[Construction of a highly efficient HBV infection cell model based on HBV co-receptor neuropilin-1].

Objective

To construct a lentiviral plasmid expressing neuropilin-1 (NRP1) and a HepG2-sodium taurocholate cotransporting polypeptide (NTCP) capable of overexpressing stable cell line NRP1 so as to study the susceptibility of this cell line to hepatitis B virus (HBV).

Methods

The plasmids pcDNA3.1-NRP1/Myc His B and pCDH-CMV-MCS-EF1-GFP-BSD were used as templates for amplification. The recombinant plasmid pCDH-NRP1-GFP-BSD was obtained using seamless cloning technology. HepG2-NTCP cells were transduced with the recombinant plasmid into lentivirus following packaging. Stable overexpression of NRP1 was obtained following screening of blasticidin S deaminase (BSD) and monoclonal antibody in HepG2-NTCP cell lines. The susceptibility of the resulting cell line to HBV was analyzed by detecting HBV-related markers post-infection. Data between groups were compared using the t-test.

Results

The restriction enzyme digestion and sequencing analysis confirmed the successful construction and identification of the recombinant plasmid containing NRP1. Western blot, RT-qPCR, and fluorescence microscopy results showed that the stable overexpression of the targeted NRP1gene had capability in HepG2-NTCP cells. Compared to the control group, NRP1-expressed stable cell lines showed approximately a two-fold increase in efficiency rate in HBV infection. Intracellular HBV covalently closed circular DNA (cccDNA), mRNA, DNA, and viral proteins, including HBsAg, HBeAg, and HBc, were significantly elevated, nearly two-fold, following HBV infection.

Conclusion

A stable monoclonal cell line overexpressing NRP1 was successfully constructed, which supports highly efficient HBV infection in vitro and provides a new tool for studying the mechanisms of chronic hepatitis B infection.