KLF15 activates the HBV surface promoter. (A) Dose-dependent effect of KLF15 on the HBV surface promoter. pS1-Luc, which contains the luciferase reporter under the expression control of the surface promoter, was co-transfected with pKLF15 or its control vector pcDNA3.1 into Huh7 cells. pRL-TK, which expresses Renilla luciferase, was also included to monitor the transfection efficiency. (B) Lack of effect of KLF15 on the cyclin D1 promoter (CCD1). The CCD1 reporter construct was co-transfected with pKLF15 or its control vector into Huh7 cells. The CCD1 promoter activity in the absence of pKLF15 was arbitrarily defined as one. (C) Effects of KLF15 on the surface promoter with mutations in the Z1/Z2 or M2 site. In the experiments shown in this figure, the relative luciferase activities were determined by comparing the luciferase activities expressed by the surface promoter in the presence of KLF15 to that in the absence of KLF15. The results represent the mean ± SD of three independent experiments.

KLF15 activates the HBV core promoter. (A, B) Dose-dependent effect of KLF15 on the HBV core promoter. Panels A and B represent the effect of KLF15 on pCP1.3x and pCP, respectively. The experiments were conducted as described in Fig. 1 legend. (C) A schematic diagram of the luciferase reporter CP and its two mutant constructs, CPm1 and CPm2. The mutated nucleotides in CPm1 and CPm2 are shown with lower-case letters. (D) Effects of KLF15 on CP-wt, CPm1 and CPm2.

KLF15 increases the expression of HBsAg and HBcAg and the replication of HBV DNA. HepG2 (A, C and D) or Huh7 (B) cells were co-transfected with pHBV1.3D and pKLF15 or its control vector (Ctrl). An aliquot of the culture medium was removed 24 hours after transfection and used for EIA to measure the HBsAg level (A and B). Cells were lysed 48 hours after transfection for Western blot analysis of HBcAg and actin (C). For qPCR analysis of HBV DNA (D), cells and the culture media were harvested 96 hours after transfection.

Endogenous KLF15 regulates HBV gene expression. (A) Reduction of KLF15 mRNA level by its siRNA. Huh7 cells transfected with pHBV1.3D, pXGH and the negative control siRNA (ctrl) or the KLF15 siRNA (KLF15) were lysted 24 hours after transfection, and the KLF15 mRNA was quantified by qRT-PCR and normalized against the GAPDH RNA internal control. (B) Reduction of HBsAg expression from the HBV genome by KLF15 siRNA. The culture media in panel A were used for HBsAg and hGH ELISAs. The amount of HBsAg was normalized against hGH, which served as the transfection control. (C–D) Luciferase reporter assays for the HBV core promoter (C) and the surface promoter (D). The reporters were co-transfected with pRL-TK and either the control siRNA or the KLF15 siRNA into Huh7 cells. Cells were then lysed 24 or 48 hours after transfection for the luciferase assay. Relative Luc Levels refers to firefly luciferase activity normalized with control Renilla luciferase activity.

Detection of KLF15 interaction with HBV core and surface promoters in electrophoretic mobility shift assays (EMSA) and ChIP assays. (A) Western blot analysis of recombinant KLF15 (KLF15) protein expressed in 293T cells and purified using an anti-FLAG affinity column. Western blot analyses were conducted using antibodies directed against KLF15, the FLAG tag, Sp1 and NF-Y. Lane 1, crude cell lysates; lane 2, unbound fraction of the affinity column; and lane 3, eluate of the bound fraction. (B) EMSA showed the specific binding of core promoter (CP35) by KLF15. Lanes 1–7, CP35 probe incubated with KLF15; lanes 2–4, competition with 100-fold excess of unlabeled competitor (CPT) CP35 (CP), CLCK1 (CK) and non-specific (NS) MLTF oligonucleotides, respectively; lane 5, supershift with 1 μg anti-KLF15 antibody; lane 6, supershift with 2 μg anti-KLF15 antibody; lane 7, supershift with 2 μg control antibody; lane 8, CP35 probe and 2 μg anti-KLF15 antibody only. The specific KLF15-DNA complex (arrow) and supershift (arrowhead) are indicated. (C) Binding of CP35 and KLF15 (lane 1) in the presence of an increasing amount (5-, 10-, 20-fold) of unlabeled CP35 (lanes 2–4) and CP35-2m (lanes 5–7). CP35-2m contained mutations in KLF15 binding sites. (D) EMSA using surface promoter (SP70) and KLF15. Lane 1, SP70 probe only; lane 2, SP70 probe plus KLF15; lanes 3–4, same as in lane 2 with the addition of 100-fold excess of unlabeled SP70 (lane 3) or 100-fold excess of non-specific MLTF oligonucleotide (lane 4). (E) Lane 1, SP70 probe only; lane 2, SP70 probe plus KLF15; lanes 3 and 4, supershift with 1 μg and 2 μg anti-KLF15 antibody. (F) Top panel: ChIP assays showed that KLF15 bound to the wild-type core promoter (CP-wt), but not to the mutant core promoter where the two potential KLF15 binding sites were mutated (Top panel). The mouse IgG was used as the control in the ChIP assay. Middle panel: ChIP assays showed that KLF15 binding to the surface promoter (SP-wt) was reduced by about 40% if Sp1 binding sites were mutated (Z1/Z2-mut). In contrast, Sp1 binding to the surface promoter was abolished by the Z1/Z2 mutations. Bottom panel: Although the M2 mutation suppressed NF-Y binding to the surface promoter, it had little effect on KLF15 binding.

