Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus

Abstract

Hepatitis B virus (HBV) is a small, double-stranded DNA virus and is the prototype of the hepadnavirus family. HBV is a human pathogen capable of causing both acute and chronic hepatitis. The World Health Organization currently estimates that 350 million people are chronically infected with HBV. Persistent HBV infection is also associated with an increased risk of cirrhosis and hepatocellular carcinoma. 1 Although a tremendous amount is known about HBV, our knowledge of the virus is by no means complete. Historically, major obstacles in the study of HBV have been the inability of the virus to infect cells in vitro, and the lack of animal model systems due to a strict virus-host range. Thus, many aspects of HBV biology have been unraveled by studying related hepadnaviruses, such as the duck hepatitis virus which is capable of in vitro infection, 2 and the woodchuck hepatitis virus which allows for the in vivo study in an animal model system. 3 The duck hepatitis virus and woodchuck hepatitis virus systems were instrumental in developing an understanding of the hepadnavirus lifecycle and remain valuable models for HBV infection. However, many significant differences exist between animal hepadnaviruses and HBV. For example, avian hepatitis viruses do not encode the X gene, 4 and major transcriptional differences between woodchuck hepatitis virus and HBV have been reported. 5 Within the last decade, several HBV expressing cell lines have been established by transfecting viral DNA into liverderived human cell lines and by selecting novel cell lines containing stably integrated HBV genomes. [6][7][8] The most widely used are the HepG2 2.2.15 cell line (2.2.15) derived from the HepG2 hepatoblastoma cell line and HB611 derived from the HuH6 hepatoma cell line. These and other cell lines have led to considerable progress in the study of HBV in vitro. However, there are some inherent drawbacks which preclude the use of these cell lines in studying some aspects of HBV biology. (1) Many HBV expressing cell lines were created using constructs containing strong heterologous promoters proximal to the HBV genome. The effect those promoters have on HBV transcription and replication is unclear but could differ substantially from what occurs in a natural infection in vivo in which HBV gene expression is driven solely by endogenous HBV promoters. (2) Cell lines commonly used to study HBV contain multiple copies of integrated HBV DNA. Unlike retroviruses, which integrate viral DNA into the host genome, hepadnavirus genomes are not integrated routinely but, instead, are maintained in the nucleus of infected cells in vivo as a pool of episomal covalently closed circular (CCC) DNA molecules. 9 Although the integration of HBV DNA in human liver has been reported, 10 it is not an obligatory part of the HBV lifecycle. HBV does not encode any machinery for integration into the host genome, and integration is not required for HBV replication. In addition, when integrated HBV DNA is found,

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

“…Baculovirus stocks and titers were prepared in Sf21 insect cells as described in Delaney and Isom. 69 Baculovirus was diluted in DMEM/F12 (control medium), calcium-free DMEM, or calcium-free DMEM medium supplemented with a designated concentration of EGTA to a final volume of 0.5 ml. Incubation of cultures with baculovirus for less than 40 min resulted in decreased gene transfer efficiencies, whereas no difference was observed if cultures were incubated for X1 h (data not shown).…”

A selection system for identifying accessible sites in target RNAs

“…To test the effectiveness of sRz in cell culture, HepG2 cells were maintained in minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum in a humidified incubator at 37 8C with 5% CO 2 + These cells were cotransfected with pBB4+5HBV1+3 (a 1+3ϫ unit length HBV DNA plasmid construct; see Delaney & Isom, 1998) and either pLSCLIP, pLSCLIPmRz408, pLSCLIPsRz777, or pLSCLIPsRz885 (pLSCLIP denotes the CLIP cassette in the LacSwitch vector, from Stratagene)+ pLSCLIPsRz777 and pLSCLIPsRz885 were constructed by annealing reverse complementary oligonucleotides (CLAW437/CLAW438 and CLAW397/CLAW398, respectively) and then inserting them into the Bgl II site of pLSCLIP+ pLSCIPmRz408 was constructed the same way with oligonucleotides CLAW435/ CLAW436+ However, these oligonucleotides were inadvertently synthesized so that the 59 flanking region contained mismatches; subsequent testing in vitro showed that this ribozymes had approximately 15% of the catalytic activity of the sRz408, which was equivalent to the activity of mRz247, and it was therefore included in the experiments as a "intermediateactivity" comparison+ Finally, we also employed the mRz CLIP that had been constructed previously (using the same procedures)+ HepG2 cells were transfected using FuGENE6 transfection reagent (Boehringer Mannheim)+ A total of 5 mg of DNA (0+5 mg pBB4+5HBV1+3 and 4+5 mg of the pLSCLIP constructs) in 10 mL FuGENE6 was used to transfect a 60-mm dish of cells seeded 24 h prior to transfection+ The reagent/ DNA mixture was incubated in serum-containing medium for 24 h+ All transfections were done independently in triplicate+ Control transfections using b-galactosidase showed a transfection efficiency of approximately 30%+…”

Initiation of hepatitis B virus genome replication and production of infectious virus following delivery in HepG2 cells by novel recombinant baculovirus vector

“…This amino acid exchange in the polymerase sequence (YMDD to YPDD) also resulted in an exchange in the surface protein (IWMM to IRMM). The previously described procedure for baculovirus transduction of HepG2 (Delaney & Isom, 1998) was modified slightly. Briefly, HepG2 cells were transduced at 80 % confluency (1610 5 -2610 5 cells cm 22 ).…”

“…Another possibility that can be used to trigger intracellular HBV replication is to use systems based on the transduction of HepG2 cells with recombinant adenovirus or baculovirus carrying the HBV genome (Delaney & Isom, 1998;Ren & Nassal, 2001). Transduction of hepatoma cells with HBV-loaded baculovirus has led to higher HBV replication levels compared with both transfection approaches and stable cell lines such as HepG2.2.15.…”

“…Transduction of hepatoma cells with HBV-loaded baculovirus has led to higher HBV replication levels compared with both transfection approaches and stable cell lines such as HepG2.2.15. Moreover, cccDNA was synthesized in transduced HepG2 cells, although the origin of its formation was not clearly explained (Delaney & Isom, 1998).…”