Reviews in . - . - - . . . MEDICALVIROLOGY VOL. 2: 161-167 (1992)

Hepatitis Delta Virus: from Structure to Disease Expression J. Taylor Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA

F. Negro Division of Gusfroenterology, Molinette Hospital, Corso Brumnte 88, Italy

M. Rizzetto* Institute of Internal Medicine, University of Twin School of Medicine, Corso A. Dogkotti 14, I0126 Turin, Italy

STRUCTURE OF VIRION

The virions of the hepatitis delta virus (HDV) consist of a lipid-containing envelope inside of which are the RNA genome and the only known protein of HDV, the delta antigen (HDAg). By means of filtration and by elec- tron microscopy it has been estimated that the average diameter of the particles is about 38-41 nm, just smaller than the 42nm infectious particles of hepatitis B virus

The envelope of HDV is substituted with the same surface proteins that are used by HBV in the assembly of its infectious particle. HBV encodes three co-carboxy- terminal roteins that are incorporated into the virus envelope! However, for HDV the relative amounts of the three proteins is quite different; it is almost as if only the smallest protein, the S protein, is present for HDV.4a5 In support of this interpretation, it is possible to use co-transfedion and assemble particles with only the S proteint6 however, the infectivity of such particles has not yet been determined. This is an important issue because for HBV, natural infections involve an interaction between a receptor and a domain called preS1, that is not present on the S protein but is unique to the largest of the three surface protein^.^

Several approaches, including electron microscopy have been used without success to detect any kind of core or nucleocapsid structure within HDV particle^.'^^'^ However, for a small fraction (15%) of particles assembled via transfection of cultured cells, a symmetrical core structure has been claimed.' Such a structure is even analogous to that seen within HBV?

As discussed later there are two co-amino-terminal forms of the HDAg. The small form is essential for genome replication' and apparently the large form is essential for a~sembly.2.~ Even though the small form will not function by itself in such assembly, it may cooperate with the large form during virus assembly."6

(HBV).',~

'Author to whom correspondence should be addressed.

ISSN 1052-9276/92/030161-07 $08.50 0 1992 by John Wiley & Sons, Ltd.

GENOMIC RNA AND RNAS INVOLVED IN REPLICATION Since 1986 a series of experiments have clarified much of the structure and replication of HDV. In many ways HDV has proven to be unique relative to other infectious agents of animals, and yet, it has shown many similarities to certain infectious agents of

The genome is a single-stranded RNA that is not only very small, only about 1700 nucleotides, but is in the form of a circle, and is able to fold onto itself by base pairing (involving 70% of all nucleotides), to form an unbranched rodlike ~tructure. '~"~*'~*'~

Replication occurs in the nucleus of an infected ceIl.l7 The enzyme involved is probably the host RNA polymerase 11." The mechanism is via RNA-directed RNA synthesis with the synthesis of two species of complementary RNA. The first, called the antigenome, is an exact complement of the genome, and it is also ~ircular.'~ The other is a smaller complementary RNA. It is polyadenylated, found in the cytoplasm, and acts as the mRNA for the delta Since this open reading frame is on the complement of the genome, we could say that HDV is in this respect, like a negative-strand RNA virus.

Both the genome and the antigenome contain a single site at which self-cleavage can occur?o*21 This reaction can be reversed to produce a self-ligation?' As presented in a subsequent section, these two ribozyme activities have been incorporated into a model of genome replication that explains how replication can lead to the production of new circular RNA specie^.'^ This model is like the so-called rolling-circle models that have been used to explain the replication of those infectious agents of plants that resemble HDV."," However, this model incorporates that unique aspect of HDV replication, the synthesis of not only the antigenomic RNA but also a polyadenylated mRN A.

THE TWO DELTA ANTIGENS As mentioned earlier, on the antigenome of HDV there is an open reading frame for the one known protein, the

Accepted 10 June 1992

I62 J. TAYLOR ETAL.

