 |
| Figure 1 - Electron microscope image of HDV purified from serum of infected woodchuck. |
History
The first clue to the existence of HDV came from an analysis of liver
biopsy samples taken from patients in Italy who had a more severe form
of hepatitis B virus (HBV) infection (79). It was observed that the nuclei
in liver biopsies from such patients often reacted with an antibody present
in their serum. This was called the d-antid reaction. It was initially
interpreted as an indicator for a variant form of HBV that could cause
a more severe form of HBV infection. To the contrary, later studies showed
that this protein was part of a separate infectious agent that had an RNA
genome and used the envelope proteins of HBV for assembly and release of
infectious particles from the liver into the serum (80).
Now HBV is the prototype of a group of related animal viruses that are called hepadnaviruses. One of these, woodchuck hepatitis B virus (WHV) is very similar to HBV, and so it was reasonable to test whether it could replace HBV as helper for HDV. It was soon found that in the presence of WHV, HDV readily replicated in woodchucks (74). Later, it was found that cultured primary woodchuck hepatocytes could be infected with HDV (98).
Helper Hepadnavirus
Hepadnaviruses are grossly inefficient in their assembly of particles.
Only one particle in 100-10,000 particles may be infectious and contain
a core structure with the required relaxed circular DNA genome and the
reverse transcriptase. The other particles contain no core structure. They
appear to be largely spheres and filaments with a 22 nm diameter. They
are composed of lipid and a mixture of the three forms of the hepadnavirus
envelope proteins. In the serum of an infected human the titer of infectious
particles might be as high as 109/ml. The non-infectious empty particles
might reach 1013/ml. It is sometimes argued that as far as the HBV is concerned
such an excess might help the virus avoid being cleared by a humoral response
of the host. Independent of the truth of this argument, it would seem that
HDV can redirect the assembly to package its own RNA and delta protein.
Titers of 1012/ HDV particles/ml of serum have been claimed (75).
There are data that at the peak of an acute infection, when HDV assembly and release is at its maximum, there is a corresponding major drop in the amount of released HBV. It is controversial as to whether this correspondence always occurs (70).
Infection
There is good evidence that HBV uses a host receptor that interacts
with the preS1 region, in the N-terminal domain that is unique to the large
form of the envelope protein. HDV, which is packaged by HBV envelope proteins,
seems to use the same preS1 domain (92). The embarrassment is that neither
for HBV nor HDV do we yet know the identity of the host receptor(s).
Now since HDV replication is dependent upon HBV acting as its helper virus one can imagine three possible time schedules for when the helper virus is provided: before, during or after. (i) A superinfection is defined as when a patient who is chronically infected with HBV, becomes infected with HDV. Since there are around 400 million chronic HBV individuals world wide, such a superinfection is quite frequent. It usually leads to an HDV infection that is also chronic. (ii) If the patient is simultaneously infected with both HBV and HDV, this is referred to as a coinfection. Such an infection can only go chronic for HDV if it also goes chronic for HBV. (iii) The third possibility is controversial. It is that a patient might be infected with HDV and then at some time afterward, be infected with HBV, which will now act as helper and rescue HDV from the HDV infected cells. This is called a latent HDV infection and there is evidence that it can be achieved in an experimental infection of woodchucks (70). It was once considered as theoretically possible for patients with HDV (and HBV) who receive a liver transplant, however, there is recent evidence that in practice, it does not occur (90).
HDV particles
Several early studies have tried to determine the physical properties
of the HDV particle. The sedimentation value, the density under different
conditions, and the average diameter have been determined (8, 83). By electron
microscopy, the particles are roughly spherical with a diameter of about
38 nm (39)(Figure 1).
|
| Figure 1 - Electron microscope image of HDV purified from serum of infected woodchuck. |
The outer envelope of the particle is composed of a mixture of the three
forms of the HBV surface proteins (8). This outer envelope, like that of
the HBV particles, must contain lipid since treatment with nonionic detergent,
removes the envelope proteins, and releases what behaves like a ribonucleoprotein
particle (RNP), containing the genome RNA and multiple copies of the delta
protein. The size and density of these RNP particles has been determined.
They are roughly spherical with an average diameter of 19 nm (83). It has
been deduced that in this RNP there are about 70 molecules of delta antigen
per molecule of genomic RNA (83). When treated with vanadyl ribonucleosides
the RNA can be eluted from such particles, but the size of the residual
delta protein structure is largely unchanged (24, 83).
