Antiviral Activity of Interferon Alpha-Inducible Protein 27 Against Hepatitis B Virus Gene Expression and Replication

Abstract

Despite the availability of effective vaccines, hepatitis B virus (HBV) is still a major health issue, and approximately 350 million people have been chronically infected with HBV throughout the world. Interferons (IFNs) are the key molecules in the innate immune response that restrict several kinds of viral infections via the induction of hundreds of IFN-stimulated genes (ISGs). The objective of this study was to confirm if interferon alpha-inducible protein 27 (IFI27) as an ISG could inhibit HBV gene expression and DNA replication both in cell culture and in a mouse model. In human hepatoma cells, IFI27 was highly induced by the stimulation of IFN-alpha (IFN-α), and it potentiated the anti-HBV activity. The overexpression of IFI27 inhibited, while its silencing enhanced the HBV replication in HepG2 cell. However, the knocking out of IFI27 in HepG2 cells robustly increases the formation of viral DNA, RNA, and proteins. Detailed mechanistic analysis of the HBV genome showed that a sequence [nucleotide (nt) 1715-1815] of the EnhII/Cp promoter was solely responsible for viral inhibition. Similarly, the hydrodynamic injection of IFI27 expression constructs along with the HBV genome into mice resulted in a significant reduction in viral gene expression and DNA replication. In summary, our studies suggested that IFI27 contributed a vital role in HBV gene expression and replication and IFI27 may be a potential antiviral agent for the treatment of HBV.

Keywords: EnhII/Cp; IFI27; ISGs; antiviral activity; hepatitis B virus.

FIGURE 1

Interferon alpha (IFN-α) induces interferon alpha-inducible protein 27 (IFI27) expression and the role of IFI27 in IFN-elicited anti-hepatitis B virus (HBV) response in human hepatoma cells. (A–D) HepG2 and Huh7 cells were treated with IFN-α (100 ng/ml) and incubated for 0, 6, and 12 h. (A,B) The treated HepG2 and Huh7 cells were subjected to western blotting or immunoblotting using an anti-IFI27 antibody, where β-actin was taken as a normal internal reference control (bottom panels). (C,D) The total HBV RNA was isolated from the treated cells, and the expression of IFI27 of mRNA was determined by using qRT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was used as a loading control to normalized the data. (E–G) pCAGGS-HA-IFI27 or pCAGGS (empty vector) plasmid was transfected into HepG2.2.15. After 24 h of incubation, the cells were treated with IFN-α (100 ng/ml). (E,F) The relative expression level of IFI27 mRNA and total HBV mRNA was examined by qRT-PCR. GAPDH was used as an internal control. (G) HBV intracellular DNA expression level was determined by qPCR. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

FIGURE 2

Overexpression of IFI27 inhibits HBV gene expression in HepG2 cells. (A–F) Human hepatoma cells (HepG2) were co-transfected with pHBV1.3 plasmid, pSV-β-gal, and pCAGGS-HA-IFI27 or pCAGGS (empty vector) at different concentrations for 48 h, as indicated in the artwork. (A) The IFI27 mRNA expression was detected by qRT-PCR. GAPDH was used as an internal control. (B) The expression levels of IFI27 protein were determined by western blotting using anti-IFI27 and anti-HA antibodies, respectively. β-Actin expression was used as a loading control. (C) The total HBV RNA was extracted and subjected to northern blot analysis. The rRNAs (28S and 18S) were used as the internal control. (D) Intercellular HBV 3.2 kb mRNA was detected by qRT-PCR. GAPDH mRNA expression was used to normalized the data. (E,F) Secreted HBsAg (E) and HBeAg (F) in the supernatants were determined by using ELISA. (G,H) HepG2 cells were transfected with pHBV1.3 plasmid vector together with pCAGGS-IFI27 or pCAGGS (empty vector) in a dose-dependent manner for 96 h. (G) The HBV intracellular core-associated and extracellular HBV DNA (H) from the supernatant were extracted and measured by qPCR. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

FIGURE 3

Knockdown of IFI27 enhances HBV gene expression in HepG2 cells. (A–F) HepG2 cells in a 12-well plate were transfected with HBV1.3 (800 ng) and pSV-β-gal (200 ng), and 24 h later, the cells were transduced with lentivirus expressing IFI27-targeting shRNAs (shIFI27-4 and shIFI27-5) or scrambled control (shControl). The lentivirus-transduced cells were collected after 3 days post-transduction. (A) The levels of IFI27 mRNAs were determined by qRT-PCR. Data were normalized to GPADH mRNA expression. (B) Expression levels of IFI27 protein were analyzed by western blotting using an IFI27-specific antibody. β-Actin was used as a loading control. (C) HBV total RNA from transducing cells was extracted and HBV transcripts were analyzed by northern blot. The 28S and 18S rRNAs were used as an internal control. (D) The HBV 3.2-kb mRNA was subjected to qRT-PCR. GAPDH mRNA expression was used as a loading control. (E,F) Expression of secreted HBsAg (E) and HBeAg (F) was detected using ELISA. (G,H) The HBV intracellular core-associated (G) and extracellular HBV DNA (H) from the supernatant were extracted 4 days post-transduction and subjected to qPCR. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

