Preprint Brief Report Version 1 Preserved in Portico This version is not peer-reviewed

HepG2-NTCP Subclones Exhibiting High Susceptibility to Hepatitis B Virus Infection

Version 1 : Received: 8 July 2022 / Approved: 14 July 2022 / Online: 14 July 2022 (12:06:08 CEST)

How to cite: Zahoor, M.A.; Kuipery, A.; Mosa, A.I.; Gehring, A.J.; Feld, J.J. HepG2-NTCP Subclones Exhibiting High Susceptibility to Hepatitis B Virus Infection. Preprints 2022, 2022070220 (doi: 10.20944/preprints202207.0220.v1). Zahoor, M.A.; Kuipery, A.; Mosa, A.I.; Gehring, A.J.; Feld, J.J. HepG2-NTCP Subclones Exhibiting High Susceptibility to Hepatitis B Virus Infection. Preprints 2022, 2022070220 (doi: 10.20944/preprints202207.0220.v1).

Abstract

HepG2 cells reconstituted with Hepatitis B virus (HBV) entry receptor sodium taurocholate co-transporting polypeptide (NTCP) are widely used as a convenient in vitro cell culture infection model for HBV replication studies. As such, it is pertinent that HBV infectivity is maintained at steady-state levels for accurate interpretation of in vitro data. However, variations in HBV infection efficiency due to imbalanced NTCP expression levels in the HepG2 cell line may affect experimental results. In this study, we performed single cell cloning of HepG2-NTCP-A3 parental cells via limiting dilution and obtained multiple subclones with increased permissiveness to HBV. Specifically, one subclone (HepG2-NTCP-A3/C2) yielded more than 4-fold higher HBV infection compared to the HepG2-NTCP-A3 parental clone. In addition, though HBV infectivity was universally reduced in the absence of polyethylene glycol (PEG), subclone C2 maintained relatively greater permissiveness under PEG-free conditions, suggesting the functional heterogeneity within parental HepG2-NTCP-A3 may be exploitable in developing a PEG-free HBV infection model. The increased viral production correlated with increased intracellular viral antigen expression as evidenced through HBcAg immunofluorescence staining. Further, these subclones were found to express different levels of NTCP, albeit with no remarkable morphology or cell growth differences. In conclusion, we isolated subclones of HepG2-NTCP-A3 which support efficient HBV production and thus provide an improved in vitro HBV infection model.

Keywords

Hepatitis B virus; HepG2-NTCP cells; Immunofluorescence; Sodium taurocholate cotransporting polypeptide (NTCP) Receptor; Subcloning; Limiting dilution; Myrcludex B

Subject

LIFE SCIENCES, Virology

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