Global Alert and Response (GAR)

Hepatitis D


The hepatitis Delta virus

- Morphology and physicochemical properties
- Genome and proteins
- Antigenicity
- Stability

The genome of HDV is unrelated to the genomes of hepadnaviruses, of which hepatitis B virus (HBV) is a member. HDV is therefore not a defective-interfering particle of HBV, and should be considered as a satellite virus, a natural subviral satellite of HBV.10, 11, 14, 25

Important parallels can be drawn between HDV and certain subviral agents of plants, especially the viroids, with respect to genome structure and replication mechanisms. Because of the many differences however, HDV has been classified into the separate genus Deltavirus.13, 18, 25

The genome of HDV was cloned and sequenced in 1986.27 HDV is a replication defective, helper (HBV) dependent ssRNA virus that requires the surface antigen of HBV (HBsAg) for the encapsidation of its own genome. The envelope proteins on the outer surface of HDV are entirely provided by HBV.16, 18, 25

The outer envelope of HDV particles actually contains lipid and all three forms (S, M, and L) of HBV surface antigen (HBsAg), but predominantly the major form of HBsAg with very few middle (pre S1) and large (pre S2) proteins. This proportion (95:5:1 of S:M:L) is different from that found in HBV particles.13, 14, 25

There is no evidence that the HBV-derived envelope proteins are additionally modified when they become the envelope of HDV.21

The internal, nucleocapsid structure of HDV is composed of the viral single stranded RNA genome and about 60 copies of delta antigen, the only HDV-encoded protein, in its large and small forms.13, 25

Synthesis of HDV results in temporary suppression of synthesis of HBV components.13

HDV does not infect established tissue culture cell lines. Complete viral replication cycles in vitro are limited to primary hepatocytes, generally of woodchucks or chimpanzees, that are coinfected with a hepadnavirus or cotransfected with hepadnavirus cDNA. When experimental conditions meet these requirements, infectious HDV particles are produced.13, 14, 21

In nature, HDV has only been found in humans infected with HBV. Experimentally, it can be transmitted to chimpanzees and woodchucks in the presence of HBV or woodchuck hepatitis virus (WHV), respectively.13, 18, 19, 21, 24, 25

Click here for: Hepatitis Delta virus replication cycle
Click here for: Genome replication model
Click here for: A model for RNA editing of HDV

Morphology and physicochemical properties
HDV virions are 36 - 43 nm, roughly spherical, enveloped particles with no distinct nucleocapsid structure. They do not have distinct spikes on their outer surface and are possibly icosahedral.13, 14, 25

When the virus particle is disrupted with nonionic detergents, an internal nucleocapsid is released and HDAg becomes detectable.13, 25

The 19 nm nucleocapsid contains about 60 copies of HDAg in its two forms (24 and 27 kDa) and HDV genomic RNA.25

The buoyant density of HDV particles is 1.25 g/cm3 in CsCl gradients.13, 14, 18

Click here for: Schematic representation of viral particles found in serum of HBV - HDV infected people

Genome and proteins
The HDV genome is a single, negative stranded, circular RNA molecule nearly
1.7 kb in length containing about 60% C+G.13, 14, 18, 25

HDV RNA is the only animal virus known to have a circular RNA genome.13

A high degree of intramolecular complementarity allows about 70% of the nucleotides to be basepaired to each other to form an unbranched, double-stranded, stable, rod-shaped structure.10, 13, 14, 18

So far, about 14 different HDV isolates from different parts of the world have been sequenced, and all range from 1670 to 1685 nucleotides in length. Based on sequence similarities, HDV isolates can be classified into three genotypes.13

Genotype I is the most predominant one in most areas of the world, and is associated with a broad spectrum of chronic HDV disease. Originally found in a Japanese isolate, genotype II has been found recently to predominate in Taiwan. Disease associated with genotype II might be less severe than genotype I. Genotype III is associated with outbreaks in Venezuela and Peru. It is responsible for more severe disease in the northern South American regions.5, 10, 11, 13

The genome contains several sense- and antisense open reading frames (ORFs), only one of which is functional and conserved. The RNA genome is replicated through an RNA intermediate, the antigenome.13, 14, 18

The genomic RNA and its complement, the antigenome, can function as ribozymes to carry out self-cleavage and self-ligation reactions.13, 18, 25

A third RNA present in the infected cell, also complementary to the genome, but 800 b long and polyadenylated, is the mRNA for the synthesis of the delta antigen (HDAg).14, 18, 25

The one and only protein expressed by HDV, the hepatitis delta antigen HDAg, is not exposed on the virion outer surface, but is present in the internal nucleocapsid.13, 14, 18

The protein is seen as two species, of 24kD and 27kD. The two species are identical, but the 27kD protein has a 19 aa longer C-terminus. The short form (195 amino acids, HDAg-S), synthesized first, is required for RNA replication; the long form (214 amino acids, HDAg-L), becoming detectable after prolonged replication, suppresses viral RNA replication and is required for packaging of the HDV genome by HBsAg.13, 14, 18, 21, 23