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The genomic RNA is defined as that RNA present within the released virus
particles. It is a small (1679 nt) single-stranded RNA with a circular
conformation (18, 45, 102). From the nucleotide sequence it is predicted
to fold on itself, by Watson and Crick base-pairing, to form an unbranched
rod-like structure (Figure 2). About 70% of the nucleotides in this RNA
are involved in this structure (47, 102). Electron microscopic studies
of naked HDV RNA support the prediction of a rod-like folding (45). Also,
in electrophoresis the RNA behaves as if it were double-stranded (52).
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| Figure 2 - The three RNAs of HDV. |
Small and Large Delta
Antigens
There are two main forms of the delta protein, 195 and 214 aa in length,
with a common N-terminus (Figure 3). As explained later, the small form
is synthesized first, and later, as a consequence of events including post-transcriptional
RNA-editing, there can be translated the large form.
 |
| Figure 3 - Features on the two forms of the delta antigen. |
These proteins have both unique and shared functions. There are three shared properties. The first is a domain located at amino acids 12-60, which can act as a site for dimerization and even facilitate multimerization. The region is almost entirely alpha-helical in structure and from a solved crystal structure, it would seem to make dimers via what is called an anti-parallel coiled-coil interaction (106). Some of the predictions from this structure have been tested by the design of specific mutants (64) and of peptide inhibitors (26).
The second shared property is a bi-partite nuclear localization signal (49). Finally there is a bi-partite RNA binding domain (49). This RNA binding domain seems have some specificity for the rod-like folding of the genomic and antigenomic HDV RNAs. However, in vitro, when offered non-HDV RNA that is 100% double-stranded RNA, it will bind strongly (16).
In terms of unique functions, the small protein is essential for genome replication. In contrast, the large protein actually inhibits replication, and can act as a potent dominant negative inhibitor of replication supported by the small protein (17). In addition, the large protein has another unique role, in that it is essential for the late steps in virus assembly, as mediated by the envelope proteins of the helper virus (12, 82).
From a search for host proteins which interact in a two-hybrid assay with the delta protein, there was found a protein, subsequently named DIPA, for delta interacting protein A. It was further considered that this protein has features and properties which might indicate it was even an evolutionary precursor to the delta protein. And, it was proposed that the HDV genome arose via an interaction between the mRNA for DIPA and a hypothetical viroid-like RNA (9). However, such interpretations remain controversial (10, 57). There have been additional reports of host proteins which bind to the delta protein. For example, nucleolin has been reported. However it is not clear whether such binding is direct rather than RNA-mediated, nor is it clear that the binding has biological relevance (54). A similar claim has been made for interactions with glyceraldehyde 3-phosphate dehydrogenase (55).
There is evidence that both the small and large forms can be phosphorylated (3, 67). Possibly the large undergoes more phosphorylation. The significance of this modification for HDV replication is not known.
Farnesylation
Soon after it was found that the large delta protein is needed for
assembly of new virus particles and that the large delta protein contains
at a cysteine four amino acids in from its novel C-terminus, the potential
for isoprenyl modification was predicted and confirmed (34). Many host
proteins have such sites and by becoming modified, the proteins increase
in their ability to interact with lipid membranes. We now know that >80%
of the large delta protein gets so modified with a 15-carbon farnesyl group,
and that this modification is necessary (but not sufficient) for the protein
to be able to be assembled by the HBV envelope proteins (32, 65). The story
is not yet complete but sites have been mapped on the small HBV envelope
protein that must be present for the interaction with the HDV protein (43,
44).
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Stoichiometry
Attempts have been made to determine the stoichiometry of delta proteins
per molecule of HDV genomic RNA in various contexts. In virions it has
been determined as around 70, and in the ribonucleoprotein particles isolated
from transfected cells, at around 30 (83). A more recent study finds that
round 100-200 copies of delta protein per genomic RNA is the molar ratio
not only for particles but also within cells, under several different conditions
of HDV repication (38).
In contrast, in terms of what is needed to initiate replication there are data to indicate that the ratio can be as low as about 1:1 (23). Our interpretation is that the role of the small delta protein in initiation is quite different from its role later on, during the accumulation of processed HDV RNA species.
