Nucleotide sequence stability of the genome of hepatitis delta virus.

Cultured cells were cotransfected with a fully sequenced 1,679-base cDNA clone of human hepatitis delta virus (HDV) RNA genome and a cDNA for the genome of woodchuck hepatitis virus (WHV). The HDV particles released were able to infect a woodchuck that was chronically infected with WHV. The HDV so produced was passaged a total of six times in woodchucks in order to determine the stability of the HDV nucleotide sequence. During a final chronic infection with such virus, liver RNA was extracted, and the HDV nucleotide sequence for the 352-base region, positions 905 to 1256, was obtained. By means of PCR, we obtained double-stranded cDNA both for direct sequencing and also for molecular cloning followed by sequencing. By direct sequencing, we found that a consensus sequence existed and was identical to the original sequence. From the sequences of 31 clones, we found 32% (10 of 31) to be identical to the original single nucleotide sequence. For the remainder, there were neither insertions nor deletions but there was a small number of single-nucleotide changes. These changes were predominantly transitions rather than transversions. Furthermore, the transitions were largely of just two types, uridine to cytidine and adenosine to guanosine. Of the 40 changes detected on HDV, 35% (14 of 40) occurred within an eight-nucleotide region that included position 1012, previously shown to be a site of RNA editing. These findings may have significant implications regarding both the stability of the HDV RNA genome and the mechanism of RNA editing.     

Primary human hepatocytes are susceptible to infection by hepatitis delta virus assembled with envelope proteins of woodchuck hepatitis virus

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) share the HBV envelope proteins. When woodchucks chronically infected with woodchuck hepatitis virus (WHV) are superinfected with HDV, they produce HDV with a WHV envelope, wHDV. Several lines of evidence are provided that wHDV infects not only cultured primary woodchuck hepatocytes (PWH) but also primary human hepatocytes (PHH). Surprisingly, HBV-enveloped HDV (hHDV) and wHDV infected PHH with comparable efficiencies; however, hHDV did not infect PWH. The basis for these host range specificities was investigated using as inhibitors peptides bearing species-specific pre-S (where S is the small envelope protein) sequences. It was found that pre-S1 contributed to the ability of wHDV to infect both PHH and PWH. In addition, the inability of hHDV to infect PWH was not overcome using a chimeric form of hHDV containing WHV S protein, again supporting the essential role of pre-S1 in infection of target cells. One interpretation of these data is that host range specificity of HDV is determined entirely by pre-S1 and that the WHV and HBV pre-S1 proteins recognize different receptors on PHH.     

Hepatitis delta antigen. Antigenic structure and humoral immune response

Hepatitis delta virus (HDV) is a small RNA virus that is dependent on helper functions provided by hepatitis B virus. The hepatitis delta Ag (HDAg) is the only protein known to be made from the viral genome, from an ORF with a coding capacity of 214 amino acids. The immunogenic epitopes of HDAg and the immune response to it were mapped by the use of synthetic peptides, antipeptide antibodies, and human mAb. Antipeptide sera covering approximately 60% of the linear sequence reacted with liver-derived HDAg. Antisera from HDV-infected humans, chimpanzees, and woodchucks reacted with from 2 to 13 of 15 peptides. The epitopes of two human anti-HD mAb were mapped to overlapping but distinct epitopes in the region around residues 106-123. Sera from infected humans, chimpanzees, and woodchucks were also tested by competition with the mAb. Use of the peptides and antipeptide sera defined one region in the sequence (residues 52-93) which is immunodominant in the immune response to HDAg. Reactivity of both peptides and antipeptide antibodies was very broad, covering most or all of the linear sequence. Competition assays also provided information on conformational epitopes, as well as the sequential epitopes defined by direct assays. The peptides and antipeptide antibodies should be useful in new assay development, in dissecting the anti-HD response in terms of chronic vs self-limited infection, and in studying the role of anti-HD in infection and recovery.     

Apparent helper-independent infection of woodchucks by hepatitis delta virus and subsequent rescue with woodchuck hepatitis virus.

