Taxonomic Structure of the Family

Family

Hepadnaviridae

Genus

Orthohepadnavirus

Genus

Avihepadnavirus

Virion Properties

Morphology

Hepadnaviruses are spherical, occasionally pleomorphic, 40-48 nm in diameter and with no evident surface projections after negative staining. The outer, detergent-sensitive envelope contains the surface proteins and surrounds an icosahedral nucleocapsid core which is composed of one major protein species, the core protein, and encloses the viral genome (DNA), the viral polymerase, and associated cellular protein(s).

In the case of Hepatitis B virus (HBV), the majority of nucleocapsid cores are around 34 nm diameter and contain 240 core protein subunits (triangulation number T = 4), while a minority are approximately 30 nm in diameter and consist of only 180 subunits (T = 3). Hepadnavirus infection induces overproduction of surface proteins which are secreted as pleomorphic lipoprotein particles together with virus to the blood. In the case of HBV, these form 17-22 nm spherical particles and filaments (Fig. 1).

Physicochemical and Physical Properties

The virion S_20w is approximately 280S. The buoyant density of virions in CsCl is approximately 1.25 g/cm³. Estimates of the buoyant density of particles lacking cores are 1.18-1.20 g/cm³. Virus-derived cores (lacking envelopes) have densities of approximately 1.36 g/cm³.

Nucleic Acid

The genome consists of a single molecule of circular DNA that is partially single-stranded, and not covalently closed. The size of the genome ranges from 3.0-3.3 kb in different family members; the viral DNA has an S_20w of about 15 and a G+C content of about 48%. One strand (negative sense, i.e., complementary to the viral mRNAs) is full-length, the other varies in length. In orthohepadnaviruses, the full-length negative sense DNA has a nick with an 8-9 base terminal redundancy at a unique site corresponding to a position 242 nts downstream from the unique 5-end of the positive sense strand (Fig. 2); while for avihepadnaviruses the nick in the negative sense DNA is about 50 nts from the 5-end of the positive strand. The 5-end of the negative sense DNA has a covalently attached terminal protein, and the 5-end of the positive sense DNA has a covalently attached 19 nts, 5-capped oligoribonucleotide primer. The 3-end of the positive strand terminates at a variable position in different molecules, creating a single stranded gap that may account for 60% of the HBV genome but is usually very short in avihepadnaviruses. Thus, the circular configuration is maintained by basepairing of overlapping cohesive 5-ends of each strand.

Proteins

1.

Virions and empty subviral particles may contain two or three envelope proteins, with common C-termini and differing N-termini due to different sites of translation initiation. Typically, virions contain a small (S) trans-membrane protein, (in many species) an intermediate sized M protein, and a large (L) protein which is myristylated at the N-terminus. In many cases more than one form of each of the above proteins occur due to alternative patterns of glycosylation. For HBV, virions and filaments are enriched in L proteins and empty spheres consist predominantly of S proteins, while for Duck hepatitis B virus (DHBV), L and S proteins are distributed evenly between particle types.

2.

The core protein has a large N-terminal domain and a small RNA-binding domain at the carboxyterminus. Core protein above a threshold concentration can self assemble via dimers to complete nucleocapsids in the absence of other viral components.

3.

The polymerase protein consists of an N-terminal domain (TP) with a DNA primer function, a spacer region of variable size, a reverse transcriptase and an RNase H domain. The TP domain is covalently attached to the 5-end of the minus strand of viral DNA.

4.

Most members of the family contain a fourth ORF (‘X’ gene) situated downstream of the S gene and partly overlapping the cohesive 5-terminal region. This codes for a non-structural protein that can function as a promiscuous transcriptional activator.

5.

Host proteins contained within virions include a protein kinase which phosphorylates the RNA binding domain of the core protein, heat shock protein Hsc 70 and heat shock protein Hsp 90 which at least in the case of DHBV appears to be part of a multicomponent chaperone complex involved in replication and nucleocapsid assembly.

6.

The core protein also exists in a secreted soluble form (‘e’ antigen) if co-expressed with the upstream pre-core sequence which can be translated from an additional start codon upstream of the core start codon.

Lipids

Lipid constitutes 30 to 40% of the viral envelope or of the empty particles. It is derived from a host membrane compartment intermediate between the ER and Golgi, and includes phospholipids, cholesterol, cholesterol esters and triglycerides.

Carbohydrates

Demonstrated in particles and virions of orthohepadnaviruses as N-linked glycans of the complex types.

Genome Organization and Replication

The hepadnavirus genome contains the following major ORFs; precore/core (preC/C), polymerase (P), env or surface (preS/S), and in the case of orthohepadnaviruses, a fourth ORF, the X gene.