Classification and Genotypes
It is inappropriate that we refer to HDV as a virus (22). Actually
because of its helper requirement HDV does not satisfy the definition of
a virus. It should be called a subviral agent. Also since it shows no recognized
sequence homology to the genome of its helper virus, it should be called
a satellite of HBV. In full, it should be called a "subviral satellite
agent with HBV as its natural helper virus".
No other agent quite like HDV has been described and so for. The viroids and virusoids of plants are in some ways similar, and yet still distinct. Thus, for classification purposes, HDV was initially left out by itself as "unclassified". More recently, it has been adopted and classified together with the hepadnavirus family.
Now that many nucleotide sequences of HDV isolates have been obtained it has been possible to use computer analyses and divide such isolates into three groups, referred to as genotypes (11). Genotypes I and II are much more common, for example in parts of Asia and Europe. Originally it seemed there were some locations where the genotype was preferentially of either genotype I or II. More recently, such specificity has become more cloudy. Genotype III is the least common, having only been reported in isolates from South America.
Replication
In nature, HDV only replicates in the liver of an infected individual.
We presume that this hepatotropism is largely due to the availability of
the (as yet unidentified) receptor on such liver cells. We know that in
experimental systems, where the replication is initiated in a receptor-independent
manner, for example by transfection, it is possible to achieve HDV genome
replication in non-liver cells (97).
The replication of the HDV genome is considered to take place largely in the nucleus. In support of this interpretation the delta protein and the genomic and antigenomic RNAs do seem to be predominantly localized to the nucleus (2, 6, 20, 98). If the delta protein is expressed in the absence of HDV replication, it localizes to the nucleolus (50). However, after the initiation of genome replication the protein moves out into the nucleoplasm with a tendency to localize preferentially to discrete sites that are called "speckles" which contain specific host proteins, such as SC35, a known RNA splicing factor (6). The current thinking is that the delta speckles are sites of accumulation of delta antigens and processed RNAs but not sites of HDV RNA transcription (20).
During HDV genome replication there usually appear variant forms of the small delta protein, including the large delta antigen. This appearance correlates with the loss of colocalization of small delta protein with the SC35 speckles (2, 6, 20).
Recent studies have applied cell fractionation to better determine the intracellular distribution of delta antigen and RNAs. Such studies indicate that delta antigen and antigenomic RNA are largely nuclear, but surprisingly, the genomic RNA is >50% in the cytoplasm (38).
Replication Systems
There are a lot of experimental systems for studying parts of the HDV
replication cycle but very few that cover the whole cycle. The only examples
of the latter, are infections of primates or of woodchucks. In the woodchuck
infections the helper function is carried out by woodchuck hepatitis B
virus (74). There has been no success in using other hepadnaviruses (such
as duck hepatitis B virus) to provide the helper function.
It is difficult but possible to culture primary hepatocytes from primates and woodchucks. Such cells can be infected by HDV and as a superinfection it is theoretically possible to achieve a full cycle of HDV replication.
Injection of HDV particles into a mouse can lead to infection in a small fraction of hepatocytes (69), but there is no evidence as to how this is initiated. That is, whether or not it is receptor-mediated.
Transfection Systems
If one totally obviates the need for receptor-mediated uptake of HDV
sequences, for example by using a transfection procedure, then replication
of the HDV genome can be achieved in many different types of cultured mammalian
cells. Such transfection can be initiated with both DNA (46) and RNA sequences
(33, 62).
Not surprisingly, when HDV sequences are transfected into the liver of a chimpanzee chronically infected with HBV, there ensues what looks like an HDV superinfection (93). Similar studies have been reported for transfection of woodchucks already infected with WHV (77, 105).
There is a report that with mice made transgenic for HDV DNA sequences, HDV genome replication can be detected in multiple tissues, including the liver (72). And, when the same DNA sequences are directly injected into the skeletal muscle of a mouse, again the accumulation of processed HDV RNA sequences can be detected (38, 72).
