abolished with excessive nonlabeled probe (NLP). Nuclear extract from Hep3B cells transfected with empty plasmid was the negative control (NEC). Lane 1, NEC with probe A; lane 2, NEC with probe B; lane 3, NEC with probe C; lane 4, NEP53 with probe A; lane 5, NEP53 with probe B; lane 6, NEP53 with probe C; lane 7, NEP53 with probe A and NLP corresponding to probe A; lane 8, NEP53 with probe B and NLP corresponding to probe B; lane 9, NEP53 with probe B and NLP corresponding to probe C. (B, Left) EMSA of binding of avian p53 to probe A region in ALV-J LTR. Lane 1, probe A; lane 2, NEC with probe A; lane 3, NEP53 with probe A; lane 4, supershift with IgG in NEP53 with probe A; lane 5, supershift with p53 antibody (PAb 240); lane 6, NEP53 with probe A and NLP corresponding to probe A. (B, Right) EMSA of avian p53 binding to probe C region in ALV-J LTR. Lane 1, probe C; lane 2, NEC with probe C; lane 3, NEP53 with probe C; lane 4, supershift band with IgG in NEP53 with probe C; lane 5, supershift with p53 antibody (PAb 240); lane 6, NEP53 with probe C and NLP corresponding to probe C.

FIG 3

p53 inhibits ALV-J replication. (A) ALV-J infection and replication in control and p53 knockout CEF cells. ALV-J replication was measured over 5 days by p27 ELISA. Error bars represent ± SD for triplicate experiments. (B) ALV-J infection and replication in control and p53 knockout DF-1 cells. Reexpression of p53 in control and p53 knockout DF-1 cells. ALV-J replication was measured over 10 days by p27 ELISA. Error bars represent ± SD for triplicate experiments.

FIG 4

Acetylation status of ALV-J genome-bound H3 and H4 histones correlates with ALV-J viremia levels in chickens in clinical settings. (A, Left) ChIP of liver nuclear extracts from 6 ALV-J-positive chickens with ALV-J infection by using specific antibodies to AcH3, AcH4, HDAC1, or control IgG, and specific PCR primes for ALV-J promoter. (A, Right) ALV-J quantification in the serum samples from 12 ALV-J-positive chickens with active (AcH3-AcH4 positive/HDAC1 negative, 4 cases; AcH3-AcH4 positive/HDAC1 positive, 3 cases) or suppressed (AcH3-AcH4 negative/HDAC1 positive, 5 cases) ALV-J replication was performed by ALV-J proviral genome copies quantitative analysis by quantitative PCR (qPCR). (B) ALV-J LTR-mediated reporter overexpression in DF-1 cells with 1 μM SAHA treatment. Thirty-six hours after transfection, cells were harvested and lysed for assessment of luciferase activity. (C, Left) ALV-J infection and replication in control and p53 knockout DF-1 cells with SAHA treatment. ALV-J replication was measured over 10 days by p27 ELISA. Error bars represent ± SD for triplicate experiments. (C, Middle) p53 expression level in each group was confirmed by Western blot analysis. (C, Right) p27 levels in the supernatant at 6 days postinfection (dpi) were analyzed to determine the SAHA-mediated regulation fold on ALV-J replication in control and p53 knockout DF-1 cells.