HDAg.'6*24 However, not all HDV RNAs have the same sequence; some encode a 195 amino acid delta antigen, while others, because of a change in the termination codon, make a longer species of 2 14 amino acids. 24.25.26 HDV has apparently exploited a unique editing trick to allow the synthesis of two proteins from what is initially one sequence. The input genome encodes the small HDAg which then enables genome replication to begin. Then, because of an RNA editing reaction in the nucleus of the infected cells, some of the genomes are specifically altered, leading to the synthesis of the larger form of the HDAg.25*26 This in turn, if hepadnavirus is present, leads to the assembly of new virus particle^.^^^^^.^^

The small and large forms of the HDAg share a common amino-terminus. In addition to the biological properties men- tioned above, both forms show nuclear localisation?' are highly basic,I5 and exhibit an HDV-specific RNA binding ability.27,28 Apparently this binding requires the rodlike struc- ture of the HDV RNA.29 Also, both in uifro and in vim, it is possible to demonstrate dimerisation of the delta

With a surprising amount of success, it has been possible to use a one-dimensional approach and map domains on these two proteins. Lai and co-workers have mapped a dimerisation domain near to the amino-terminus, an RNA binding domain in the central region, and between these, a bipartite nuclear localisation signal." Some other properties cannot be explained as simply as this; the most important example is the ability of the large protein to interfere with the ability of the small to support genome rep l ica t i~n .~~ In this dominant negative effect the one-dimensional approach does not apply; the 19 extra amino acids on the large form do not simply act as a signal. Glenn and White" have shown that other even smaller changes are sufficient to change the small form into one that can act as a dominant negative inhibitor. In other words, it is as if there is some three-dimensional alteration of the shared domain that is involved.

MODEL OF GENOME REPLICATION The replication of HDV is in many ways like that of the plant a ents known as viroids, virusoids and satellite RNAS:""~ '~~~~ the replication is in the nucleus, the enzyme is probably a redirected host RNA polymerase 11," and both self-cleavage and self-ligation are involved. However, the situation for HDV is more complex; primarily it is necessary to explain the synthesis of a polyadenylated mRNA and also the regulation of such synthesis relative to that of full length antigenomic RNA. A model has been ro osed for HDV that explains such regulation (Figure

:)?'the model invokes the ability of delta antigen to bind to rodlike RNA and thereby participate in the sup- pression of certain polyadenylation events." However, there remain lots of unsolved questions regarding this replication. Where does it initiate and why is there ten times more genomic relative to antigenomic RNA? Are there more roles for the delta antigen in this process? What is needed to redirect the host polymerase? Alternatively, what do cell RNAs lack in that they are not normally templates for such RNA-directed RNA synthesis.

With the RNA of certain plant agents sometimes not even a single nucleotide can be changed without suffering a loss of replicating ability." In contrast for HDV certain changes on the HDV genome can be made without loss of replicating ability; a deletion of 2 bases did not interfere' and even an insertion of 30 bases was acceptable.'" Such results offer promise for the use of modified HDV genomes as vectors for the delivery of specific nucleotide sequences, such as biologically active RNAs.'~

FROM VIRION STRUCTURE TO DISEASE EXPRESSION

From the clinical standpoint, HDV infection is associated with an array of presentations ranging from the sym tom- less, healthy ~ a m e r ' ~ , ' ~ to end-stage liver cirrhosi8 and hepatocellular carcinoma.'' Assuming that HDV is pathogenic, in which way does it cause liver disease, and why is this so heterogeneous? Evidence that HDV is directly cytotoxic has been presented, whereas other studies seem to suggest an immune-mediated pathogenesis of delta hepatitis. An attempt to reconcile the views on how HDV causes liver disease is presented below.

EVIDENCE THAT HDV IS DIRECTLY CYTOTOXIC

As mentioned above, nuclei of infected hepatocytes are packed with a huge number of copies of genomic HDV RNA, usually several hundreds of thousand^.'^,'^ The possibility of an interaction with the host nuclear content is therefore likely. HDV replication might interfere with the physiological functions of the cellular RNA pol ymerases" or even with the mechanisms of DNA replication and cell division.

Indeed, evidence that HDV may have a direct cytotoxic effect on hepatocytes came from the initial experimental transmission studies in the chimpanzee animal model.40 A strict time relationship was consistently found between the intrahepatic expression of HDAg and the liver enzyme elevations in all animals inoculated with HDV. Interest- ingly, this association was observed before the appearance of a specific anti-HD response. When quantitative data became available,41 it was, however, clear that the extent of liver cell necrosis was not correlated with the HDV replication level (in terms of serum HDV RNA), suggesting a more complex mechanism of damage.