When using HDV DNA in transfections, the sequences can be as little as unit-length, with or without flanking non-HDV sequences. When using linear HDV single-stranded RNA, there is at the outset an essential caveat relating to the provision of the small delta protein which is needed for genome replication. Three choices can be distinguished. First, the recipient cells can be already expressing this protein (33). Second, the RNA can be admixed with delta protein prior to transfection (23). A variation of this is that natural RNP complexes of delta protein with genomic RNA, as isolated from virions, can be used to transfect cells and initiate genome replication (4). Recently, a third and surprising choice has been shown. The greater than unit length HDV RNA can be co-transfected with a mRNA that is capped and polyadenylated and can be directly translated to make small delta protein. This is called a 2-RNA combination (62). It is also possible to transfect mouse hepatocytes in vivo using an intravenous injection of either HDV sequences or the 2-RNA combination (15).
Transcription and Replication
For other RNA viruses the terms "transcription" and "replication" have
specific and distinct meanings. Such differences have not been maintained
for HDV.
Three HDV RNA species are readily distinguished during HDV genome replication:
these are the genome, the antigenome, and the mRNA. Note however that these
three are each processed RNAs rather than primary transcripts. The antigenome
is an exact complement of the genome (Figure 2). It is moreover circular
and possesses its own ribozyme, related to but different from the ribozyme
of the genome (81).
|
| Figure 2 - The three RNAs of HDV. |
At the peak of replication in an infected liver there are about 100,000
copies of the genomic RNA per average liver cell (18, 38). The antigenomic
RNA is about 10-times less abundant. The third RNA is about 500-times less
abundant. It also is complementary to the genome, but at about 800 nt,
it is less than full-length (Figure 2). It has a 5'-end that is located
at nt 1630 (36)(Figure 4) and capped (37). At the 3'-end there is a poly(A)
sequence, which seems to be like host mRNA species in that near to the
3'-end is the sequence AAUAAA (40), which is considered to be a signal
for polyadenylation. This structure and location of this RNA indicates
that it is the mRNA for the small delta protein. The posttranscriptional
modifications of 5'-capping and 3'-polyadenylation may be considered as
indirect evidence that the host polymerase II is involved in the transcription.
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Initiation
As mentioned above, the majority of the HDV RNAs detected in infected
cells are processed to unit-length (99.8%) and in most cases these are
circular in conformation. Being already processed to circles, such RNAs
thus leave no clue as to where their RNA transcription might have initiated.
In contrast, the much less abundant mRNA species has a 5'-end that is not
only largely focused to one site, nt 1630, but also capped (37)(Figure
4). Several labs have thus concluded that this site represents a site of
initiation (1, 40). Furthermore, they have presumed to look in the adjacent
RNA sequence and predicted RNA structure, for what might be acting as a
promoter for such transcription. Without question, mutagenesis has shown
that the nearby region is important, but the putative promoter has not
been found (1, 36, 104). Maybe a determining feature is that this site
is just 10 nt from the one end of the predicted rod-like folding of the
template RNA (36).
 |
| Figure 4 - The folding at the top of the rodlike genomic RNA together with the 5'-end of the antigenomic mRNA. |
Some studies have found that either genomic RNA or antigenomic RNA can
be used to initiate replication in transfected cells (23). In contrast,
there are claims of specific differences (89) and equally puzzling claims
that the effect of the large delta protein as an inhibitor depends upon
which RNA strand is used for the initiation (61).
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Polymerase
What is the host polymerase which copies the HDV RNA into RNA? This
is not a simple question because all the known mammalian host polymerases
normally use DNA templates to transcribe RNA. With this caveat, there are
three candidate classes of RNA polymerases (pol). (i)There are the multi-subunit
enzymes known as pol I, II, and III (25). (ii) Another candidate is the
nucleus-encoded single-subunit polymerase that acts to transcribe RNA in
mitochondria (100). (iii) We can speculate about one more candidate: recently
a true RNA-directed RNA polymerase (RDRP) has been cloned and sequenced
from tomato (85). This polymerase sequence has thus been seen to be present
in all plants and some animals. Furthermore there is good genetic evidence
for its role in post-transcriptional gene silencing (PTGS). However, at
this time, even though there are at least two examples of PTGS in mammalian
cells (94, 103), a putative mammalian equivalent of the RDRP has not (yet)
been found.
Several studies have tried to assemble in vivo or in vitro data to identify the polymerase used in HDV transcription (1, 28, 30, 59, 63). While the interpretations favor pol II, the data are not definitive.
