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Once packaged, reverse transcription is initiated The second potential HBV nucleocapsid-associated
through priming by the polymerase from a specific process is envelopment by HBV envelope glycoproteins
tyrosine residue within the N-terminal, TP domain of residing in the ER membrane [Figure 2]. Interestingly,
the polymerase [63,64] [Figure 1a]. A bulge region within ε mechanisms exist that may limit envelopment of
supplies the template for the first 3-4nt of the (-)-DNA capsids containing immature HBV genome; however,
strand before translocation to a matching acceptor motif these mechanisms remain incompletely understood.
in the 3’ direct repeat 1* (DR1*). [120] This strand is then For example, it has been suggested that only mature
extended until completion, resulting in a unit length rcDNA-containing nucleocapsids are enveloped, while
(-)-DNA strand copy of the pgRNA that contains an ssDNA or RNA containing nucleocapsids are not
additional 10nt terminal redundancy (r). The majority of secreted from the cell. [135] Studies utilizing an RNase
pgRNA is degraded during DNA synthesis by the RNase H H-deficient polymerase, which renders the virus unable
activity of polymerase, with the remaining bases serving to initiate (+)-DNA strand synthesis, have suggested
as the 5’ primer for synthesis of the (+)-strand. [121] Direct that only completion of the (-)-DNA strand is required for
extension of this primer from its 5’ position results in envelopment, [136] and specific mutations in core protein
a double-stranded linear form of the genome that is can allow envelopment of immature nucleocapsids. [137]
replication incompetent. [122] This double-stranded linear The mechanisms associated with this selectivity are
form does, however, seem to play a role as the main unknown, although the phosphorylation state of core
form of HBV DNA that can be integrated into the host protein, likely influenced by the nucleic acid species
genome. [123] Instead of direct extension of the RNA primer inside the capsid, could be playing a role. Specifically,
from its 5’ location, successful rcDNA formation can studies have shown that core protein isolated from DNA-
occur only after the RNA primer is translocated to the 3’ containing capsids is dephosphorylated (after the prior
DR2 sequence. Once on DR2, the RNA primer is extended phosphorylation required for pgRNA packaging and
towards the 5’ end of the (-)-DNA strand. Because r on reverse transcription) in a specific C-terminal region,
the other end has the same sequence, exchange of the while immature nucleocapsids remain phosphorylated
[50]
two ends allows (+)-strand synthesis to continue. As at at least 6 different sites. The overall secretion of
with the previous translocations, additional cis- infectious HBV Dane particles has been hypothesized
[138]
acting elements are likely playing a role in long-range to be as little as 1-10 virions per cell per day, which,
base-pairing, which allows the close juxtaposition of because of the large number of cells in the liver, can
these donor and acceptor sites that can otherwise account for high in vivo HBV titers, but can hinder in vitro
research requiring isolation of large amounts of infectious
be separated by kilobases of sequence. [124,125] In virus. Secretion of Dane particles was originally thought
addition, recent evidence has suggested a role for core to follow the same secretory pathway as the much more
protein in regulating DNA synthesis, as mutations in core abundant SVP, with the envelope proteins residing within
protein inhibit the synthesis of the (+)-strand of DNA. [126] the ER-golgi intermediate compartment from where
The complex process of reverse transcription has been they could bind the DNA-containing capsid, enter the
reviewed in more detail elsewhere. [30,31,68]
lumen, and be secreted from the cell. Recent evidence
has suggested, however, that mature HBV virions are
Replication occurs in core particles in the cytosol of secreted from the cell using a pathway that is dependent
an HBV-infected hepatocyte, and the final product of on proteins involved in the endosomal sorting complex
DNA synthesis is the encapsidated, partially double- required for transport (ESCRT) pathway, which forms
stranded rcDNA with the polymerase still bound to the multivesicular bodies. [139] One characteristic that needs to
5’ end of the (-)-DNA strand [Figure 2]. This nucleocapsid be considered regardless of the pathway of HBV secretion
can then proceed in one of two directions. The first is is the seemingly contradictory conformations of L-HBsAg,
shuttling of the nucleocapsid back to the nucleus to with the domains required for both interaction with the
amplify and maintain a stable pool of cccDNA. [127,128] nucleocapsid and hNTCP being needed on opposite sides
The levels of envelope proteins influence this recycling, of the membrane. This is addressed by the fact that nearly
with decreased amounts of HBsAg promoting shuttling half of L-HBsAg changes transmembrane conformation
of the nucleocapsid to the nucleus. [129] In particular, after translation, to expose the preS domains within the
levels of L-HBsAg directly influence shuttling back to ER lumen. [140]
the nucleus, [130] and these findings correlate well with
the early establishment of a cccDNA pool, followed by MODEL SYSTEMS USED IN THE STUDY OF HBV
identification of secreted infectious HBV. [127] The result is
a pool of cccDNA that contains a fluctuating number of Each member of the hepadnavirus family has a narrow
copies (typically less than 10) of cccDNA per cell, [131-133] host range that is thought to be defined primarily by
which can be maintained in the cell for years. Additionally, the interaction between the virus and a specific cell-
it has been suggested the half-life of a single cccDNA surface receptor that is present on host hepatocytes. [11,90]
molecule is between 33 and 57 days, [132,134] underscoring Available cell culture systems for studying the life cycle
the role of cccDNA in maintaining HBV persistence. of the Hepadnaviridae are limited. Typically, members of a
170 Hepatoma Research | Volume 2 | July 1, 2016