Introduction Hepatitis B virus (HBV) requires host cellular machinery such as cyclophilins to support its ongoing propagation (Phillips et al , Gastroenterology 2014) these host proteins represent ideal candidates for therapeutic interventions as they are generally expected to have a lower frequency of drug-resistance and antiviral efficacy across genotypes. We have also recently described a role for the host protein Osteopontin (OPN), a pro-fibrogenic downstream effector of the Hedgehog pathway, in enhancing HCV replication (Choi et al , Clinical Sciences 2014). Whilst increased levels of blood OPN have similarly been reported in chronic Hepatitis B (CHB), its role in viral replication remains unknown. This study aimed to evaluate the role of OPN in HBV replication, HBsAg secretion and HBV-driven liver injury.
Method Stably (HepG2215), transiently (HUH-7) transfected and infected (HepaRG) cell lines, producing full HBV virions and HBsAg particles were cultured over 72 h in the presence/absence of several concentrations of recombinant OPN (recOPN). In addition, secreted OPN was neutralised using OPN-specific aptamers. Cells and supernatants were harvested at baseline, 24, 48 and 72 h. Intracellular OPN mRNA and HBV-DNA levels were quantitated by Real-Time qPCR. HBsAg levels were measured by ELISA. OPN levels were also measured by ELISA in cell culture supernatants and in sera of controls and HBeAg (+) CHB patients who were either treatment naive or treated with potent antiviral agents. In addition, expression of OPN was assessed in explanted livers from healthy and HBV-cirrhotic patients.
Results Serum levels and intrahepatic expression of OPN were significantly increased in CHB patients compared to controls (up to 7 fold; p < 0.05). In vitro, mRNA and protein levels of OPN were highly correlated with intracellular HBV-DNA (r = 0.858; p < 0.001/r = 0.997; p = 0.003), secreted HBV-DNA (r = 0.968; p < 0.001/r = 0.818; p = 0.047) and secreted HBsAg (r = 0.738; p = 0.001/r = 0.89; p = 0.018). The relationship between OPN and HBV replication was further confirmed following treatment with recOPN which showed a significant increase in intracellular and secreted HBV-DNA by an additional 1.3 Log10 copies/mL and amplified HBsAg secretion rates by 2 fold.
Conclusion These data confirm that OPN is upregulated in the blood and livers of CHB patients. We also show that OPN directly augments HBV replication, thus identifying a novel therapeutic target.
Disclosure of interest None Declared.
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