Hepatitis B virus (HBV) surface antigen interacts with and promotes cyclophilin a secretion: possible link to pathogenesis of HBV infection

Abstract

Cyclophilin A (CypA), predominantly located intracellularly, is a multifunctional protein. We previously reported decreased CypA levels in hepatocytes of transgenic mice expressing hepatitis B virus (HBV) surface antigen (HBsAg). In this study, we found that expression of HBV small surface protein (SHBs) in human hepatoma cell lines specifically triggered CypA secretion, whereas SHBs added extracellularly to culture medium did not. Moreover, CypA secretion was not promoted by the expression of a secretion deficient SHBs mutant, suggesting a close association between secretion of CypA and SHBs. Interaction between CypA and SHBs was observed by using coimmunoprecipitation and glutathione S-transferase pull-down assays. Hydrodynamic injection of the SHBs expression construct into C57BL/6J mice resulted in increased serum CypA levels and ALT/AST levels, as well as the infiltration of inflammatory cells surrounding SHBs-positive hepatocytes. The inflammatory response and serum ALT/AST level were reduced when the chemotactic effect of CypA was inhibited by cyclosporine and anti-CD147 antibody. Furthermore, higher serum CypA levels were detected in chronic hepatitis B patients than in healthy individuals. In HBV patients who had received liver transplantation, serum CypA levels declined dramatically after the loss of HBsAg as a consequence of liver transplantation. Taken together, these results indicate that expression and secretion of SHBs can promote CypA secretion, which may contribute to the pathogenesis of HBV infection.

FIG. 1.

Increased secretion of CypA in HBV replicating cells. (A) Comparison of CypA levels in the cell lysate and culture supernatant between HepG2.2.15 cells and HepG2 cells. The left panel shows Western blot analysis of CypA within cells. GAPDH was used for normalization of sample loading. The right panel shows Western blot analysis of CypA in supernatant. (B) Comparison of CypA protein levels in the cell lysate and culture supernatant between Huh7 cells transiently transfected with HBV replicating construct C8-1.3 and pUC19 vector. The left panel shows Western blot analysis of CypA within cells. The right panel shows Western blot analysis of CypA in supernatant.

FIG. 2.

CypA secretion was specifically promoted by the expression of SHBs. (A) Supernatants of HA-SHBs or vector transfected Huh7 cells were collected 48 h after transfection, and AFP levels were determined by enzyme-immunoassay. Mock, mock-transfected cells. Media, fresh culture medium. (B) Serum albumin and complement component 3 (C3) level in serum samples of HBsAg expressing transgenic mice (Tg) and C57BL/6J control mice (C57). (C) CypA secretion was not affected by expression of HBeAg and LHBs. Supernatants and cells were harvested 48 h after transfection. Extracellular and intracellular CypA were analyzed by Western blotting. LHBs expression was analyzed by Western blotting with anti-preS1 antibody. GAPDH was used for normalization of sample loading. HBsAg and HBeAg in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA) and are showed in the right panel.

FIG. 3.

Impact on CypA secretion by SHBs added to culture medium of Huh7 cells. Supernatants and cells were collected 2 days after yeast-derived recombinant SHBs (150 ng/ml) was added to the medium of Huh7 cells. (A) Western blot analysis of intracellular CypA. Actin was used for normalization of sample loading. The results are represented as means ± the standard deviations (SD) in the right panel. (B) Western blot analysis of CypA in supernatant. The results are represented as means ± the SD in the right panel.

FIG. 4.

Induction of CypA secretion was dependent on SHBs secretion. Secretion-deficient SHBs construct (N77) and wild-type SHBs construct (N65) were transfected into Huh7 cells. The left panel shows Western blot analysis of CypA in supernatant and intracellular expression of CypA and SHBs. GAPDH was used for normalization of sample loading. The right panel shows the ELISA results of SHBs secretion in supernatants.

FIG. 5.