Hepatitis delta virus (HDV) is a subviral agent of humans which is dependent upon hepatitis B virus as a helper for transmission. HDV can be experimentally transmitted to woodchucks by using woodchuck hepatitis virus (WHV) as the helper. We used this model system to study two types of HDV infections: those of animals already chronically infected with WHV and those of animals without any evidence of prior exposure to WHV. At 5 to 10 days after infection with HDV, liver biopsies of these two groups of animals indicated that around 1% of the hepatocytes were infected (HDV antigen positive). Moreover, similar amounts of replicative forms of HDV RNA were detected. In contrast, by 20 days postinfection, the two groups of animals were quite different in the extent of the HDV infection. The animals chronically infected with WHV showed spread of the infection within the liver and the release of high titers of HDV into the serum. In contrast, the animals not previously exposed to WHV showed a progressive reduction in liver involvement, and at no time up to 165 days postinfection could we detect HDV particles in the serum. However, if these animals were inoculated with a relatively high titer of WHV at either 7 or even 33 days after the HDV infection, HDV viremia was observed. Our data support the interpretation that in these animals, hepatocytes were initially infected in the absence of helper virus, HDV genome replication took place, and ultimately these replicating genomes were rescued by the secondary WHV infection. The observation that HDV can survive in the liver for at least 33 days in the absence of coinfecting helper virus may be relevant to the reemergence of HDV infection following liver transplantation.     

Core antigen and antibody in woodchucks after infection with woodchuck hepatitis virus

The woodchuck hepatitis virus is a naturally occurring hepatitis B-like virus that infects the eastern woodchuck. Direct immunofluorescence staining for woodchuck hepatitis virus core antigen in liver biopsies demonstrated the presence of this antigen in 14 of 17 chronically infected woodchucks, and in 8 of 10 woodchucks undergoing acute infections. Fluorescent localization of woodchuck hepatitis virus core antigen was typically cytoplasmic, and this was confirmed further by electron microscopy. Experimental infection with woodchuck hepatitis virus was achieved in four of four woodchucks inoculated with serum from chronic carrier woodchucks. All infected animals developed a self-limited disease characterized by seroconversion to antibodies against the major viral antigens (core and surface antigens); naturally acquired acute infection demonstrated a similar course. A chimpanzee seronegative for all markers of hepatitis B virus developed a subclinical infection after inoculation with woodchuck hepatitis virus.     

Woodchuck hepatitis virus is a more efficient oncogenic agent than ground squirrel hepatitis virus in a common host.

Chronic infection with hepatitis B viruses (hepadnaviruses) is a major cause of hepatocellular carcinoma (HCC), but the incubation time varies from 1 to 2 years to several decades in different host species infected with indigenous viruses. To discern the influence of viral and host factors on the kinetics of induction of HCC, we exploited the recent observation that ground squirrel hepatitis virus (GSHV) is infectious in woodchucks (C. Seeger, P. L. Marion, D. Ganem, and H. E. Varmus, J. Virol. 61:3241-3247, 1987) to compare the pathogenic potential of GSHV and woodchuck hepatitis virus (WHV) in chronically infected woodchucks. Chronic GSHV infection in woodchucks produces mild to moderate portal hepatitis, similar to that observed in woodchucks chronically infected with WHV. However, HCC developed in GSHV carriers about 18 months later than in WHV carriers. Thus, although both viruses are oncogenic in woodchucks, GSHV and WHV differ in oncogenic determinants that can affect the kinetics of appearance of HCC in chronically infected animals.     

Replication of human hepatitis delta virus in primary cultures of woodchuck hepatocytes.

