Abstract
Hepatitis B virus (HBV) infection and its sequelae remain a major public health burden, but both HBV basic research and the development of antiviral therapeutics have been hindered by the lack of an efficient in vitro infection system. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the HBV receptor. We herein report that we established a NTCP-complemented HepG2 cell line (HepG2-NTCP12) that supports HBV infection, albeit at a low infectivity level following the reported infection procedures. In our attempts to optimize the infection conditions, we found that the centrifugation of HepG2-NTCP12 cells during HBV inoculation (termed “spinoculation”) significantly enhanced the virus infectivity. Moreover, the infection level gradually increased with accelerated speed of spinoculation up to 1,000g tested. However, the enhancement of HBV infection was not significantly dependent upon the duration of centrifugation. Furthermore, covalently closed circular (ccc) DNA was detected in infected cells under optimized infection condition by conventional Southern blot, suggesting a successful establishment of HBV infection after spinoculation. Finally, the parental HepG2 cells remained uninfected under HBV spinoculation, and HBV entry inhibitors targeting NTCP blocked HBV infection when cells were spinoculated, suggesting the authentic virus entry mechanism is unaltered under centrifugal inoculation. Our data suggest that spinoculation could serve as a standard protocol for enhancing the efficiency of HBV infection in vitro.
Highlights
Hepatitis B virus (HBV) is a hepatotropic enveloped DNA virus that causes transient and chronic hepatitis B in humans [1]
Primary human hepatocytes (PHHs) and the HepaRG cell line can be used for certain HBV infection experiments, the primary human hepatocytes (PHHs) are costly with limited supply, and their genetic background and susceptibility to HBV infection vary from donor to donor [5]; in these regards, the HepaRG system does have advantages over PHH but time-consuming cell proliferation and differentiation steps are required prior to infection [6]
Microscopic imaging demonstrated a marked pattern of cell surface expression of Na+-taurocholate cotransporting polypeptide (NTCP) in HepG2-NTCP12 cells, which is consistent with the localization of endogenous NTCP in hepatocytes [21]
Summary
Hepatitis B virus (HBV) is a hepatotropic enveloped DNA virus that causes transient and chronic hepatitis B in humans [1]. Primary human hepatocytes (PHHs) and the HepaRG cell line can be used for certain HBV infection experiments, the PHHs are costly with limited supply, and their genetic background and susceptibility to HBV infection vary from donor to donor [5]; in these regards, the HepaRG system does have advantages over PHH but time-consuming cell proliferation and differentiation steps are required prior to infection [6]. The reconstitution of NTCP expression in commonly used hepatocyte-derived cells (i.e. HepG2 and Huh7) confers permissiveness of cells to HBV infection, fostering novel mechanistic and therapeutic studies on the early steps of the HBV life cycle, including receptor-mediated HBV entry, uncoating, and first round cccDNA formation, etc. Further optimization and standardization of the protocol for HBV infection in NTCP-expressing cells is warranted
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