Pathomorphological observations in human^^'-^^ and in the woodchuck animal m ~ d e l ~ ~ . ~ ' are suggestive of a direct cytotoxic effect of HDV on hepatocytes. These include a cytoplasmic eosinophilia and microvesicular steatosis without concomitant inflammatory infiltrate. According to immunohistochemical and in sitcc hybridisation studies, however, the eosinophilic degeneration is rarely associated with the replication of HDV at the cell leve1.49~50 Moreover, genetic and environmental factors have been blamed for the peculiar occurrence of the microvesicular steatosis, often observed in epidemics of severe delta hepatitis within certain geographical area^.^^,^' Steatosis may be accounted

HEPATITIS DELTA VIRUS: FROM STRUCTURE TO DISEASE EXPRESSION 163

1 2 3 4 5 6 7

+ + +

Figure I. A model for the regulation of RNA processing during the replication of the HDV genome. This is a representation of the transcription of antigenomic RNA (solid line) from a rod-like circular template of genomic RNA (broken line). As indicated in step 1, RNA synthesis is assumed to initiate 5 nucleotides kom the top of the rod, just as is known for the mRNAI9. In step 2, this transcript proceeds beyond the polyadenylation signal (open square), polyadenylation site (open rectangle) and self-cleavage site (open circle). In step 3, this nascent RNA is processed by the cellular machinery to release both the mature mRNA for the delta antiqen, and a downstream RNA. As indicated in step 4, the self-cleavage of this downstream RNA leads to its stabilisation.” The transcription can thus continue, as in step 5, and pass for a second time through the regions directing the polyadenylation and self-cleavage. However, the nascent RNA now has the ability to fold into the rod-like structure. And, the delta antigen is able to specifically bind to this structure.” Thus, when the level of newly synthesised delta antigen reaches a sufficient level, such binding can suppress the polyadenylation process” and allow only the self-cleavage, as indicated in step 6. This will release a unit length species, which as indicated in step 7, will fold into the rod-like structure, bring the two ends together, and undergo a self-ligation event,” to make a circular antigenomic RNA species. As indicated in step 7, the nascent RNA transcript may continue on to make more arcular RNA products. The model as shown, refers only to the synthesis of antigenomic RNAs from a genomic RNA template. However, a similar model is proposed for the synthesis of genomic RNA horn an antigenomic RNA template, where the processing will, however, be less complex in that polyadenylation is not involved. The diagram is from reference,” and is reproduced with permission from the Journal of Virology.

for by a derangement in the secretory pathway of the hepatocyte. Interestingly, a speculative mechanism of pathogenesis has been postulated on the basis of a signifi- cant sequence similarity between two conserved regions of HDV RNA and two regions of the human signal recognition particle (SRP) RNA,” a small cytoplasmic RNA. SRP RNA is part of a ribonucleoprotein responsible for the correct targeting of secretory proteins to the membrane of the endoplasmic reticulum. As a consequence of this sequence homology, replicative intermediates of HDV RNA could theoretically anneal to the corresponding SRP RNA regions, thus impairing its function in cell secretion processes. Alternative, hypothetical interactions between HDV RNA or HDAg and the SRP have been proposed:’ although they remain highly speculative.

A recent study has shown that in vitro expression of the small isoform of HDAg is associated with cy to t~x ic i ty .~~ Indeed, necrotic cells expressing this viral protein resembled morphologically the apoptotic bodies seen in vivo, characterised by a pyknotic nucleus and an eosinophilic, shrunken cytoplasm. This observation is not shared by other investigators who have successfully transfected in vitro cell lines with cloned HDV8e54 without noticing any cytopathic effect. The possibility exists,

however, that in these experiments a non-pathogenic HDV clone has been selected. Unfortunately, evidence from in uitro infection, although successful, was not conclusive” due to the very small number of HDV-infected hepato- cytes, too small to allow for the identification of specific cytotoxic changes.