We obtained two lines of evidence that monolayer cultures of primary woodchuck hepatocytes support replication of the genome of human hepatitis delta virus (HDV). (i) From a Northern (RNA blot) analysis of the HDV-related RNA in infected cultures, both genomic and antigenomic 1.7-kilobase RNA species were detected at 11 days after infection. The ratio of genomic RNA to antigenomic RNA was 2:1 to 10:1, comparable to that previously reported in studies of experimentally infected chimpanzees and woodchucks. (ii) Replication in culture was also demonstrated by in situ hybridization with a strand-specific probe. Such studies showed that only a small fraction of the cultured cells supported replication and that in such cells the relative and absolute levels of the HDV RNAs were comparable to those in liver cells infected in vivo. Furthermore, as with the in vivo studies, the HDV RNAs were predominantly localized to the nucleus. In summary, we demonstrated that cultured cells supported both the early events of HDV adsorption and penetration and the intermediate events of genome replication.     

Oligomerization of hepatitis delta antigen is required for both the trans-activating and trans-dominant inhibitory activities of the delta antigen.

Two forms of hepatitis delta antigen (HDAg) have different roles in the replication cycle of hepatitis delta virus (HDV); the small forms trans activates HDV RNA replication, whereas the large form suppresses it but is needed for virion assembly. To understand the mechanism of these regulatory activities, we studied the possible HDAg oligomerization and its role in HDV replication. In this report, we provide direct biochemical evidence for the in vitro and in vivo formation of homodimers and heterodimers between these two HDAg species. By deletion mutagenesis, we showed that this protein interaction is mediated by the leucine zipper-like sequence residing in the N-terminal one-third of HDAg. Furthermore, site-specific mutants with various substitutions on two of the leucine residues in this stretch of sequence had reduced or no ability to form HDAg dimers. Correspondingly, the small HDAg with mutations in the leucine zipper-like sequence had reduced abilities to trans activate HDV RNA replication. Similar mutations on the leucine zipper-like sequence of the large HDAg also resulted in loss of the ability of large HDAg to inhibit HDV RNA replication. The in vivo biological activities of both forms of HDAg (trans activation and trans-dominant inhibition of HDV RNA replication, respectively) correlated with the extent of HDAg oligomerization in vitro. Thus, we conclude that the small HDAg participates in HDV RNA replication as an oligomer form and that the large HDAg inhibits HDV RNA replication as a result of its complex formation with small HDAg. A "black sheep" model for the mechanism of trans-dominant inhibition by the large HDAg is presented.     

Hepadnavirus infection of peripheral blood lymphocytes in vivo: woodchuck and chimpanzee models of viral hepatitis.

The peripheral blood lymphocytes (PBL) of five hepatitis B virus (HBV)-infected chimpanzees and 17 woodchuck hepatitis virus (WHV)-infected woodchucks were examined for the presence of viral DNA and RNA. HBV DNA was detected in the PBL of three of three chronically infected chimpanzees but in neither of two animals with acute HBV infection. WHV DNA was found in the PBL of 11 of 13 chronically infected woodchucks and in the PBL and bone marrow of 1 of 4 woodchucks with antibody to WHV surface antigen. Viral DNA in the PBL and bone marrow was episomal, primarily existing as multimers with some monomeric forms. Integrated HBV DNA was detected in the PBL of one chronically infected chimpanzee, but only for a brief period. Viral RNA was also detected in the PBL, although less frequently than was DNA. HBV RNA in chimpanzee PBL existed as 3.8- and 7.5-kilobase species, while 2.3- and 3.8-kilobase WHV RNA was found in woodchuck PBL. Subfractionation of PBL isolated from the chronically infected chimpanzees demonstrated that HBV DNA and RNA were located in B and T cells. No HBV DNA was detected in the macrophages. These results, along with the recent reports of HBV nucleic acids in the PBL of human patients, suggest that infection of PBL may be a general phenomenon associated with the pathology of hepadnaviruses.     

Functional study of hepatitis delta virus large antigen in packaging and replication inhibition: role of the amino-terminal leucine zipper.