Interaction between SHBs and CypA. (A) In vitro-translated SHBs was incubated with an equal amount of GST or GST-CypA fusion protein. SHBs bound to GST or GST-CypA was detected by Western blotting. (B) SHBs was immunoprecipitated from the cell lysate and supernatant of HA-SHBs-transfected Huh7 cells by a horse anti-HBsAg polyclonal antibody. Precipitated HA-SHBs was detected by Western blotting with anti-HA antibody, while coprecipitated CypA was determined by anti-CypA antibody. Horse normal IgG was used as a negative control for the immunoprecipitation assay. (C) Immunofluorescence assay of SHBs (red) and CypA (green) was performed in the SHBs-overexpressing cells (SHBs cell) and the vector control cells (vector cell). DAPI (4′,6′-diamidino-2-phenylindole) was used to stain the nucleus.

FIG. 6.

Increased serum CypA levels in HBsAg expressing transgenic mice. (A) Higher serum CypA levels in HBsAg transgenic mice (n = 13) than in C57BL/6J control mice (n = 10) (P < 0.001, Student t test). The left panel shows a representative Western blot image of serum CypA. The right panel shows the relative density of Western blot images. (B) Real-time PCR analysis of CypA mRNA levels in liver tissues of HBsAg transgenic mice and C57BL/6J control mice. GAPDH was used as an internal control for normalization.

FIG. 7.

Serum HBsAg, CypA, ALT/AST, and infiltration of inflammatory cells in the mouse liver after hydrodynamic injection with HA-SHBs plasmid. (A) Serum HBsAg levels in C57BL/6J mice 4 days after hydrodynamic injection with HA-SHBs plasmid or vector. (B and C) Serum CypA (B) and ALT/AST levels (C) in SHBs expression mice, VSV-G expression mice, and vector control mice. Serum CypA and ALT/AST levels were increased significantly in HA-SHBs injected mice (*, P < 0.05 [Student t test]). (D and E) Immunohistochemical staining of HBsAg in serial liver tissue sections (400× magnification) of HA-SHBs-injected C57BL/6J mice (D) and H&E staining (E). Infiltration of inflammatory cells was observed around HBsAg-positive hepatocytes. (F and G) Immunohistochemical staining of HBsAg (F) and H&E staining (G) of serial liver tissue sections from vector injected control mice. (H and I) Immunohistochemical staining of VSV-G protein (H) and H&E staining (I) of serial liver tissue sections from VSV-G-injected control mice.

FIG. 8.

Impact of Cs or anti-CD147 antibody treatment on serum HBsAg, CypA, and ALT/AST levels and infiltration of inflammatory cells in the mouse liver hydrodynamically injected with HA-SHBs plasmid. (A) Serum HBsAg levels of mice injected with Cs or anti-CD147 antibody 4 days after hydrodynamic injection of HA-SHBs plasmid or vector. (B and C) Serum CypA (B) and ALT/AST (C) levels in Cs- or anti-CD147 antibody-treated C57BL/6J mice with hydrodynamic injection of HA-SHBs or vector. (D and E) Immunohistochemical staining and H&E staining of liver tissue sections (400× magnification) of mice receiving NS and HA-SHBs. Infiltration of inflammatory cells is shown around HBsAg-positive hepatocytes. (F and G) Immunohistochemical staining of HBsAg (F) and H&E staining (G) of liver tissue sections of Cs and HA-SHBs-injected C57BL/6J mice. (H and I) Immunohistochemical staining of HBsAg (H) and H&E staining (I) of liver tissue sections of mice receiving isotype control antibody and HA-SHBs. (J and K) Immunohistochemical staining of HBsAg (J) and H&E staining (K) of liver tissue sections of anti-CD147 antibody and HA-SHBs injected C57BL/6J mice. (L and M) Immunohistochemical staining (L) and H&E staining (M) of liver tissue sections from mice receiving vector DNA which were used as negative control. Serial sections were used in the present study. Cs, cyclosporine.

FIG. 9.

Increased serum CypA levels in natural HBV infections. (A) Serum CypA levels were compared among chronic hepatitis B patients and healthy individuals. Serum CypA was increased significantly in chronic hepatitis B patients (P < 0.01, Student t test). The left panel shows a representative Western blot image of serum CypA. The right panel shows the semiquantification of the relative densities of dot blot images. (B) Dot blot analysis of serum CypA levels pre- and posttransplantation in HBV-infected patients who underwent liver transplantation. In 13 of 15 patients, the serum CypA level was decreased markedly after liver transplantation.