The preC/C ORF codes for two distinct products: one is the core protein forming the protein shell of the nucleocapsid, the other, made by translation of the joint preC/C ORF, is the precore protein which is targeted into the cell’s secretory pathway, processed at both ends and eventually found in the serum of infected individuals as e antigen. Both products are translated from genomic, terminally redundant 3.5 kb transcripts with slightly different 5-ends. The longer precore mRNAs contain the preC initiation codon, the shorter core mRNA lacks it. The P-ORF covers some 80% of the genome and encodes the viral replication enzyme P, which is also an indispensable component in the assembly process (see below). P protein is translated from the same genomic RNA that directs synthesis of core protein by internal initiation. The env or surface gene consists of three in-phase ORFs, termed in 5- to 3-direction preS1, preS2 and S. S can be separately expressed to give the small or S protein; cotranslation of preS2/S yields the middle or M protein, that of the entire preS1/preS2/S gene the large or L protein. Thus the S domain is common to all three forms of surface protein. As for the preC/C ORF, this is achieved by the generation of mRNAs with staggered 5-ends in which the initiator codons of the preS1, the preS2 or the S region are the first to be encountered by translating ribosomes. L protein is translated from a 2.4 kb mRNA, M and S from a set of 2.1 kb transcripts. All viral transcripts are 3-terminally colinear, ending after a unique polyadenylation signal located in the C gene. The X gene encodes a pleiotropic transcriptional activator that appears to be required for establishment of infection with Woodchuck hepatitis virus (WHV), and has been implicated in one proposed mechanism for hepadnavirus carcinogenesis. The DNA sequence of HBV has 2 enhancer regions (ENHI and ENHII), two 11-base direct repeat sequences, DR1, DR2, a polyadenylation signal (TATAAA) and putative glucocorticoid-responsive elements (GRE). The 5-end of the negative strand is located within DR1, the 5-end of the positive strand is at the 3 boundary of DR2 (Fig. 2).

Replication can be considered in two stages; an incoming or afferent arm in which the input viral genome enters the nucleus and is converted to covalently closed supercoiled DNA (cccDNA), and an outgoing or efferent arm in which RNA transcripts from the cccDNA are reverse transcribed in the cytoplasm and the resulting genomic DNA is encapsidated and secreted.

There is evidence that the infectious DNA-containing virion binds to its target cell via interaction of the L protein with cellular receptor(s) that are not yet fully characterised. The nucleocapsid is presumably delivered to the cytoplasm, and via mechanisms currently under study the viral genome gains access to the nucleus. As a result of host cell polymerase action to repair the single-stranded gap, removal of the terminal protein and oligoribonucleotide from the negative and positive strands respectively, supercoiling and DNA ligation, the viral genome is converted to covalently closed, circular (supercoiled) DNA (ccc DNA) by action of cellular enzymes. cccDNA, in the form of a histone-associated mini chromosome, provides a stable template for transcription.

Genomic and sgRNA’s are transcribed by host RNA polymerase II into a number of RNA size classes, some of which also show micro heterogeneity at the 5-end but all of which terminate at a common 3 polyadenylation site. The largest of these is translated to form precore protein and serves as the RNA template (“pregenome”) for reverse transcription. A slightly shorter RNA encodes the core protein and (by internal initiation) the polymerase protein. The polymerase protein associates with a specific encapsidation signal () on pregenomic RNA and this preassembly complex triggers assembly of core protein dimers into complete nucleocapsids.

Concurrently, the different classes of sgRNA’s are translated to produce the various surface gene products (L, M and S) which dimerise in the ER, oligomerise and bud into the lumen of a post ER/pre-Golgi compartment to give rise to empty particles.

Reverse transcription of pregenomic RNA takes place within cytoplasmic immature cores. This process uses the terminal protein domain of polymerase as primer for first strand synthesis and a short undigested oligoribonucleotide derived from the RNA template as the second strand primer, and, like retroviruses, first strand synthesis appears to be discontinous with translocation of a short segment of newly synthesised (-) DNA from the 5-end to the 3-end of the RNA template. A second template switch during (+) DNA synthesis leads to the formation of open circular genomes with a less than full length (+) strand, maintained by overlapping cohesive ends. Nucleocapsids containing partly reverse transcribed DNA may then either associate with cytoplasmically located pre S domains of the L envelope protein followed by budding into the ER as maturing virions, or alternatively may cycle back into the nucleus, thereby increasing the pool of cccDNA. While integration of viral DNA into the host genome is not required for replication and appears to be an infrequent event, integrated viral DNA, often containing deletions, inversions and reduplications is found in hepatocellular carcinoma (HCC) cells in culture and in patients as well as in apparently normal livers from chronic carriers.

Antigenic Properties

Three principal antigens have been identified for hepadnaviruses, designated surface, core and e antigen. These are abbreviated HBsAg, HBcAg, HBeAg for the HBV-related antigens, DHBsAg, DHBcAg, DHBeAg for DHBV-related antigens, etc while the corresponding antibodies are designated anti-HBs, anti-HBc, anti-DHBc etc. HBsAg is involved in neutralization. It cross-reacts to a limited extent with the analogous antigens of WHV and GSHV, but not with DHBsAg. PreS antigens may bear specific neutralisation determinants. S proteins are sufficient to stimulate protective immunity.