The large hepatitis delta antigen (HDAg) has been found to be essential for the assembly of the hepatitis delta virion. Furthermore, in a cotransfection experiment, the large HDAg itself, without the hepatitis delta virus (HDV) genome and small HDAg, could be packaged into hepatitis B surface antigen (HBsAg) particles. By deletion analysis, it was shown that the amino-terminal leucine zipper domain was dispensable for packaging. The large HDAg could also help in copackaging of the small HDAg into HBsAg particles without the need for HDV RNA. This process was probably mediated through direct interaction of the two HDAgs as a mutated large HDAg whose leucine zipper domain was deleted such that it could not help in copackaging of the small HDAg. This mutated large HDAg did not suppress HDV replication, suggesting that this effect is probably also via protein interaction. These results indicated that functional domains of the large HDAg responsible for packaging with HBsAg particles and for the trans-negative effect on HDV replication can be separated.     

Immunogenic domains of hepatitis delta virus antigen: peptide mapping of epitopes recognized by human and woodchuck antibodies.

Hepatitis delta virus (HDV) is a defective RNA virus which is dependent on hepatitis B virus for essential helper functions. Only a single highly basic phosphoprotein, HDV antigen (HDAg), is expressed by the HDV genome during infection in humans. Antibody directed to HDAg is important in the diagnosis of HDV infection, and it is likely but not yet proven that the immune response to HDAg provides significant protection against subsequent exposures to HDV. In an effort to map the antigenic domains of HDAg, 209 overlapping hexapeptides, spanning the entire 214 amino acid residues of the protein, were synthesized on polyethylene pins and probed by enzyme-linked immunosorbent assay with sera containing high titers of anti-HD antibodies. Domains recognized by antibodies present in serum from human chronic carriers of this virus included residues 2 to 7, 63 to 74, 86 to 91, 94 to 100, 159 to 172, 174 to 195, and 197 to 207. Antibody from an acutely superinfected woodchuck recognized similar epitopes, as well as a domain spanning residues 121 to 128. Together, residues in these antigenic domains constitute 41% of the HDAg molecule. Oligopeptides 15 to 29 residues in length and representing epitopes of HDAg found to be dominant in humans (residues 2 to 17, 156 to 184, and 197 to 211) were synthesized in bulk and found to possess significant antigenic activity by microdilution enzyme-linked immunosorbent assay. The reactivity of peptide 197-211 with human sera confirms that the entire 214 amino acids of HDAg are expressed during infection in vivo. In addition, these results suggest that synthetic peptides may be useful reagents for development of new and improved diagnostic tests for HDV infection.     

The large delta antigen of hepatitis delta virus potently inhibits genomic but not antigenomic RNA synthesis: a mechanism enabling initiation of viral replication

Hepatitis delta virus (HDV) contains two types of hepatitis delta antigens (HDAg) in the virion. The small form (S-HDAg) is required for HDV RNA replication, whereas the large form (L-HDAg) potently inhibits it by a dominant-negative inhibitory mechanism. The sequential appearance of these two forms in the infected cells regulates HDV RNA synthesis during the viral life cycle. However, the presence of almost equal amounts of S-HDAg and L-HDAg in the virion raised a puzzling question concerning how HDV can escape the inhibitory effects of L-HDAg and initiate RNA replication after infection. In this study, we examined the inhibitory effects of L-HDAg on the synthesis of various HDV RNA species. Using an HDV RNA-based transfection approach devoid of any artificial DNA intermediates, we showed that a small amount of L-HDAg is sufficient to inhibit HDV genomic RNA synthesis from the antigenomic RNA template. However, the synthesis of antigenomic RNA, including both the 1.7-kb HDV RNA and the 0.8-kb HDAg mRNA, from the genomic-sense RNA was surprisingly resistant to inhibition by L-HDAg. The synthesis of these RNAs was inhibited only when L-HDAg was in vast excess over S-HDAg. These results explain why HDV genomic RNA can initiate replication after infection even though the incoming viral genome is complexed with equal amounts of L-HDAg and S-HDAg. These results also suggest that the mechanisms of synthesis of genomic versus antigenomic RNA are different. This study thus resolves a puzzling question about the early events of the HDV life cycle.     

