Hepatitis B Virus (HBV) Surface Antigen Interacts with and Promotes Cyclophilin A Secretion: Possible Link to Pathogenesis of HBV Infection

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.Hepatitis B virus (HBV) infects more than 350 million people worldwide and is a major cause of chronic viral hepatitis and hepatocellular carcinoma (25). Three morphologically distinct forms of viral particles exist in the sera of HBV-infected patients, namely, the 22-nm-diameter spherical particles, tubular particles, and 42-nm-diameter virions (19). Strikingly, the subviral particles (spheres and tubules), containing only viral envelop glycoproteins and host-derived lipids, typically outnumber the virions by a factor of 1,000- to 10,000-fold (6, 11). There are three HBV envelop glycoproteins collectively known as HBV surface antigen (HBsAg), including the large (LHBs), middle (MHBs), and small (SHBs) surface proteins. Among them, SHBs is the most abundant viral envelop protein in virion and subviral particles. Although excess HBsAg subviral particles have been suggested to sequester the neutralizing antibody against HBV and contribute to a state of immune tolerance, thereby enabling the survival of infectious virions and leading to persistent infections (6, 27), the biological and pathological significance of the overproduction of HBsAg subviral particles still remains elusive.HBsAg has been proved extremely effective in inducing protective antibodies (anti-HBs) and thus has been used as the prophylactic vaccine. Thus far, most studies on HBsAg have focused on the development of hepatitis B vaccines (41), identification of HBsAg-interacting membrane proteins as potential host HBV receptors (9, 13), and characterization of the impact of naturally occurring HBsAg mutations on its antigenicity (12, 43). However, specific interactions between HBsAg and host intracellular factors have not been extensively studied.To address this issue, SHBs-secreting cell lines and lineages of HBV transgenic mice persistently expressing HBsAg were used in our previous studies (28, 34, 44). We found that the level of cyclophilin A (CypA) decreased markedly in the livers of HBsAg transgenic mice but increased significantly in their sera (44). CypA is a multifunctional cellular protein. It is the major binding protein for the immunosuppressive drug cyclosporine (Cs) (14) and exhibits peptidyl-prolyl cis-trans isomerase activity (35). Recently, it was found CypA played important roles in regulating inflammatory responses and viral infections. Regarding these newly recognized physiological functions, CypA was speculated to be involved in HBV infection. In the present study, the mechanism and clinical implications of elevated secretion of CypA induced by SHBs were explored in detail, including studies in cell cultures, hydrodynamic injected mouse models, and chronic hepatitis B patients. Our findings indicate that expression and secretion of SHBs can trigger the secretion of CypA, which may induce liver inflammation and contribute to the pathogenesis of HBV infection.