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hepatocytes. [83-86] Importantly, the requirement for HBx even in the absence of replicating HBV, and potentially
was also confirmed in primary human hepatocytes contribute to HCC development or maintenance.
directly infected with either wild-type or HBx-deficient
HBV. [28] HBV LIFE CYCLE
The lack of a single accepted model for studies of HBV Studies have shown that the species specificity and
and HBx has created some confusion about the overall hepatotropic nature of HBV are due to at least two
consequences of HBx expression for HBV replication and different layers of cellular factors. The first is the
hepatocyte physiology. HBx-related studies have often hepatocyte-specific expression of the recently described
been performed in transformed or immortalized cell HBV receptor, human sodium taurocholate cotransporting
lines and with different levels of HBx expression, leaving peptide (hNTCP/SLC10A1) [Figure 2]. hNTCP is only
the impact of HBx on a normal hepatocyte incompletely expressed on human hepatocytes, and mouse NTCP
understood. While HBx is generally considered to have cannot be bound by HBV, which correlates with the
[87]
oncogenic potential, it is yet to be determined if it is inability of HBV to directly infect mouse hepatocytes. [42]
directly oncogenic or simply acts as a co-factor in HCC The second level of cell-specificity of an HBV infection
development, as both effects have been demonstrated is controlled by hepatocyte-specific transcription factors
in different HBx-tg mouse models. [88-91] It is important such as HNF1α and HNF4α; these control post-entry,
to recognize that a strongly oncogenic HBx would not downstream stages of the HBV life cycle. Evidence for
be consistent with the biology of HBV-associated HCC, the additional role of intracellular factors for controlling
which involves decades of a chronic HBV infection, and the cell-specificity of an HBV infection comes from the
it is more likely that HBx plays a cofactor role in the observation that humanized-mouse NTCP, in which
development of HBV-induced liver cancer. The hypothesis the binding residues from mouse NTCP are replaced
that HBx-induced subtle changes in hepatocyte by hNTCP, allows binding of HBV to the receptor but
physiology sensitize cells to other oncogenic signals, does not result in a productive HBV infection when
while facilitating HBV replication, is more consistent expressed in mouse cells. [103] Studies using hepatitis
with the biology of HBV-associated HCC. Peripheral D virus (HDV), which is a satellite virus requiring HBV
[92]
evidence for the oncogenic potential of HBx comes from envelope proteins for entry into a cell, demonstrated
the fact that hepadnavirus-associated HCC seems to be that the 75 aa at the N-terminal portion of the PreS1
restricted to mammalian hepadnaviruses, which each domain of L-HBsAg are required residues responsible for
express a form of the X protein. Avian hepadnaviruses, binding to the viral receptor. [104] In addition, it was shown
which do develop a chronic infection but do not cause that N-myristylation of the PreS1 domain is required
HCC, either do not express an X protein or express a for infectivity, but not HBV virion assembly. [105] In fact, a
highly divergent form. [78,93] myristylated peptide consisting of only the first 47 aa of
the preS1 domain is able to bind to hNTCP and inhibit
HBx is a multifunctional protein that can modulate the binding of HBV. [41] Additional studies have suggested
many hepatocyte signaling cascades and factors that a role for heparin sulfate proteoglycans in the initial
have also been linked to mechanisms that underlie stages of HBV binding to hepatocytes, [106] including the
cellular transformation. For example, HBx can modulate recent identification, using an RNAi-based screen in Huh7
calcium, [84,85] apoptosis, [83,86] and proliferation signals, cells stably expressing hNTCP, of glypican 5 as an HBV
among other pathways, and can activate numerous and HDV entry factor. [107]
transcription factors, including activator proteins 1
[94]
[95]
and 2 (AP-1 and AP-2), nuclear factor of activated T cells Although amino acid sequences of both preS1 and hNTCP
[96]
(NFAT), and nuclear factor kappa-light-chain-enhancer that affect binding of HBV to hNTCP are known, the lack
of activated B cells (NF-κB). [97-99] HBx can also regulate of an effective model system that mimics a robust natural
cellular signaling factors, such as Wnt/β-catenin, [100] infection has hampered a complete understanding of
p53, [101] and Akt, [86,102] that have been implicated in HCC. aspects of the HBV life cycle immediately following
Recently, modulation of miRNA expression has also been receptor-binding. The observation that preS1 binds
included in the functions of HBx. It is possible that the to clathrin heavy chain and the adapter protein AP-2
many functions attributed to HBx could actually be the in immortalized primary human hepatocytes, and that
result of a few fundamental upstream HBx functions knockdown of these proteins inhibits infection, suggests
that can affect multiple downstream cellular signal- that the HBV-hNTCP complex may enter the cell through
transduction pathways in a context-dependent manner. clathrin-mediated endocytosis. [108] Once in the cell, the
Interestingly, while HBV replication in established HBV- HBV DNA is delivered into the nucleus by mechanisms
associated HCCs is typically absent, a number of groups that remain unclear. One potential mechanism is the
have shown that these tumors can still express HBx from active transport of the nucleocapsid through nuclear
fragments of the HBV genome that have integrated into pores. [109] Another potential mechanism involves CTD
the host genome. The presence of HBx in these cells phosphorylation of the core protein, which is thought
could mean that HBx might be active in these HCC cells, to expose nuclear localization signals, leading to
[49]
168 Hepatoma Research | Volume 2 | July 1, 2016