In all systems studied, including the one where a cytotoxic effect was reported, the in oitro expression of the small form of the HDAg, as detected by immuno- fluorescence, gives rise to a peculiar nucleolar distri- bUtion.8,53,54,55 However, these in vitro findings were not confirmed in vivo, where the immunofluorescence pattern consistent with the predominant expression of the small form of the HDAg, as identified by a human polyclonal anti-HD antiserum, did not correspond to the occurrence of morphological damage of the infected hepa tocy te~ .~~

The presence of HDV has been correlated also with hepatocytic nuclear dysplasia: giant nuclei, ‘sanded nuclei and binucleated hepatocytes have all been shown to be associated with HDV repl i~at ion.~’ ,~~ It has been observed that the HDAg expression may be associated with the expression of the c-myc proto-~ncogene.~~ Interestingly, progression towards the development of hepatocellular carcinoma seems accelerated in patients dually infected

164 J. TAYLOR ET AL.

with HBV and HDV with respect to those who carry the HBV alone.38 Both direct and indirect mechanisms could be involved.

Although a direct interaction between viral products and cellular constituents provides an attractive hypothesis to explain the mechanism of HDV-associated damage, the wide variability of the clinical spectrum of human HDV disease implies a more complex pathogenesis. The occur- rence of healthy HDV carriers has been established on the basis of both experimental and epidemiologic data. More than half of the chimpanzees at the US National Institutes of Health colony were shown to be chronic HDV carriers with minimal lesions at h i ~ t o l o g y . ~ ~ Interestingly, they progressed to this healthy chronic carriage after a full- blown, often severe acute delta he pa ti ti^.^' In humans, the existence of healthy HDV carriers was inferred from evidence of many (60%) subclinical cases among HDV infected individuals in a highly endemic area?5 It should be emphasised that these variations in pathogenicity may still depend on viral factors, rather than on the variability of the host immune system, as discussed below. One should mention, among these virus-related factors, the possibility of sequence heterogeneity, which has been recognised to occur extensively for HDV.I6 In an anecdotal report, antigenic diversity (a single protein of M, 28 000 revealed by specific antibodies, in place of the forms of 24 000 and 27 000 usually observed) was noted in the HDV associated with an epidemic of fulminant hepatitis in Bangui, characterised by spongiocytic histological aspects.47 No sequence data have been reported so far concerning this intriguing isolate. On the other hand, very little is known about the relationship between the sequence of any of the different isolates of HDV and the variation in disease expression.

THE INTERACTION WITH THE HELPER HEPADNAVIRUS

Both in uitroI7 and in uiuo evidence6' has been presented showing that HDV replication can proceed in the absence of the hepadnavirus. However, to spread from cell to cell and express its own pathogenic potential, HDV clearly needs some kind of genetic information from the hepadnavirus. As described above, in uifro co-transfection s t ~ d i e s ~ ~ ~ ~ ~ ~ ~ ~ ~ have identified these sequences as the envelope-encoding region of the co-infecting hepadnavirus. Therefore, the presence of a productive HBV infection is a mandatory requirement to maintain the HDV life cycle.

In the clinical setting, the highest levels of HDV replication were found in patients with the highest levels of replication of the co-infecting HBV.64 These patients also had the worst clinical outcome. Recent observations from liver transplant patients show that full expression of the pathogenic potential of HDV requires HBV replication. The virologic follow up of patients who underwent liver transplantation for end-stage HDV-associated liver disease showed that HDV infection may recur soon after grafting without signs of HBV reinfection or evidence of liver damage. The reappearance of liver disease was preceded by

reactivation of HBV replication and by the intrahepatic spreadin of HDV, previously limited to a few hepato- cytes.61*6g,66 Therefore, although not required to establish HDV reinfection of the graft, cooperation with HBV seems nevertheless necessary to allow for disease expression. The critical role of HBV may explain why hepatitis was prevented in patients in a series where the patients were given prophylaxis with anti-HBs, thus preventing the development of disease even in the presence of HDV reinfection.66

The data obtained from studies of transplant patients allow one to speculate that, besides HDV co-infection with HBV or HDV superinfection of HBV carriers, a third pathobiological mechanism of delta hepatitis is HBV superinfection allowing reactivation of a clinically latent HDV infection. If this were the case, an unrecognised reservoir of HDV might exist in healthy HBsAg-negative individuals. Upon superinfection with HBV, HDV would be rescued and allowed a full life cycle, with production of infectious virions, cell-to-cell spread and disease expression.

