Molecular biology of hepatitis B virus infection

Abstract

Human hepatitis B virus (HBV) is the prototype of a family of small DNA viruses that productively infect hepatocytes, the major cell of the liver, and replicate by reverse transcription of a terminally redundant viral RNA, the pregenome. Upon infection, the circular, partially double-stranded virion DNA is converted in the nucleus to a covalently closed circular DNA (cccDNA) that assembles into a minichromosome, the template for viral mRNA synthesis. Infection of hepatocytes is non-cytopathic. Infection of the liver may be either transient (<6 months) or chronic and lifelong, depending on the ability of the host immune response to clear the infection. Chronic infections can cause immune-mediated liver damage progressing to cirrhosis and hepatocellular carcinoma (HCC). The mechanisms of carcinogenesis are unclear. Antiviral therapies with nucleoside analog inhibitors of viral DNA synthesis delay sequelae, but cannot cure HBV infections due to the persistence of cccDNA in hepatocytes.

Keywords: Hepatitis B virus; Pathogenesis; Replication.

Figure 1. Genome structure, genes and mRNAs of hepatitis B virus

The relaxed-circular DNA genome of HBV with a complete minus strand and incomplete plus strand is shown (inner circle), along with the major mRNAs, all of which end at a common polyadenylation signal located in the core open reading frame. Dashed lines at the 5′ end of mRNAs indicate the use of staggered starts sites for preCore/core and M/S, as described in the text. All open reading frames have a clockwise direction. The single stranded gap in the plus strand can be filled in in vitro by the viral RT, which is covalently attached to the 5′ end of minus strand DNA. In avihepadnaviruses, the plus strand is generally complete except for DR2, which remains as an RNA/DNA hybrid (Lien et al., 1987). DR1 and DR2 represent ~11 base direct repeats that have an important role in viral DNA synthesis. Minus strand synthesis exhibit a short terminal redundancy created during reverse transcription of pregenomic RNA (Figure 2).

Figure 2. Replication of HBV DNA

Reverse transcription begins with binding of the pol protein to the 5′ copy of a stem-loop structure epsilon (ε) located in the terminal redundancy of pregenome RNA (A), which facilitates packaging into viral nucleocapsids. Then, following copying of 3 nucleotides from a bulge in the side of epsilon, the polymerase translocates to the copy of the 11 nt sequence, DR1, located in the 3′ terminal redundancy. The basis for the translocation is unclear but is sufficient to align the nts copied from epsilon with a complementary sequence in DR1. Reverse transcription then continues to the 5′ end of the pregenome (B, C), with the template being degraded by an RNase H encoded within the pol protein. The terminal 18 nts including the CAP and DR1 are not degraded. Normally this oligonucleotide translocates to DR2, where it can anneal because of the identity of DR1 and DR2 (D). Plus strand synthesis then initiates and extends to the 5′ end of the minus strand. Circularization then occurs, facilitated by the short terminal redundancy on the minus strand (E). Plus strand synthesis stops before completed (F), perhaps because nucleocapsids containing incomplete plus strands are packaged into viral envelopes, preventing access to dNTPs. For avihepadnaviruses, the plus strand is generally complete except for DR2. About 10% of the time, the RNA primer of plus strand synthesis fails to translocate, resulting in in situ priming to produce double stranded linear DNA (dsl DNA) (G). Both rcDNA and dslDNA can be converted to cccDNA when transferred to the nucleus, though the pathways are different and, for dsl DNA, generally lead to defective cccDNA. Dsl DNA is a preferred substrate for integration into host DNA.

Figure 3. HBV replication cycle

The figure shows a model for the life cycle of hepadnaviruses, as described in the text. Envelope proteins are shown in yellow, DNA containing capsids in blue and RNA containing capsids in red. Upon infection mediated by the entry receptor, NTCP, virus nucleocapsids are transported to the nucleus, where rcDNA is converted to cccDNA. cccDNA does not undergo semiconservative DNA synthesis (Tuttleman et al., 1986a). Early in infection, when envelope protein concentrations are low, newly made nucleocapsids, with their enclosed viral DNA, are transported to the nucleus to amplify cccDNA copy number to up to 50 per hepatocyte (Tuttleman et al., 1986b). At the same time, envelope proteins enter the ER and assemble into subviral particles (SVP) or transfer to MVBs where virion assembly is believed to occur. When sufficient envelope is present, nucleocapsids are directed to their secretory pathway and cccDNA amplification ceases. Mature virions might exit cells through exosomes (for details and references see the text). Virus with dsl DNA can also infect hepatocytes. cccDNA is formed from dsl DNA by non-homologous recombination, resulting in a loss of sequences and, generally, rendering this cccDNA unable to support virus replication (Yang and Summers, 1995). Dsl DNA may also integrate into host DNA via non-homologous recombination; this pathway does not appear to have a role in the virus life cycle. The figure does not show “empty” HBV virions produced by infected hepatocytes because the pathway for their assembly and secretion is not yet known (Ning et al., 2011}.

Figure 4. Liver structure

A highly idealized 2D representation of a portion of a 3D liver lobule is shown representing some of the major cell types that are present. Blood enters through the portal vein and hepatic artery and exits at the central vein, after flowing through sinusoids in which hepatocytes are separated from blood via a fenestrated epithelium though which virus can pass. Bile produced by hepatocytes is released into bile canaliculi formed by the apical interfaces of hepatocytes and flows to the bile ducts via the Canals of Hering. Bile eventually flows to larger ducts before leaving the liver. Historically, progenitor cells residing in bile ducts and/or the Canals of Hering were thought to emerge under conditions of stress as ovoid cells (oval cells) and to differentiate into small transitional hepatocytes and finally mature hepatocytes (paths shown as dashed lines), as well as to contribute to formation of foci of altered hepatocytes, some of which appear to be preneoplastic. Recent studies questioned the role of oval cells in hepatocyte replacement, suggesting instead that hepatocytes themselves are able to evolve and repopulate the liver even under extreme stress to the normal hepatocyte population. And, by inference, to give rise to foci of altered hepatocytes and HCC. A possible resolution of some of these divergent observations is suggested by the very recent report that mature hepatocytes themselves give rise to oval cells that have the capacity to re-differentiate to hepatocytes (Tarlow et al., 2014). Thus, at present, some of the illustrated pathways are still uncertain (indicated with ? mark). (Figure adapted from (Seeger et al., 2013))

Figure 5. Stages of chronic HBV infection

Chronic HBV infection is thought to go through immune tolerant, immune clearance, and immune control (no disease progression) phases. In this latter phase, the degree of ongoing liver damage can vary from none to significant, depending on how completely virus replication is blocked (see text). HCC incidence generally rises after the immune clearance phase, which may not be clinically apparent but can still be inferred to have occurred because of the greatly reduced virus titers that come after it, reflecting immune control or restriction of virus replication. Loss of immune control may also occur after a period of disease inactivity, leading to enhanced virus replication and exacerbation of liver damage. (Figure adapted from (Yim and Lok, 2006))