Early events of hepatitis B virus infection of HepG2 cells
Objective To explore the mode of hepatitis B virus (HBV) adsorption and penetration into HepG2 cells, and to identify the limiting factors of HBV natural infection infinite cell line. Methods HBV adsorbed to HepG2 cells at 4℃ or 37℃, then HBV DNA within trypsinization solution and HepG2 cells were detected by polymerase chain reaction (PCR). After synchronization of cells at low temperature, another group of HepG2 cells were inoculated with HBV. Subsequently, culture supernate and cells were collected at various phases. Aliquots of collected cells were partitioned into nuclear and cytoplasmic components. HBV DNA and antigens were detected by PCR and enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescence (IIF). HBV covalently closed circular DNA (cccDNA) was examined by selective PCR. Results HBV was merely able to bind to HepG2 cells surface at 4℃. However, HBV could penetrate into cytoplasm at 37℃ and HBV DNA was positive both in trypsinization solution and HepG2 cells by PCR. After incubation at 37℃, HBV DNA could be detected in HepG2 cells at 4?h-24?h, however, viral DNA became weakly positive and negative at 48?h and 96?h, respectively. Antigens and DNA of HBV in culture supernate had been negative after incubation at 37℃ and HBV congregated in nucleus. Viral DNA within the nucleus increased from 4?h to 24?h after incubation, but became weakly positive at 48?h, and disappeared at 96?h. HBV DNA was detected in both nucleoplasm and nuclear membrane at 8?h after inoculation. cccDNA was all negative. Conclusion HBV infected HepG2 cells by co-receptors mode. The failure of uncoating viral genome maybe the main limiting factor that lead to HepG2 cells retractile to HBV replicative infection.