FIGURE 4

IFI27 knockout (IFI27-KO) in HepG2 cells promotes HBV replication. (A–F) IFI27-deficient HepG2 cell line (HepG2 IFI27-KO) was generated by the CRISPR/Cas9 system using a guide RNA; the sequence is already shown in the methodology. HepG2 parent cells (wild-type) and the IFI27 knockout HepG2 cell line were transfected with HBV1.3 (800 ng) and pSV-β-gal (200 ng) and harvested after 48 h. (A) Western blot analysis was used to confirm IFI27 protein expression using an anti-IFI27 antibody and β-actin was used as a loading control. (B) Total RNA from HepG2 parent cell and IFI27-KO cells was extracted and detected with northern blotting. The 28S and 18S rRNAs were used as the internal control. (C) The HBV 3.2-kb mRNA was separately subjected to qRT-PCR. GAPDH was used as a loading control. (D,E) The secreted HBsAg (E) and HBeAg (F) proteins were analyzed using ELISA. (F,G) HepG2 parent cells and the IFI27 knockout HepG2 cell line were transfected with pHBV1.3 and harvested after 96 h. The HBV intracellular core-associated (F) and extracellular HBV DNA (G) from the supernatant were purified and subjected to qPCR. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

FIGURE 5

IFI27 represses HBV gene expression by inhibiting EnhII/Cp promoter activity. (A) HepG2 cells plated in a 24-well plate were co-transfected with reporter plasmids pGL3-EnhI/Xp, pGL3-EnhII/Cp, pGL3-SP1, and pGL3-SP2 (200 ng each) together with pCAGGS-HA-IFI27 or pCAGGS (250 ng). Luciferase activities of Firefly relative to Renilla were determined. pRL-TK (50 ng) was used as a control of transfection efficiency. (B) HepG2 cells were co-transfected with reporter plasmid pGL3-EnhII/Cp-Luc and/or pCAGGS-HA-IFI27 or pCAGGS at different concentrations for 2 days (48 h). Luciferase assay was measured and normalized with control (pRL-TK). (C) HepG2 cells were co-transfected with pGL3-pCMV-Luc (200 ng) along with pCAGGS-IFI27 or pCAGGS. Luciferase assay was measured and normalized with pRL-TK. (D) The schematic diagram of serial deletion mutants of the EnhII/Cp reporter. (E) IFI27 and their effect on serial deletion of HBV EnhII/Cp promoter. (F) HepG2 cells were seeded in 10 cm dish and then co-transfected with pHBV 1.3 (5 μg) and pCAGGS-HA-IFI27 or pCAGGS (5 μg) for 48 h. The CHIP assay was achieved using an anti-HA antibody to examine the binding capacity of IFI27 to EnhII/Cp. The amplification of extracted chromatin DNA was done by PCR and determined by agarose gel electrophoresis. Normal IgG is used as a negative control (IgG), while RNA polymerase II acts as a positive control (RNAPII) in this experiment. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ns (non-significant).

FIGURE 6

IFI27 suppresses HBV replication and gene expression in vivo. (A–F) Two groups of C57BL/6 mice (n = 10) were co-injected with pHBV1.3 along with pCAGGS-HA-IFI27 or pCAGGS. After 4 days of hydrodynamic injection, the mice were sacrificed and the livers and blood were processed for analyses. (A) The expression level of IFI27 was determined by western blot using anti-HA antibodies, and the levels of β-actin serve as loading controls (lower panel). (B,C) The titer of secreted HBsAg (B) and HBeAg (C) proteins expressed in mice blood was measured using ELISA. (D) The level of HBV DNA from sera was also evaluated using qPCR. (E) Total HBV RNA was isolated from the liver tissues, and the levels of HBV pgRNA were measured by qRT-PCR. The level of mice GAPDH (mGAPDH) was used as an internal control. (F) Immunohistochemical staining was used to determine the HBcAg levels in the liver. **P ≤ 0.01, ****P ≤ 0.0001.

FIGURE 7

Schematic representation of IFN-α and IFN-stimulated gene (ISG) induction pathway. The type I IFN (IFN-α) is cascaded via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, resulting in the expression of a large spectrum of activated transcriptional ISGs. IFI27 is an ISG that suppresses viral gene expression, prevents transcript production, and decreases the accumulation of viral replicative intermediates.