HBeAg and HBcAg proteins share common sequences and epitopes but also contain epitopes which distinguish these two proteins from each other. The HBeAg is a Mr 16 10³ truncated derivative of HBcAg. It is found as a soluble antigen in the serum of patients. HBcAg has been found to cross-react more strongly with the WHV core antigen than is seen with the corresponding surface antigens. In much of the earlier literature the term surface antigen or HBsAg is used arbitrarily to refer to either the antigenic specificity, various protein products of the preS1/preS2/S gene, or the empty 17-22 nm HBsAg-expressing particles. The term “antigen” should not be used if “protein” or “particle” is intended. Similar considerations apply to the use of “core antigen”.

Biological Properties

All hepadnaviruses show narrow host specificity. In vitro, replication of many hepadnaviruses has only been demonstrated following transfection of tissue culture cells by cloned DNA, resulting in the production of infectious virus. Replication of several hepadnaviruses has been achieved following inoculation of primary hepatocytes with serum that contains virus.

Hepadnavirus infections in vivo possess characteristic features:

1.

They are markedly hepatotropic; although viral antigens and nucleic acid replicative intermediates can also be detected in some extrahepatic sites (eg. pancreas, spleen, kidney, white blood cells) particularly with avihepadnaviruses, the liver remains the main site of virus production.

2.

Infection may be transient or persistent, the outcome depending on factors including host age and dose of inoculum. Persistent infections are normally long lived and can be accompanied by high levels of virions and subviral particles in the circulation.

3.

Empty virus-like particles, composed of excess virus envelope material, are present in much greater numbers than complete virions in most individuals and at most stages of infection.

4.

Virus replication is generally thought to be non cytopathic, and different degrees of ongoing liver damage in different individuals are thought to be governed by different degrees of immune-mediated damage to infected hepatocytes.

5.

In ortho-but not avi-hepadnavirus infections, persistent virus infection confers a significantly increased rate of primary hepatocellular carcinoma, and a number of direct and indirect mechanisms have been described.


Family	*Hepadnaviridae*
Genus	*Orthohepadnavirus*
Genus	*Avihepadnavirus*

1.	Virions and empty subviral particles may contain two or three envelope proteins, with common C-termini and differing N-termini due to different sites of translation initiation. Typically, virions contain a small (S) trans-membrane protein, (in many species) an intermediate sized M protein, and a large (L) protein which is myristylated at the N-terminus. In many cases more than one form of each of the above proteins occur due to alternative patterns of glycosylation. For HBV, virions and filaments are enriched in L proteins and empty spheres consist predominantly of S proteins, while for Duck hepatitis B virus (DHBV), L and S proteins are distributed evenly between particle types.
2.	The core protein has a large N-terminal domain and a small RNA-binding domain at the carboxyterminus. Core protein above a threshold concentration can self assemble via dimers to complete nucleocapsids in the absence of other viral components.
3.	The polymerase protein consists of an N-terminal domain (TP) with a DNA primer function, a spacer region of variable size, a reverse transcriptase and an RNase H domain. The TP domain is covalently attached to the 5-end of the minus strand of viral DNA.
4.	Most members of the family contain a fourth ORF (‘X’ gene) situated downstream of the S gene and partly overlapping the cohesive 5-terminal region. This codes for a non-structural protein that can function as a promiscuous transcriptional activator.
5.	Host proteins contained within virions include a protein kinase which phosphorylates the RNA binding domain of the core protein, heat shock protein Hsc 70 and heat shock protein Hsp 90 which at least in the case of DHBV appears to be part of a multicomponent chaperone complex involved in replication and nucleocapsid assembly.
6.	The core protein also exists in a secreted soluble form (‘e’ antigen) if co-expressed with the upstream pre-core sequence which can be translated from an additional start codon upstream of the core start codon.

1.	They are markedly hepatotropic; although viral antigens and nucleic acid replicative intermediates can also be detected in some extrahepatic sites (eg. pancreas, spleen, kidney, white blood cells) particularly with avihepadnaviruses, the liver remains the main site of virus production.
2.	Infection may be transient or persistent, the outcome depending on factors including host age and dose of inoculum. Persistent infections are normally long lived and can be accompanied by high levels of virions and subviral particles in the circulation.
3.	Empty virus-like particles, composed of excess virus envelope material, are present in much greater numbers than complete virions in most individuals and at most stages of infection.
4.	Virus replication is generally thought to be non cytopathic, and different degrees of ongoing liver damage in different individuals are thought to be governed by different degrees of immune-mediated damage to infected hepatocytes.
5.	In ortho-but not avi-hepadnavirus infections, persistent virus infection confers a significantly increased rate of primary hepatocellular carcinoma, and a number of direct and indirect mechanisms have been described.