One in vivo approach used in attempts to identify and characterize the polymerization of HDV RNA, is the application of pol II inhibitors, especially amanitin, to cells in which HDV genome replication has been initiated either with DNA sequences or the above-mentioned 2-RNA strategy. Another approach has been to carry out transcription in vitro, either with nuclear extracts or purified pol II to which is added HDV RNA as template. The third approach is like a combination of the first two. It is to initiate replication via transfection and then make nuclear extracts and characterize the in vitro transcription that can be achieved from what are now endogenous HDV RNA templates. The results so far, for the in vivo and vitro approaches, have usually been interpreted as evidence for pol II copying the HDV RNA. However, a personal opinion, is that the data are not yet good enough.
The role of the small delta protein in the transcription process has not yet been clarified. It has been claimed that there is no obligate requirement but such studies are flawed (59).
In summary, the identification of the host polymerase(s) involved in HDV genome replication is not complete or watertight. At this time pol II is generally regarded as the best candidate. This is based in vivo and in vitro experiments, which provide indirect and direct evidence, respectively (66). There is also circumstantial evidence, such as the mRNA that has a 5'-end at a specific location and is post-transcriptionally processed, with a 5'-cap and a 3'-poly(A), just as is typical of pol II transcripts (37). This being said, there is the partially contrary report which interprets pol II as being needed for mRNA and genomic RNA synthesis with a second and different polymerase being needed for genomic RNA synthesis (63).
Ribozymes and Ligation
As mentioned earlier, both the HDV genome and antigenome contain a
short sequence that in vitro will carry out a site-specific cleavage reaction
(48, 87). These two ribozymes have been trimmed to 84 nt in length and
still function in vitro. They are related to each other in sequence and
predicted folding (81). A crystal structure has been determined for the
genomic ribozyme (27)(Figure 5). While HDV is the only animal "virus" with
(not just one, but two) ribozyme activities there are numerous plant agents
which contain ribozyme activities (95). Such plant ribozymes do not show
any detected similarity to HDV in primary sequence or secondary structure.
However, in terms of chemical mechanism they are similar in that cleavage
produces ends with a 5'-OH and a 2'-,3'-cyclic monophosphate (87). The
cleavage reaction is usually found to depend upon the presence of magnesium
ions, although at lower pH it becomes independent of added divalent metal
ion (101).
 |
| Figure 5 - Genomic ribozyme of HDV. Two-dimensional representation derived from three-dimensional crystal structure (27). |
Somehow, after ribozyme cleavage, the unit-length HDV RNAs are converted to circular forms. This is also true for many of the subviral plant agents. In a way this outcome is deceptive because the RNAs that are not circularized are at a significant survival disadvantage. For example, it has been shown that in a cell extract a circular RNA can be 300-times more stable than the corresponding linear species (76).
How the linears become circularized is an issue. For some the RNAs of some plant agents the ribozyme cleavage is followed, at significant efficiency, by a protein-independent ligation. This has not been demonstrated for an HDV RNA, except in vitro as a low efficiency event (88). Instead, it is considered that in vivo a host RNA ligase activity is needed to convert the linear to a circle (78).
While being in a circular conformation does have survival advantage for an RNA species, it is not sufficient. The HDV circle still contains the full sequence of a ribozyme and you might think it could cleave again. In truth, it can be made to recleave, but under natural conditions this circle folds into a conformation, the unbranched rod-like structure, which is incompatible with active ribozyme activity (53). Even then, the circle is susceptible to host endonuclease activity; such specific cleavage has been recently detected on HDV RNAs in the liver of an infected animal (13).
It has been suggested that the delta proteins can act in vivo to facilitate cleavage by the two HDV ribozymes (42) and also act likewise in vitro (41). However, the interpretation of such observations is controversial since it is clear that cleavage in vitro does not require the delta proteins (52).
Translation
As mentioned earlier, the 800 nt polyadenylated species is considered
to be a mRNA for the translation of the delta protein (Figure 2). At the
same time, there are in the infected cell other polyadenylated RNA species
with 5'-ends upstream of nt 1630 (37). These might also act as mRNAs. We
might be detecting the 800 nt species because of its specific size and/or
because of some specific stabilization advantage, such as 5'-capping (37).