Nucleolar targeting of hepatitis delta antigen abolishes its ability to initiate viral antigenomic RNA replication

Hepatitis delta virus (HDV) is a small RNA virus that contains one 1.7-kb single-stranded circular RNA of negative polarity. The HDV particle also contains two isoforms of hepatitis delta antigen (HDAg), small (SHDAg) and large HDAg. SHDAg is required for the replication of HDV, which is presumably carried out by host RNA-dependent RNA polymerases. The localization and the HDAg and host RNA polymerase responsible for HDV replication remain important issues to be addressed. In this study, using recombinant SHDAg fused with a heterologous nucleolar localization sequence (NoLS) to confine its subcellular localization in nucleoli, we aimed to study the effect of SHDAg subcellular localization on HDV RNA replication. The initiation of genomic RNA synthesis from antigenomic template was hardly detectable when SHDAg was fused with the NoLS motif and localized mainly in nucleoli. In contrast, the initiation of antigenomic RNA synthesis was not affected. Drug treatment to release a SHDAg-NoLS mutant from nucleoli could partially restore the replication of HDV genomic RNA from antigenomic RNA. This also recovered the cointeraction between SHDAg and RNA polymerase II. These data strongly suggest that nuclear polymerase (RNA polymerase II) is involved in the synthesis of genomic RNA and that the synthesis of antigenomic RNA can occur in nucleoli. Our results support the idea that the replication of HDV genomic RNA or antigenomic RNA is likely to be carried out by different machineries in different subcellular localizations.     

Packaging of hepatitis delta virus RNA via the RNA-binding domain of hepatitis delta antigens: different roles for the small and large delta antigens.

Hepatitis delta virus (HDV) is composed of four specific components. The first component is envelope protein which contains hepatitis B surface antigens. The second and third components are nucleocapsid proteins, referred to as small and large hepatitis delta antigens (HDAgs). The final component is a single-stranded circular RNA molecule known as the viral genome. In order to study the mechanism of HDV RNA packaging, a four-plasmid cotransfection system in which each viral component was provided by a separate plasmid was employed. Virus-like particles released from Huh-7 cells receiving such a cotransfection were found to contain HDV RNA along with three proteins. Therefore, the four-plasmid cotransfection system could lead to successful HDV RNA packaging in vitro. The system was then used to show that the large HDAg alone was able to achieve a low level of HDV RNA packaging. Analysis of a variety of large HDAg mutants revealed that the RNA-binding domain was essential for viral RNA packaging. By increasing the incorporation of small HDAg into virus-like particles, we found a three- to fourfold enhancement of HDV RNA packaging. This effect was probably through a direct binding of HDV RNA, independent from that of large HDAg, with the small HDAg. The subsequent RNA-protein complex was packaged into particles. The results provided insight into the roles and functional domains of small and large HDAgs in HDV RNA packaging.     

Hepatitis delta virus antigen is methylated at arginine residues, and methylation regulates subcellular localization and RNA replication

Hepatitis delta virus (HDV) contains a circular RNA which encodes a single protein, hepatitis delta antigen (HDAg). HDAg exists in two forms, a small form (S-HDAg) and a large form (L-HDAg). S-HDAg can transactivate HDV RNA replication. Recent studies have shown that posttranslational modifications, such as phosphorylation and acetylation, of S-HDAg can modulate HDV RNA replication. Here we show that S-HDAg can be methylated by protein arginine methyltransferase (PRMT1) in vitro and in vivo. The major methylation site is at arginine-13 (R13), which is in the RGGR motif of an RNA-binding domain. The methylation of S-HDAg is essential for HDV RNA replication, especially for replication of the antigenomic RNA strand to form the genomic RNA strand. An R13A mutation in S-HDAg inhibited HDV RNA replication. The presence of a methylation inhibitor, S-adenosyl-homocysteine, also inhibited HDV RNA replication. We further found that the methylation of S-HDAg affected its subcellular localization. Methylation-defective HDAg lost the ability to form a speckled structure in the nucleus and also permeated into the cytoplasm. These results thus revealed a novel posttranslational modification of HDAg and indicated its importance for HDV RNA replication. This and other results further showed that, unlike replication of the HDV genomic RNA strand, replication of the antigenomic RNA strand requires multiple types of posttranslational modification, including the phosphorylation and methylation of HDAg.     

