Hepatitis B Virus Infection of a Mouse Hepatic Cell Line Reconstituted with Human Sodium Taurocholate Cotransporting Polypeptide

Abstract

c 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...

Sodium taurocholate cotransporting polypeptide is the limiting host factor of hepatitis B virus infection in macaque and pig hepatocytes

“…To investigate whether cccDNA is established in these cells, we extracted total intracellular HBV DNA, digested the relaxed circular DNA pool by T5 exonuclease, and quantified the nuclease‐resistant fraction using cccDNA‐specific primers. This method has been used reliably before and has been verified in comparison with Southern blot analyses (Bingqian Qu, personal communication). cccDNA could only be detected in infected hepatocytes from human, cynomolgus macaque, rhesus macaque, and pig hepatocytes (Fig.…”

“…Although the specific replication step at which this restriction occurs is still poorly understood, a recent study suggests that it occurs after hNTCP‐mediated entry but before cccDNA transcription and that it is due to a missing host dependency factor rather than presence of an active murine restriction factor . This missing host factor is probably not species‐specific, because one peculiar mouse liver cell line (AML12) supports the full HBV replication cycle, including cccDNA formation after hNTCP expression . To overcome this peculiar limitation in murine cells, more thorough investigations of the AML12 cell line and particularly its differences to other murine cell lines will be crucial.…”

“…(18) This missing host factor is probably not species-specific, because one peculiar mouse liver cell line (AML12) supports the full HBV replication cycle, including cccDNA formation after hNTCP expression. (26,29) To overcome this peculiar limitation in murine cells, more thorough investigations of the AML12 cell line and particularly its differences to other murine cell lines will be crucial.…”

Modification of Three Amino Acids in Sodium Taurocholate Cotransporting Polypeptide Renders Mice Susceptible to Infection with Hepatitis D Virus In Vivo

“…Recently, studies reported that AML12, a mouse hepatocyte cell line derived from transforming growth factor ␣ (TGF-␣)-transgenic CD-1/ICR mice (25,26) supported HBV cccDNA formation (27) while cell lines from other mice did not (23,(27)(28)(29). NTCP-complemented AML12 cells were reported to be susceptible to highdose HBV inoculation without robust cccDNA formation (30). Therefore, it will be interesting to test whether the NTCP editing in this specific mouse line could result in successful HBV infection in vivo.…”

Robust Human and Murine Hepatocyte Culture Models of Hepatitis B Virus Infection and Replication

“…The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as the HBV receptor (also the hepatitis delta virus [HDV] receptor) has been a landmark advance in HBV research in recent years (11). Human hepatocellular carcinoma cell lines HepG2 and Huh-7 and an immortalized mouse hepatocyte cell line, AML12, expressing NTCP have been shown to be susceptible to HBV infection and replication, albeit inefficiently (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). AML12 appears to be the only murine hepatocyte cell line known to support HBV infection and replication when expressing NTCP (13).…”