The Role Of IL2、ASGR1and Cav-1in Hepatitis B Virus Entry

Background: Due to lack of effective hepatitis b virus infection model in vitro, the early steps progress of hepatitis b virus entry was very slow, affect the basic and clinical research on hepatitis b virus related disease. More studies have shown that receptors play a role in the progress of virus enter cells.Aim:To establish a high copy serum infected cell model and study the effect of cell receptors in hepatitis B virus entry.Methods:We collected HBeAg positive hepatitis B patients serum (HBV DBA>108copies/mL), unvaccinated HBsAg negative patients serum, and2.2.15cell supernants. After centrifugation to remove celluar debris, hepatitis B virus was concentrated and purified Then PEG precipitation and sucrose dendsity gradient centrifugation in high copy HBV-DNA serum and2.2.15cell supernants, remove virus in serum and obtain virus particles in cell supernants. In this study, we simulated the entry of HBV into HepG2cells using HBeAg positive HBV-DNA high-copy serum (>108copies/mL) obtained from2.2.15cells co-cultured with HepG2. DMSO treated HepG2cells as positive group.24h later, remove the medium, washed3times with PBS, trypsinized and resupended in fresh medium. We collected the supernants from HepG2cells after resupended. HBsAg in cell culture medium was detected by ELISA method, HBV DNA in supernants was detected by PCR. Virus particles in cell culture were observed by electron microscope. Histochemistry, confocal and western blot were used to detected HBcAg in cells. Then using antibody neutralization experiments to neutralization IL-2, IL-12in the serum, quantitative PCR was used to detect HBcAg in mRNA levels. ASGR1and CAV-1was reduced by RNA interference, PCR, western blot and confocal was used to observe the intracellular HBcAg.Results:Hepatitis B surface and core proteins were observed in the cytoplasm of cells exposed to high-copy serum, and hepatitis B surface protein, and virus-like particles were seen in the cell culture supernatant. We also detected interleukins2and12(IL-2, IL-2) in high-copy serum and confirmed that interleukin2acted as marker of HBV entry into HepG2cells. Asialoglycoprotein receptor1(ASGRl) and cavolin-1(CAV-1) were detected on the cell membrane of HepG2cells. Experiments suppressing the expression of these proteins suggested that these receptors may affect HBV entry.Conclusion:Taken together our findings indicate that high copy serum infected cells model can be able to mimic natural infection with HBV. The mechanism of cellular entry may involve an interaction between serum cytokines and cell receptors.