Based on the clinical experience of antiviral chemotherapy for chronic hepatitis B and the lessons learned from the therapeutic management of HIV infection, using combination chemotherapy to maximize antiviral activity and decrease the rate of emergence of drug-resistant virus is the best strategy for future chronic hepatitis B therapy (24
). Models for testing drug cross-resistance and screening new compounds with different targets of action against resistant HBV are urgently required. In this paper, we describe a new cell culture system, which is a group of three cell lines, HepG2-WT10, HepG2-SM1, and HepG2-DM2, stably transfected with wild-type, l
(−)SddC- and PCV-resistant HBV genomes.
Viral production in the cell lines was studied. It was demonstrated that the cell lines were able to produce HBV RNAs, antigens, and virions. Furthermore, this ability was stable for at least 72 cell passages without requiring G418 selection pressure. This ensures that the cell culture is easy to perform and that the drug testing condition is consistent during a long culture period and avoids the complication caused by additional culture maintenance drugs.
Under our culture condition, it was observed that it was easier to detect intracellular viral DNA than extracellular viral DNA, due to the fact that more intracellular than extracellular HBV progeny DNA was present at the sampling time points. It was noticed that the HBV production was very low in HepG2-DM2 cells, although the RNA level was not dramatically lower than that in HepG2-WT10 cells and higher than that in HepG2-SM1 cells. This very low virion production could be due to defective replication of the HBV variant with two mutations, L526M and M550V, in the polymerase, which was demonstrated by previous studies (8
The sensitivity of HBV DNA synthesis in these cell lines to l
(−)SddC and PCV was studied, and it was confirmed that this system could be used for evaluating the activities of compounds against l
(−)SddC- and PCV-resistant viruses. To further validate this cell-based model, several well-studied nucleoside analogs, l
-FMAU, and PMEA, were tested in these cells. The results here were consistent with the observation obtained from other systems, such as transient transfection and an in vitro polymerase activity assay (9
Currently there are several systems for the study of antiviral activity against l
(−)SddC-resistant HBV, such as a transient-transfection system, a system using isolated HBV polymerase from baculovirus, and stable virus-producing cell lines (1
). The transient-transfection system is suitable for initially identifying the role of mutations in the resistance phenotype. However, it is not easy to perform. Batch-to-batch variation, and lack of transfection efficiency and short-term stability are the major problems limiting its application for large-scale drug screening. The major application for the in vitro assay with a system using isolated HBV polymerase is the study of HBV replication at the HBV plus-strand DNA synthesis step, but the assay is not useful for studying the other steps of viral reproduction, such as regulation of HBV core particle assembly, HBV RNA processing (transcription and posttranscription), and cellular regulation (41
). Our cell-based system is not only able to test the HBV DNA synthesis inhibitors but also is useful for the evaluation of HBV inhibitors that target other HBV regulation processes. With the cell lines presented here, we have found that a novel natural product could actively inhibit wild-type and resistant HBV by decreasing HBV RNA and HBV antigen levels without altering in vitro HBV DNA polymerase activity (L. Fu et al., unpublished data).
In conclusion, cell lines HepG2-WT, HepG2-SM1, and HepG2-DM2, established by introducing wild-type adr and L526M and L526MM550V mutant HBV genomes, respectively, into their parental HepG2 cells were capable of stably producing HBV virions with different susceptibilities to l
(−)SddC and PCV. Using these cell lines, several compounds were tested, and the results were consistent with those from other testing systems (4
). In addition, purine analog QYL685 was identified as a promising inhibitor of resistant HBV. The establishment of these cell lines should be helpful in assessing new compounds against l
(−)SddC- and PCV-resistant virus in a more controllable fashion and thus avoiding the complication of using a transient HBV DNA transfection system.