FIG 4.
Inhibition or knockdown of Sirt2 reduces HBV replication. (A) Treatment with AGK2, a potent Sirt2 inhibitor, reduces HBV replication. Huh7 cells were mock transfected (lane 1) or transfected with 4 μg of 1.3-mer HBV WT (ayw) (lanes 2 and 3) or with 4 μg of 1.-mer HBV WT (ayw) plus 4 μg of the 3×FLAG-Sirt2.1 construct (lanes 4 and 5). Lanes 3 and 5 were treated for 72 h with AGK2 (10 μM) at the time of transfection. (B) HBV replication is decreased when Sirt2 is knocked down by shRNAs. Huh7 (lanes 1 to 7) and HepG2 (lanes 8 to 14) cells were transduced with lentivirus-like particles containing control shRNA (shControl) (lanes 3 and 10) or SIRT2-targeting shRNAs (shSIRT2-#1, shSIRT2-#2, shSIRT2-#3, and shSIRT2-#4) (lanes 4 to 7 and 11 to 14). The control (lanes 2 and 9) and transduced Huh7 (lanes 3 to 7) and HepG2 (lanes 10 to 14) cells were transiently transfected with 4 μg of 1.3-mer HBV WT (ayw) and lysed 72 h later. Lanes 1 and 8 are mock-transfected controls, which were adjusted with pcDNA3. (C) Promoter activities of EnhI/Xp, EnhII/Cp, PreS1p, and PreS2p are decreased upon Sirt2 knockdown. Freshly plated shControl-, shSIRT2-#2-, or shSIRT2-#3-transduced Huh7 cells were transfected with 2 μg of the indicated luciferase reporter vector containing null, EnhI/Xp, EnhII/Cp, PreS1p, or PreS2p regions. Lysates were prepared at 72 h posttransfection, and luciferase activity was measured. Luciferase activity relative to that in control shRNA-transduced cells is presented. Data are presented as the mean luciferase activities from four independent experiments. (D) Effects of Sirt2 knockdown or entecavir on HBV-infected cells. A total of 2 × 105 HepG2 (lane 2), HepG2-hNTCP-C9-shControl (lane 3), HepG2-hNTCP-C9-shSIRT2-#2 (lane 4), HepG2-hNTCP-C9-shSIRT2-#3 (lane 5), and HepG2-hNTCP-C9 (lanes 7 to 9) cells in 6-well plates were infected with 1.7 × 103 GEq per cell of HBV and lysed at 9 days postinfection (dpi). For Northern blotting (D, eighth panel), lysates were prepared at 5 days postinfection. Lane 1 (HepG2) and lane 6 (HepG2-hNTCP-C9) were mock infected. SDS-PAGE and Western blotting of proteins (A and D, first to fifth panels, and B, first to fourth panels), native agarose gel electrophoresis and Western blotting of core particles (A and D, sixth panels, and B, fifth panel), Southern blotting of HBV DNA (A and B, bottom panels, and D, seventh panel), and RT-PCR for Sirt2 mRNA (D, bottom panel) were performed as described in the legend of Fig. 1. Relative levels of acetylated α-tubulin, core particles, HBV RI DNA, and HBV mRNA were measured using ImageJ 1.46r. Data are presented as mean values from three independent experiments. Statistical significance was evaluated using Student's t test. P values relative to the respective control are shown.