Cloned hepatitis delta virus cDNA is infectious in the chimpanzee.

A head-to-tail trimer of a full-length cDNA clone of the hepatitis delta virus (HDV) genome was examined for infectivity by direct inoculation into the liver of a chimpanzee that was already infected with hepatitis B virus. Five weeks after inoculation, a marked elevation of serum alanine aminotransferase activity was observed, followed by the appearance of high levels of HDV RNA and antigen in both liver and serum and a high level of viral particles in the serum. A transient suppression of hepatitis B virus replication was evident during the acute phase of HDV infection. Seroconversion for antibodies to delta antigen occurred 3 weeks after the onset of the disease. These results demonstrate that a typical HDV infection can be initiated by inoculation of a susceptible animal with recombinant HDV cDNA.     

Hepatitis delta virus antigen forms dimers and multimeric complexes in vivo.

Although the hepatitis delta virus genome contains multiple open reading frames, only one of these reading frames is known to be expressed during replication of the virus. This open reading frame encodes two distinct molecular species of hepatitis delta antigen (HDAg), p24 delta and p27 delta, depending on the location of the stop codon which terminates translation. We found antibody specific for p27 delta to be capable of precipitating p24 delta in extracts of infected liver, indicating that p27 delta and p24 delta form heterologous complexes in vivo. After cross-linking with 0.05% glutaraldehyde, specific HDAg dimers were detected in antigen prepared from both the liver and serum of an HDV-infected woodchuck carrier of woodchuck hepatitis virus. Guanidine HCl-denatured HDAg extracted from liver and dialyzed against phosphate-buffered saline sedimented in rate-zonal sucrose density gradients as 15S multimeric complexes. These 15S multimers were stable in the presence of 1.2% Nonidet P-40. After RNase digestion, the 15S complex was reduced to a 12S complex without associated RNA, while boiling for 3 min in 1% sodium dodecyl sulfate-0.5% 2-mercaptoethanol further reduced the 15S complex to 3S HDAg monomers. In the absence of glutaraldehyde cross-linking, HDAg extracted from liver migrated as monomer species in reducing and nonreducing gels, suggesting that the conserved cysteine residue present in p27 delta does not play a role in the formation of either dimers or multimers. On the other hand, an amino-terminal chymotrypsin-digested HDAg fragment, with a predicted length of 81 or less amino acids, retained the ability to form dimers, consistent with the hypothesis that a coiled-coil motif present between residues 27 and 58 may play a role in HDAg protein interactions in vivo.     

Genotype-Specific Complementation of Hepatitis Delta Virus RNA Replication by Hepatitis Delta Antigen

Characterizations of genetic variations among hepatitis delta virus (HDV) isolates have focused principally on phylogenetic analysis of sequences, which vary by 30 to 40% among three genotypes and about 10 to 15% among isolates of the same genotype. The significance of the sequence differences has been unclear but could be responsible for pathogenic variations associated with the different genotypes. Studies of the mechanisms of HDV replication have been limited to cDNA clones from HDV genotype I, which is the most common. To perform a comparative analysis of HDV RNA replication in genotypes I and III, we have obtained a full-length cDNA clone from an HDV genotype III isolate. In transfected Huh-7 cells, the functional roles of the two forms of the viral protein, hepatitis delta antigen (HDAg), in HDV RNA replication are similar for both genotypes I and III; the short form is required for RNA replication, while the long form inhibits replication. For both genotypes, HDAg was able to support replication of RNAs of the same genotype that were mutated so as to be defective for HDAg production. Surprisingly, however, neither genotype I nor genotype III HDAg was able to support replication of such mutated RNAs of the other genotype. The inability of genotype III HDAg to support replication of genotype I RNA could have been due to a weak interaction between the RNA and HDAg. The clear genotype-specific activity of HDAg in supporting HDV RNA replication confirms the original categorization of HDV sequences in three genotypes and further suggests that these should be referred to as types (i.e., HDV-I and HDV-III) rather than genotypes.