The relative ratios of these two species vary from patient to patient. Two separate ORFs on different RNAs encode HDAg-S and HDAg-L. A single nucleotide at the termination codon for HDAg-S is altered by a specific posttranscriptional RNA editing event in some RNAs, so that the ORF extends for 19 additional amino acids.13

HDAg is a non-glycosylated phosphoprotein.13, 21, 25 It has an RNA-binding activity and appears to bind specifically to HDV RNA in the virus particle.28 In infected cells, HDAg is localized in the nuclei.13, 14, 25

Functional domains present in HDAg include the nuclear localization signal located within the N-terminal one-third of the protein, the RNA-binding motif present in the middle one-third of the protein and a third domain, consisting of the C-terminal 19 amino acids, possibly involved in interactions with the HBsAg during virion assembly, and in the inhibition of HDV RNA assembly.13, 14, 25

The other protein present in HDV particles is HBsAg. This protein is derived from the coinfection with HBV and is essential for HDV virion assembly and virus transmission.

Click here for: The three RNAs of HDV present in the infected cell
Click here for: Comparison of HDV genotypes

Antigenicity
The intact virus particle is reactive with anti-HBsAg antibody, but not with anti-HDAg antibody.

Despite the sequence heterogeneity observed in HDV isolates from different geographical regions, there appear to be no serological differences among these isolates. 14

All HDV are antigenically related, and antibodies to HDAg do not neutralize HDV.21

Surface epitopes unique to HDV have not been detected.

Under experimental conditions, HDV can use different hepadnaviruses as helpers. In each case, the envelope of HDV has both the physical and antigenic characteristic of the helper virus.


Stability
Because of its double-strandedness, the HDV RNA is relatively stable.

The hepatitis delta virus survives dry heat at 60°C for 30h.14

 


Hepatitis Delta virus replication cycle
To replicate efficiently, a virus requires the cooperation of the host cell at all stages of the replicative cycle: attachment, penetration, uncoating, provision of appropriate metabolic conditions for the synthesis of viral macromolecules, the final assembly of viral subunits and the release of new virions. HDV also requires the presence of a helper hepadnavirus to provide the protein components for its own envelope.

How HDV enters hepatocytes is still not known, but it may involve the interaction between HBsAg-L and a cellular receptor. The incoming HDV RNA is then transported into the nucleus, the site of genome replication, probably by the small form of delta antigen, HDAg-S. Binding of HDAg to RNA also protects the HDV RNAs from degradation. 13, 14, 25

HDV RNA replication is carried out by cellular RNA polymerase II, without a DNA intermediate, and without the help of HBV. Replication proceeds via a double rolling-circle model. The genomic strand which is of negative polarity yields an oligomeric linear structure with site-specific autocatalytic cleavage and ligation. This structure generates circles of the opposite positive strand polarity, which again replicate in the same way and produce the genomic negative RNA. The only functional open reading frame which codes for the two HDV structural phosphoproteins occurs in the antigenomic strand at one end of the HDV RNA rod. It is 800 bases long and terminates at a polyadenylation site. 10, 13, 14, 17, 18, 25

RNA transcription is regulated: initially, mRNA(s) is(are) transcribed from the incoming minus-strand genome and later, after the translation of the mRNA to make essential replication proteins, there is a switch in the mode of RNA-directed RNA synthesis to facilitate replication of the RNA genome.13, 25

Translation of the 800 b RNA transcript yields a small (p24) and a large (p27) form of HDAg. These two proteins, known as short (HDAg-S) and long form (HDAg-L) of HDAg, have very different functional roles during viral replication. The HDAg-S is a transactivator of HDV RNA replication, while the HDAg-L inhibits RNA synthesis and initiates virion assembly with HBsAg.8, 10, 13, 14, 21, 23, 25

The production of HDAg-S, as opposed to HDAg-L, depends on the extent of HDV RNA editing. A specific modification at nucleotide position 1012 from A to G changes the UAG stop codon of the transcript to UGG (tryptophan), allowing translation to continue for another 19 amino acids. The target of editing is the antigenomic strand; the adenosine is converted to guanosine via inosine by a cellular double-stranded RNA adenosine deaminase. The intracellular ratio of p24/p27 will determine the extent of viral replication, assembly and transport.5, 10, 13, 20, 25

Since HDV particles consist of HBsAg, HDAg-S and HDAg-L, and RNA, they are assembled only in the presence of the helper virus, HBV. HBsAg and HDAg-L are necessary and sufficient for virus assembly, whereas HDV RNA or HDAg-S are not required, but are certainly present, in viral particles.25 The basis of selectivity of RNA packaging in vivo is not yet clear, and although HDAg can interact with both genomic- and antigenomic-sense HDV RNA, only genomic-sense RNA is found in viral particles.13, 23, 25


The primary initiation event for HDV assembly is the interaction of HDAg-L with HBsAg, which is determined by the presence of the C-terminal 19 amino acids of HDAg-L and the prenylation of the cystein residue 211 on HDAg-L.9, 25 However, HDAg is localized in the nuclei, and HBsAg is present in the cytoplasm of the infected cells. How these two proteins in different cellular compartments come into direct contact remains a puzzling issue.12, 13