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Editing
For HDV replication to take place there must be adequate amounts of
the small delta protein. However, as mentioned earlier, there arises during
genome replication, a 19 aa longer form which we call the large delta antigen
(58, 93). We now know that this is a consequence of post-transcriptional
RNA editing. The target for the editing is the adenosine at nt 1012, in
the UAG amber termination codon in the open reading frame for small delta
protein, as located on antigenomic RNA. This adenosine is converted to
inosine by an enzyme of a class now referred to as ADAR, adenosine deaminase
acting on RNA. In humans there are at least two such activities. They have
important roles for the host cell. That one known as ADAR-1 has recently
been shown to be the natural ADAR acting on HDV (51, 84). It is worth noting
that if ADAR-2 is overexpressed, it wll also act on HDV antigenomic sequences
with both efficiency and site-specificity (84).
It is considered, but not established, that after the amber codon is modified there has to be two rounds of RNA-directed transcription which effectively replaces the UIG with UGG. On the mRNA UGG encodes tryptophan and so it is now possible to translate the long form of the delta antigen. An intriguing and simpler alternative explanation is that just like for most host RNAs, the ADAR editing can occur soon after transcription, even prior to most RNA processing. More specifically, it could be that a nascent antigenomic RNA transcript undergoes ADAR editing even prior to RNA cleavage and polyadenylation (60).
This editing site, at nt 1012, is not the only site at which HDV RNA is edited. Nor is all editing restricted to the antigenomic strand (71, 73). However, this site on the antigenome is obligatory: without it the large delta protein that is needed late in replication, is not made.
The levels of editing at nt 1012 can reach almost 50% in infected cells. Since the large protein does not support the initiation of HDV replication we would expect that only the remaining unedited RNAs are the ones that can be used in subsequent re-infections.
Recent studies have detected what might be site-specific RNA editing on HDV by a mechanism other than ADAR. It would appear that in many situations a cytidine deaminase acting on RNA, a CDAR, can efficiently target nt 137 on genomic RNA and convert C to U (38).
Variant Proteins
From the above it might seem that there are only two species of delta
protein. This is probably an oversimplification. For exmple, there are
some reproducible studies in which other open reading frames on the HDV
genome also lead to the translation of protein, and in patients, this translation
can lead to corresponding antibodies (5).
The above-mentioned variant protein only arises in a small fraction of the cells undergoing HDV replication. There are yet other variant proteins that arise during genome replication; these can be inferred from the aberrant intracellular distributions of proteins related to small delta protein that can be detected by immuno-microscopy (6).
Assembly
In a natural primate infection HBV provides the envelope proteins needed
for assembly. As mentioned earlier, in an experimental setting the envelope
proteins of WHV can be used for assembly of particles that have the new
potential to infect woodchucks. No other hepadnavirus or animal virus has
been reported to act as a source of envelope proteins.
In studies with transfected cells it is possible to achieve assembly into particles of both genomic and antigenomic RNA species (52). However, in natural infections the assembly process is specific for genomic RNA relative to antigenomic RNA. We do not know why this is. Maybe there is a packaging signal in the genomic RNA. Alternatively, the antigenomic RNA might not be accessible for it to be assembled.
It is possible to get assembly using only the small form of the HBV envelope protein but such particles are noninfectious, since it is only on the unique preS1 domain of the large envelope protein that there is the domain which interacts with the as yet unidentified HBV receptor (92).
As mentioned earlier, assembly also requires the large form of the delta protein, and moreover, this protein must be modified by farnesylation at a unique cysteine 4 residues in from the C-terminus.
It remains controversial as to whether HDV infection is directly pathogenic to liver cells (7, 35). In experimental systems expression of the small or large forms of the delta protein in a cell can cause a reduction in the growth potential or even toxicity (19). And, when the small protein is expressed in certain avian cells, it induces significant levels of apoptosis (14). A different interpretation has been made for the restricted replication of HDV in avian cells (56).
There are reports that at the peak of an HDV infection there is a suppression of HBV assembly (91). Such suppression was not seen for WHV in woodchucks super-infected with HDV (71).
Epidemiology
As mentioned earlier, HDV is only spread when there is a source of
the helper virus, HBV. If the new host receives both HBV and HDV this is
considered a coinfection. However, if the host was already chronically
infected with HBV, then addition of HDV is considered a superinfection.
A superinfection is more likely to become chronic simply because the HBV
infection is already chronic. In contrast, the chance of a coinfection
going chronic, is intrinsically limited by the chance of the helper virus
HBV infection going chronic.
