Histone deacetylase III interactions with BK polyomavirus large tumor antigen may affect protein stability

Abstract

Background: Human polyomavirus BK (BKPyV) causes associated nephropathy and contributes to urinary tract cancer development in renal transplant recipients. Large tumor antigen (LT) is an early protein essential in the polyomavirus life cycle. Protein acetylation plays a critical role in regulating protein stability, so this study investigated the acetylation of the BKPyV LT protein.

Methods: The BKPyV LT nucleotide was synthesized, and the protein was expressed by transfection into permissive cells. The BKPyV LT protein was immunoprecipitated and subjected to LC-MS/MS analysis to determine the acetylation residues. The relative lysine was then mutated to arginine in the LT nucleotide and BKPyV genome to analyze the role of LT lysine acetylation in the BKPyV life cycle.

Results: BKPyV LT acetylation sites were identified at Lys3 and Lys230 by mass spectrometry. HDAC3 and HDAC8 and their deacetylation activity are required for BKPyV LT expression. In addition, mutations of Lys3 and Lys230 to arginine increased LT expression, and the interaction of HDAC3 and LT was confirmed by coimmunoprecipitation.

Conclusions: HDAC3 is a newly identified protein that interacts with BKPyV LT, and LT acetylation plays a vital role in the BKPyV life cycle.

Keywords: Acetylation; BKPyV; HDAC3; Large T antigen; Protein stability.

Fig. 1

Acetylated residues in BKPyV LT were determined by mass spectrometry. A Immunoprecipitation of BKPyV LT protein. LT protein was transfected into HK-2 cells, and transfected cells were subjected to nuclear/cytoplasmic fractionation 48 h later. Nuclear protein was immunoprecipitated using an anti-LT antibody and analyzed by SDS-PAGE followed by Coomassie blue staining. The band corresponding to LT protein was excised and in-gel digested by trypsin. B Sequence coverage of the BKPyV LT protein, indicated in red. C K3 and K230 residues of BKPyV LT were acetylated. Digested peptides were analyzed by mass spectrometry, and the identified acetylated lysine residues are marked in red (*: The immunoprecipitated LT protein; M: protein molecular weight marker; IP: immunoprecipitation)

Fig. 2

BKPyV LT expression increased when both K3 and K230 residues were mutated to arginine. K3 or K230 in LT was single or both mutated to arginine and labeled K3R, K230R, and K3R/K230R LT. A Wild-type (WT) and mutated LT were transfected into HK-2 cells before LT protein expression was determined 48 h posttransfection. B WT and mutated LT were transfected into HK-2 cells, and LT protein expression was determined by western blotting 48 h posttransfection. C WT and mutated LT were transfected into HK-2 cells, and the nuclear/cytoplasmic fraction was separated 48 h posttransfection. LT protein was immunoprecipitated and detected by the acetyl-lysine antibody. D WT and mutated LT DNA were transfected into HK-2 cells, and LT protein expression was determined by flow cytometric analysis 48 h posttransfection. Data are representative of at least three independent experiments and are shown as the mean ± SD (* P < 0.05; ** P < 0.01; *** P < 0.001). (MFI: Mean fluorescence intensity)

Fig. 3

HDAC3 and HDAC8 are required for BKPyV LT protein expression. A The expression of class I HDACs was knocked down by siRNA, then cells were infected with BKPyV and LT protein expression was determined by immunofluorescence and flow cytometric analysis 48 h post-infection. Middle panel: Quantification of the LT expression levels in the immunofluorescence assay. Right panel: LT protein expression was determined by intracellular staining and quantified by flow cytometry. B Class I HDACs were inhibited by HDAC inhibitors, cells were infected with BKPyV, and LT protein expression was determined by immunofluorescence and flow cytometric analysis 48 h postinfection. Middle panel: Quantification of the LT expression levels in the immunofluorescence assay. Right panel: LT protein expression was determined by intracellular staining and quantified by flow cytometry. BKPyV infection without siRNA or HDAC inhibitor treatment was considered 100%. (Green, LT protein; Red, Evan’s Blue stain). Data are representative of at least three independent experiments and are shown as the mean ± SD (* P < 0.05; ** P < 0.01; *** P < 0.001). (MFI: Mean fluorescence intensity)

Fig. 4

HDAC3 interacts with BKPyV LT. A LT fused with a Myc tag (LT_myc) and HDAC3 fused with a flag tag (HDAC3_Flag) were cotransfected into HK-2 cells. Forty-eight hours later, transfected cells were immunoprecipitated with LT antibody to detect the presence of HDAC3 by western blotting. B LT fused with a Myc tag (LT_myc) and HDAC3 fused with a flag tag (HDAC3_Flag) were cotransfected into HK-2 cells. Forty-eight hours later, transfected cells were immunoprecipitated with LT antibodies to detect the LT-myc and HDAC3_Flag fusion proteins

Fig. 5

HDAC3 is critical in the WT BKPyV life cycle. Endogenous HDAC3 was knocked down by siRNA for 24 h, and the wild-type or K3R/K230R BKPyV genome was transfected into HK-2 cells. Seventy-two hours later, cells were harvested for immunofluorescent assay to determine LT protein expression. A BKpyV LT expression was determined by immunofluorescence. B Quantification of the LT expression ratio. C LT protein expression was quantified by flow cytometric analysis. Data are representative of at least three independent experiments and are shown as the mean ± SD (* P < 0.05; ** P < 0.01; ns, not significant). (MFI: Mean fluorescence intensity)