A speculation is that the genomic RNA, assembled into a ribonucleoprotein (RNP) involving both HDAg-S and HDAg-L, interacts with HBsAg already inserted in the membranes of the endoplasmic reticulum. This would then be followed by the passage of assembled particles onto the Golgi apparatus, and the release of virions from the cell, without direct toxicity.13, 25

Genome replication model

From: Modahl LE and Lai MMC. Transcription of Hepatitis Delta antigen mRNA continues throughout hepatitis Delta virus (HDV) replication: a new model of HDV RNA transcription and replication. Journal of Virology, 1998, 72(7):5449-5456,17 "Copyright (1998) American Society for Microbiology", with permission.

Proposed models of HDV RNA transcription and replication. (A) The previously accepted model of HDV RNA transcription and replication. The initial product of replication from the genomic HDV RNA template is the 0.8 kb HDAg-encoding mRNA (arrow 1). HDAg produced from this mRNA suppresses the HDV polyadenylation signal, allowing synthesis of multimeric RNA (arrow 2)., which is processed into full-length antigenomic HDV RNA (arrow 3). Subsequent rounds of replication bypass the polyadenylation signal due to the presence of HDAg and directly synthesize full-length antigenomic HDV RNA (arrow 4). (B) Proposed new model for HDV RNA transcription and replication. The syntheses of 0.8 kb mRNA (a) and 1.7 kb monomer RNA (b) are independent and occur in parallel.17, 25

 

A model for RNA editing of HDV


Reprinted by permission from Nature (Polson AG, et al. RNA editing of hepatitis delta virus antigenome by dsRNA-adenosine deaminase. Nature, 1996, 380:454-45620) copyright (1996) Macmillan Magazines Ltd. (http://www.nature.com).

A model for RNA editing of HDV. (1) Replication-competent genomes are transcribed to produce an mRNA encoding HDAg-p24. (2) HDAg-24 enables replication of the genome by RNA polymerase II, generating antigenomic RNA. (3) dsRAD (double-stranded-RNA-adenosine deaminase) acts on antigenomic RNA to convert the adenosine at the amber/W site to an inosine. (4) Like G, inosine prefers to pair with C; thus, after replication, the genome has a C at the amber/W site instead of a U. (5) The edited genome is transcribed to yield an mRNA encoding HDAg-p27. HDAg-p27, which contains a 19 amino acid extension (shaded), inhibits replication and helps packaging of the HDV genome by HBV surface antigen.20


Schematic representation of viral particles found in serum of HBV - HDV infected people

Infectious HBV particle:

 

  • 42 nm outer envelope containing lipid and three forms of HBsAg
  • 27 nm nucleocapsid containing 180 copies of core protein and reverse transcriptase and HBV DNA

Infectious HDV particle:

 

  • 36 - 43 nm outer envelope containing lipid and three forms of HBsAg
  • 19 nm nucleocapsid containing 60 copies of delta antigen and HDV genomic RNA

Empty noninfectious particles:

 

  • 22 nm filaments and spheres made of lipid and mainly one form of HBsAg

 

From: Taylor JM, Hepatitis Delta virus and its replication. In: Fields Virology, 3rd ed. Philadelphia, Lippincott - Raven, 1996:2809-2818,25 with permission (http://lww.com).


The three RNAs of HDV present in the infected cell




From: Lai MCC. The molecular biology of hepatitis Delta virus. Annual Review of Biochemistry, 1995, 64:259-286.13 "With permission, from the Annual Review of Biochemistry, Volume 64 ©1995 by Annual Reviews http://www.AnnualReviews.org ".

Schematic diagrams of the structure of HDV RNA. The antigenomic RNA and mRNA are detected only in the cells. The nucleotide numbers are according to Reference 16 and represented in genomic orientation even on the antigenomic strand. The genomic RNA is represented in clockwise orientation, while antigenomic RNA is counterclockwise. Nucleotides 688/689 and 903/904 are ribozyme cleavage sites for genomic and antigenomic RNAs respectively. The hatched boxes represent the ribozyme domain. Nucleotide 1015 (Ed) denotes RNA editing site. (A)n represents polyadenylation signal. The UV cross-linking site is indicated by a vertical line in the viroid domain.13

Comparison of HDV genotypes



From: Casey JL et al. Molecular biology of HDV: analysis of RNA editing and genotype variations. In: Viral Hepatitis and Liver Disease, Turin, Edizioni Minerva Medica, 1997:290-294, 5 with permission.

1. Genetic variability, tentative disease associations, and geographic distributions of HDV genotypes I, II, and III.5


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From: Casey JL et al. Molecular biology of HDV: analysis of RNA editing and genotype variations. In: Viral Hepatitis and Liver Disease, Turin, Edizioni Minerva Medica, 1997:290-294,5 with permission.

2. Comparison of virion formation, RNA editing and RNA replication for HDV genotypes I and III.5

 

 

 

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