Fig 2: SIRT2 regulates p53 level by targeting the p53 promoter. (A) Real-time PCR analysis of gene expression of various transcription factors associated with HBV transcription. (B) HepG2-NTCP and PHH cells were transduced with lentivirus expressing shSIRT2-1/shSIRT2-2 and shCont. The effect of SIRT2 knockdown on p53 mRNA level was analyzed by real-time PCR. (C,D) The effect of SIRT2 overexpression or SIRT2 depletion on p53 protein level in HepG2-NTCP and PHH cells was examined by Western blot. ß-actin served as the loading control. (E,F) HepG2-NTCP and Huh-7 cells were transfected with pGL3-p53 promoter and transfected with plasmids expressing SIRT2 or transduced with indicated lentivirus expressing shSIRT2. The luciferase activity was measured at 36 h post transfection using dual-luciferase reporter assay. Representative data are from at least three independent experiments. Data are shown as mean ± SD. *p < 0.05, **p < 0.01.

Fig 3: Gene silencing of SIRT2 inhibits HBV transcription and replication. HepG2-NTCP cells or PHH were infected with 1 × 103 genome equivalents/cells of HBV for 16 h and then transduced with lentivirus expressing indicated shRNA (shSIRT2-1 and shSIRT2-2) and scramble control (shCont). Cells were harvested to test the knockdown efficiency and examine the viral markers after 5 days. (A) The efficiency of SIRT2 silencing was examined via Western blot. (B–D) SIRT2 suppression decreased total HBV RNAs level and HBV 3.5-kb RNA level based on real-time PCR (B,C) and Northern blot (D). Each panel was loaded with an equal amount of total RNA and the HBV RNAs were probed with DIG-labeled single-stranded HBV RNA probe with a length of 1,000 bp. Ribosomal RNAs (28 s and 18 s) were used as loading controls. (E,F) SIRT2 knockdown suppressed HBV core DNA level as determined by real-time PCR (E) and Southern blot (F). (G,H) HBsAg and HBeAg levels were detected by ELISA. Representative data are from at least three independent experiments. Data are shown as mean ± SD. *p < 0.05, **p < 0.01.

Fig 4: SIRT2 facilitates HBV transcription and replication via repressing the binding of p53 on HBV EnI/Xp and EnII/Cp. (A,B) Effect of SIRT2 overexpression (A) and down-regulation (B) on the recruitment of p53 to HBV EnI/Xp and EnII/Cp was detected by ChIP assay. Cross-linked chromatin from HBV-infected HepG2-NTCP cells was immunoprecipitated with anti-p53 antibody followed by real-time PCR with specific HBV EnI/Xp and EnII/Cp primers. The promoter of GAPDH and MYH6 were used as internal controls. The ChIP results are expressed as % of input. (C) HepG2-NTCP cells were transfected with indicated plasmids. Dual-luciferase assay was performed to examine the activities of HBV EnI/Xp and EnII/Cp. pGL3-EnI/Xp Mut and pGL3-EnII/Cp Mut denoted the p53 binding sites in HBV EnI/Xp and EnII/Cp were mutated. (D–F) HepG2-NTCP cells were infected with HBV WT or HBV Mut (binding sites of p53 to EnI/Xp and EnII/Cp were mutated) and transduced with lentivirus expressing SIRT2. Total HBV RNAs and HBV 3.5-kb RNA levels were analyzed by real-time PCR (D) and Northern blot (E), HBV core DNA level in HepG2-NTCP cells was analyzed by real-time PCR and Southern blot (F). (G) HepG2-NTCP cells were transfected with indicated plasmids. Dual-luciferase assay was performed to examine the activities of HBV EnI/Xp and EnII/Cp. (H–J) HBV-infected HepG2-NTCP cells were transfected with plasmids containing SIRT2 or p53. Total HBV RNA and HBV 3.5-kb RNA levels were detected by real-time PCR (H) and Northern blot (I). HBV core DNA level was measured by real-time PCR and Southern blot (J). Representative data are from at least three independent experiments. Data are shown as mean ± SD. *p < 0.05, **p < 0.01.

Fig 5: SIRT2 is correlated with chronic HBV infection. (A,B) The mRNA and protein levels of SIRT2 from PBMC or serum in CHB patients and healthy individuals were analyzed by real-time PCR and ELISA, ß-actin was used as an internal control in real-time PCR. (C–E) Correlation between serum SIRT2 level and HBV viral load (C), HBsAg (D), HBeAg levels (E) and HBcrAg (F). HBcrAg level in the serum of CHB patients was detected by fully automated lumipulse chemiluminescence enzyme immunoassay system (Fujirebio Inc., Tokyo, Japan). The HBV viral load, HBsAg and HBeAg levels were log10 transformed. The correlation co-efficiency (r) and two-tailed p values were calculated via Pearson correlation. (G,H) Analysis of correlation between serum SIRT2 level and transaminase ALT, and AST levels using Pearson’s test.