At one stage it was considered that maybe 25% of the world's chronic HBV carriers (now about 400 million) were also infected with HDV. With the exception of intravenous drug users, the current percentage is probably much lower than this. The incidence of new HDV infections is now low, and the average age of those with chronic infections is progressively increasing (31).
Vaccination
Since HDV appears to use the same (unidentified) receptor as HBV, and
is strictly dependent upon HBV for assembly, it is not surprising that
appropriate vaccination against HBV will simultaneously vaccinate against
HDV.
With the intent of possibly being able to prevent chronic HBV carriers from being superinfected with HDV, some experiments have been done to vaccinate with the delta protein. Such tests have used the woodchuck model of HDV and WHV. The results are not clear as to whether such vaccination is of value.
However, the good news is that probably because of increasing levels of HBV vaccination and because of behavioral changes amongst at risk populations, the incidence of new cases is dropping. HDV infection is now being referred to as a "disappearing disease" (31).
Plant Agents
Almost soon as it was found that the small single-stranded RNA genome
of HDV was circular in conformation, it was recognized that HDV was like
certain small subviral agents of plants, that are known as viroids (22).
These plant agents have small (200-400 nt) single-stranded RNAs that are
circular in conformation and in many cases fold into an unbranched rod-like
structure. These agents replicate by RNA-directed RNA synthesis using host
polymerases. For some, the host polymerase is considered to be a nucleus-encoded
chloroplast enzyme (68) and for others, it is considered to be the nuclear
pol II (86). Only several of the viroids possess two ribozymes that are
known to function in vitro (95).
Without question, the analogy of HDV to the viroids has been and continues to be a major force in testing hypotheses regarding such things as the origin and the mechanism of replication. However, there are two major differences that must be kept in mind. The genome of HDV is several times larger than even the biggest viroid, and HDV unlike all the viroids, encodes an essential open reading frame.
There are other subviral plant agents which can be considered as slightly more sophisticated than viroids. One group is defined as subviral satellite RNAs. These RNAs replicate because of a coinfection with by a plant virus that provides the RNA polymerase needed for RNA transcription. Some of these subviral satellite RNAs also have a small single-stranded RNA genome that is circular, and for a while these agents were referred to as virusoids (22, 96). Another subgroup of subviral satellite RNAs have a small single-stranded RNA genome with a circular conformation, but they are helped by a virus that provides a reverse transcriptase rather than an RNA polymerase (21).
Model of Replication
In virology text books and lectures there is usually a demand to show
a model of replication for the viral genome. Such models have been drawn
up for HDV. The original was adapted from a model that is considered relevant
for some but not all of the plant viroids. It is known as a double rolling-circle
model. The HDV version is more complicated in that it attempts to explain
not only the circular genomic and antigenomic RNAs but also the 800 nt
polyadenylated species that is considered as a mRNA for the delta antigen
(99)(Figure 6). This model has many speculations and not surprisingly,
it has been the subject of criticism (62). However, the model continues
to be the basis for further experimentation.
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| Figure 6 - Double rolling-circle model of genome replication (29). |
Outlook
One would hope that in the near future there will be significant clarification
of the processes by which HDV and the viroids replicate. Which host polymerase(s)
is involved and how does it recognize the RNA template. There may be some
common elements to these replication schemes. We might also be able to
perceive how these agents arose. And, with some effort, we might find how
such knowledge can be usefully applied in both animal and plant situations.
HDV can be considered as a parasite of a parasite (HBV). Possibly more than any other infectious agent of animals HDV is dependent upon redirecting host functions. There are major implications for the likely possibility that the host pol II is involved in HDV transcription. Recent studies of pol II transcription make clear that there is a coordination of transcription with capping, splicing, 3'-end modification, and possibly ADAR editing (60). Maybe all of these are also coordinated in the HDV life cycle! And, of even more significance, HDV has ribozyme activities and transcription that is RNA-directed.
Acknowledgments
Some of the results mentioned above are unpublished studies from my
lab. They represent the works of Jinhong Chang, and Severin Gudima. I would
also like to thank them for helpful comments on this document. Michael
Massimino helped in the initial placement of this document on the web.
This file was last updated March 11, 2002.
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Comments
If you have any comments, questions, or corrections, please forward
them to JM_Taylor@fccc.edu.
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