Strain differences in the helper HBV may be equally important. Levels of HDV replication are usually higher in patients with HBV infection expressing the HBeAg in serum, as compared to those seen among HBsAg carriers with anti-HBe. Preliminary observations have suggested that HBeAg-minus HBV is less efficient in supporting HDV expression than the wild-type HBV, although the mechanisms for this defective helper function, as well as the pathobiological consequences, are undefined.67

HOST FACTORS INFLUENCING THE CLINICAL EXPRESSION OF HDV An additional factor influencing the pathogenesis of delta hepatitis is represented by the response of the host immune system. The mechanisms of interplay between the expression of the two forms of HDAg and the immune system are totally unknown.

Large variations in clinical expression of HDV disease have been observed in the woodchuck animal model!' The same inoculum induced in a battery of animals a liver disease of varying histological severity. Disease variations were also observed upon the second and third passage of this inoculum in other woodchucks. These data have been interpreted as consistent with individual variations in the immune response to the same pathogen, although other explanations are possible.

The presence of immune-mediated liver damage may correlate with an increased probability of clearing the HDV infection. In one study, the degree of cellular infiltration within the portal tracts correlated with the number of HDAg positive hepatocytes, and inflammation and HDAg expression showed their highest expression in those cases destined to resolve.43

Autoimmune reactions may occur in a significant proportion of patients with chronic delta hepatitis, although the pathogenetic significance of this phenomenon is obscure. About 50% of them have serum auto- antibodies directed against an antigen present in both

HEPATITIS DELTA VIRUS FROM STRUCTURE TO DISEASE EXPRESSION 165

human thymocytes and the basal cell layer of rodent fore- stomach.j7 This antigen has been extracted from rat thymus and fore-stomach and characterised6' as a protein of M, 46 000, but it is unrelated to keratin.69 Fifteen to 20% of the patientd7 have an autoantibody reacting with the human liver and kidney microsomes (LKM,) that resembles the liver-kidney microsomal antibody characteristic7' of type 2 autoimmune hepatitis (LKM,). Interestingly, a strong epidemiological relationship has been noted between chronic hepatitis C and the presence of antibodies against the LKM, antigen." A possible role of the major histocompatibility complex haplotype has been hypothe- sised to account for the aberrant autoimmune rea~tion.~' The time relationship between the appearance of anti- bodies against LKM, and seroconversion to anti-HD7' points to a causal link between the immunologic derangement and HDV infection. However, there are no apparent differences, from the clinical point of view, between autoantibody-positive and negative patients with chronic delta hepatitis. In conclusion, although it is evident that host factors are involved in the pathogenesis of delta hepatitis, the fine mechanisms of the interaction between HDV infection and the specific immune response are largely unknown and speculative.

CONCLUDING REMARKS Most of the data supporting the view that HDV is directly cytotoxic have been gathered from acute infection settings, both natural and experimental. On the contrary, observations suggesting a role for the host immune system come from chronically infected individuals. This distinc- tion is remarkable, since the two mechanisms might well coexist in a mutual balance throughout the natural history of delta hepatitis. During the acute phase the liver damage may be primarily explained by a direct interaction between HDV replication and/or protein expression with the cell components (the RNA polymerase I1 is likely to be one of the candidates18). The respective role of each of the three HDV RNAs and of the two forms of HDAg is still unknown. During the chronic phase, the damage mediated by the host immune system seems to play a major role, mainly as the expression of the immune elimination of infected hepatocytes. Finally, the role played by the differ- ent strains of the co-infecting hepadnavirus might lie in the level of replication and cell-to-cell spread of HDV, although more data are needed to evaluate this possibility.

ACKNOWLEDGEMENTS The authors wish to thank Eric J. Gowans for helpful discussion. J.T. was supported by grants a-06927 , RR-05539 and AI-26522 from the National Institutes for Health, by grant MV-7P from the American Cancer Society, and by an appropriation from the Commonwealth of Pennsylvania.

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