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Hepatitis B virus (HBV) enters hepatocytes via its receptor, human sodium taurocholate cotransporting polypeptide (hNTCP). So far, HBV infection has been achieved only in human hepatic cells reconstituted with hNTCP and not in cells of mouse origin.Here, the first mouse liver cell line (AML12) which gains susceptibility to HBV upon hNTCP expression is described. Thus, HBV infection of receptor-expressing mouse hepatocytes does not principally require a human cofactor but can be triggered by endogenous murine determinants.
Hepatitis B virus (HBV) is a hepatotropic, enveloped DNA virus replicating via an RNA intermediate (1). Infections with HBV and coinfections with hepatitis delta virus (HDV), which uses the HBV envelope for dissemination, increase the risk for liver cirrhosis and carcinoma (2). Both viruses exploit human sodium taurocholate cotransporting polypeptide (hNTCP), a hepatic bile salt transporter, as an essential entry receptor (3, 4). Hitherto, only hepatic human liver cells expressing hNTCP have been shown to become susceptible for HBV (3,5,6). In contrast, HDV infections can be established through hNTCP supplementation in nonhuman and even nonhepatic cells.Several hNTCP-expressing mouse liver cell lines are resistant to HBV infection (3, 6-8). Likewise, HBV transgenic mice cannot form covalently closed circular DNA (cccDNA), the transcriptional viral template (9). Such previous results have led to interpretations that mouse cell lines might lack a factor needed for HBV replication-a hypothesis strongly supported by recent work by alternatively, might express a restriction factor that prevents cccDNA formation. However, Cui et al. recently described a mouse liver cell line inducibly expressing HBV from an integrate (AML12HBV10) capable of forming cccDNA (10). Here, we provide evidence that AML12 cells complemented with hNTCP gain susceptibility to HBV, including cccDNA formation and antigen (Ag) expression.Generation of hNTCP-expressing cell lines. Mouse AML12 cells were tested for their ability to support HBV infection after stable hNTCP transduction. For controls, we implemented the HepG2 cell line, which becomes susceptible to HBV upon hNTCP transduction, and the mouse liver cell line mH274#26, which remains refractory (3,4,8). Stable transduction was achieved using lentiviruses encoding hNTCP. Western blot analysis (Fig. 1A) confirmed expression of hNTCP exclusively in the transduced lines. The hNTCP-expressing cells bound Myrcludex B-atto (myristoyl-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDF NPNKDHWPEANKVG-atto [MyrB-atto]), a fluorescently labeled entry inhibitor of HBV and HDV that specifically binds to NTCP (Fig. 1B) (11). This indicates correct folding and localization to the plasma membrane. Functionality of hNTCP was further confirmed by its ability to transport 3 H-labeled taurocholate (Fig. 1C) (3,(12)(13)(14).
hNTCP expression confers susceptibility to HDV infection.Cell lines were infected with HDV, and replication was analyzed by immunofluorescent staining of hepatitis delta virus antigen...