KLF15-specific RNAi constructs reduce KLF15 mRNA levels in vitro and in vivo and HBcAg expression in vivo. (A) Co-transfection of pKLF15 and various KLF15 RNAi constructs (Cons 1 to 4) or the control (Ctrl) RNAi construct into Huh7 cells. The relative KLF15 mRNA levels were measured by qRT-PCR and normalized against the GAPDH RNA. (B) Reduction of KLF15 mRNA levels in the mouse liver by the KLF15 RNAi construct. Two groups of mice (4 in each group) were co-injected with pAAV-HBV1.2 and the control RNAi construct (Ctrl) or the KLF15 RNAi construct 4 (KLF15). The plasmid pLive-SEAP was also used for co-injection to monitor the transfection efficiency. GFP-positive (GFP+) and GFP-negative (GFP−) hepatocytes were isolated from these mice 3 days after injection for RNA extraction. The KLF15 RNA was quantified by qRT-PCR and normalized against mouse 36B4 RNA. The KLF15 mRNA level in GFP negative cells isolated from mice injected with the KLF15 RNAi construct was arbitrarily defined as 100%. (C) Confocal microscopy of liver tissue sections from mice injected with pAAV-HBV1.2 and the control RNAi construct (upper panels) or KLF15 RNAi construct 4 (lower panels). Panels a and e, GFP signals expressed from the miR RNAi vector; panels b and f, HBcAg staining; panels c and g, DAPI staining; and panels d and h, merged images. (D) Western blot analysis of the HBV core protein in the mouse liver. The liver homogenates of mice hydrodynamically injected with pAAV-HBV1.2 and the control RNAi construct (lane 1), pAAV-HBV1.2 and KLF15 RNAi construct (lane 2) or phosphate-buffered saline (PBS) (lane 3) were used for Western blot analysis for the core protein and actin.

Suppression of KLF15 expression decreases HBsAg and HBV DNA levels in mouse serum. (A–B) HBsAg studies. Mice were hydrodynamically injected as described in Fig. 6 legend. Mouse sera were isolated at the time points indicated. In (A), 30 μg of the control RNAi construct (Ctrl) or the KLF15 RNAi construct (KLF15) was injected; and in (B), 50 μg were used. The time points with statistically significant differences are labeled with asterisks. (C) HBV DNA studies. Mouse sera from day 5 after injection as shown in panels A and B were first treated with TURBO DNase (Ambion) for the removal of free DNA prior to the extraction of encapsidated HBV DNA, which was quantified by qPCR.

The CPm2 mutations in the HBV core promoter reduced HBV DNA level in mice. Mice were injected with pAAV-HBV1.2-CPm2 (n=5), which contains the CPm2 mutations, or with the parental wild-type (WT) plasmid pAAV-HBV1.2 (n=6). The plasmid pLive-SEAP was used for the co-injection to monitor the transfection efficiency. HBV DNA copy numbers in the mouse sera were measured by qPCR three days after injection and normalized against the SEAP activity. The short bars represent the median values of each group.