trans-dominant inhibition of human hepatitis delta virus genome replication.

Infection with hepatitis delta virus (HDV) is an important cause of acute and chronic liver disease and can be rapidly fatal. Sequencing of the HDV RNA genome has revealed variability at the C-terminal end of the delta antigen reading frame. One genome type (termed the S genome) synthesizes a 24-kDa protein thought to be required for genome replication. Another genome type (termed the L genome) extends the reading frame by 19 amino acids as a result of a single base change. Replication of the S and L genomes was studied in cultured fibroblasts. While the S genome efficiently initiated genome replication, the L genome did not. Moreover, in a codelivery experiment, L genome RNA inhibited replication of the S genome. Potent trans inhibition was also observed following cotransfection of the S genome and a plasmid encoding the larger delta antigen. Mutational analysis indicated that the inhibitory activity was not a simple function of the large delta antigen reading frame's extra length. Implications for the viral life cycle, clinical infection, and potential treatment are discussed.     

Role of two forms of hepatitis delta virus antigen: evidence for a mechanism of self-limiting genome replication.

The replication of the RNA genome of hepatitis delta virus is greatly facilitated by the presence of the only known virus-coded protein, the delta antigen. Most, if not all, infections are characterized by the presence of two electrophoretic forms of the delta antigen. These forms correspond to polypeptide lengths of 195 and 214 amino acids which are encoded by genomes with different nucleotide sequences. We used cDNA transfections to investigate the functions of these two forms of the delta antigen. We found that only the small form of delta antigen supported hepatitis delta virus genome replication and that the large form acted as a dominant negative repressor of such replication. This inhibition was potent. For example, the amount of genome replication was reduced eightfold when as little as 10% of the delta antigen was present as the large form. One interpretation of our results is that the delta antigen normally functions as part of a multimeric structure. In addition, our data suggest that synthesis of the large form, either during genome replication in cultured cells or even during infection in animals, may suppress delta replication, possibly leading to a self-limiting infection.     

Initiation of replication of the human hepatitis delta virus genome from cloned DNA: role of delta antigen.

Beginning with three partial cDNA clones of the RNA genome of human hepatitis delta virus (HDV), we assembled the complete 1,679-base sequence on a single molecule and then inserted a trimer of this into plasmid pSLV, a simian virus 40-based eucaryotic expression vector. This construct was used to transfect both monkey kidney (COS7) and human hepatocellular carcinoma (HuH7) cell lines. In this way we obtained replication of the HDV RNA genome and the appearance, in the nucleoli, of the delta antigen, the only known virus-coded protein. This proved both that the HDV genome could replicate in nonliver as well as liver cells and that there was no requirement for the presence of hepatitis B virus sequences or proteins. When the pSVL construct was made with a dimer of an HDV sequence with a 2-base-pair deletion in the open reading frame, genome replication was reduced at least 40-fold. However, when we cotransfected with a plasmid that expressed the correct delta antigen, the mutated dimer achieved a level of genome replication comparable to that of the nonmutated sequence. We thus conclude that the delta antigen can act in trans and is essential for replication of the HDV genome.     

Assembly of hepatitis delta virus particles.

Hepatitis delta virus (HDV) is a subviral satellite of hepatitis B virus (HBV). Since the RNA genome of HDV can replicate in cultured cells in the absence of HBV, it has been suggested that the only helper function of HBV is to supply HBV coat proteins in the assembly process of HDV particles. To examine the factors involved in such virion assembly, we transiently cotransfected cells with various hepadnavirus constructs and cDNAs of HDV and analyzed the particles released into the medium. We report that the HDV genomic RNA and the delta antigen can be packaged by coat proteins of either HBV or the related hepadnavirus woodchuck hepatitis virus (WHV). Among the three co-carboxy-terminal coat proteins of WHV, the smallest form was sufficient to package the HDV genome; even in the absence of HDV RNA, the delta antigen could be packaged by this WHV coat protein. Also, of the two co-amino-terminal forms of the delta antigen, only the larger form was essential for packaging.     

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.     

Ribonucleoprotein complexes of hepatitis delta virus.

Human hepatitis delta virus (HDV) is a subviral satellite agent of hepatitis B virus (HBV). The envelope proteins of HDV are provided by the helper virus, HBV, but very little is known about the internal structure of HDV. The particles contain multiple copies of the delta antigen and an unusual RNA genome that is small, about 1,700 nucleotides in length, single stranded, and circular. By using UV cross-linking, equilibrium density centrifugation, and immunoprecipitation, we obtained evidence consistent with the interpretation that delta antigen and genomic RNA form a stable ribonucleoprotein (RNP) complex within the virion. Furthermore, electron-microscopic examination of the purified viral RNP revealed a roughly spherical core-like structure with a diameter of 18.7 +/- 2.5 nm. We also isolated HDV-specific RNP structures from the nuclei of cells undergoing HDV genome replication; both the genome and antigenome (a complement of the genome) of HDV were found to be in such complexes. From the equilibrium density analyses of the viral and nuclear RNPs, we were able to deduce the number of molecules of delta antigen per molecule of HDV RNA. For virions, this number was predominantly ca. 70, which was larger than for the nuclear RNPs, which were more heterogeneous, with an average value of ca. 30.     

Organization and Expression of the Hepatitis Delta Virus Genome

The RNA genome of human hepatitis delta virus (HDV) is an unusual small circular single-stranded species that can fold on itself to form an unbranched rod-like structure. This RNA is replicated in the nucleus by RNA-directed RNA synthesis coupled with RNA processing events. During processing events a subgenomic, polyadenylated RNA that is complementary to the genome and expressed in the cytoplasm as the small form of the delta antigen, a 195-amino-acid protein essential for genome replication is produced. The strategies of RNA virus genome organization and expression are very diverse; those used by HDV seem unique among animal viruses, although there are some distant similarities with those used by some plant pathogens.     

A single antigenomic open reading frame of the hepatitis delta virus encodes the epitope(s) of both hepatitis delta antigen polypeptides p24 delta and p27 delta.

On the basis of the complete nucleotide sequence of the single-stranded, covalently closed circular hepatitis delta virus RNA genome (K.-S. Wang, Q.-L. Choo, A. J. Weiner, J.-H. Ou, R. C. Najarian, R. M. Thayer, G. T. Mullenbach, K. J. Denniston, J. L. Gerin, and M. Houghton, Nature [London] 323:508-514, 1986 [Author's correction, 328:456, 1987]), five long open reading frames (ORFs) encoding polypeptides containing a methionine proximal to the amino terminus were expressed in bacteria. Only polypeptides encoded by the antigenomic ORF5 cross-reacted with antisera obtained from patients with hepatitis delta virus infections. Immunological analysis of viral extracts and the recombinant ORF5 polypeptides synthesized in bacteria and yeast cells revealed that ORF5 encodes the immunogenic epitope(s) shared by both hepatitis delta viral polypeptides p27 delta and p24 delta and probably represents the complete structural gene for p27 delta and p24 delta. We also present evidence that ORF5 encodes the hepatitis delta antigen, an antigen originally found in the nuclei of hepatocytes of infected individuals (M. Rizzetto, M. G. Canese, S. Arico, O. Crivelli, F. Bonino, C. G. Trepo, and G. Verme, Gut 18:997-1003, 1977). A comparison of the primary structure of the predicted hepatitis delta antigen polypeptides with that of the core antigen of the hepatitis B virus shows that these polypeptides are very dissimilar.     

Relating structure to function in the hepatitis delta virus antigen.

Hepatitis delta virus expresses two forms of a single protein, the small (delta Ag-S) and large (delta Ag-L) antigens, which are identical except for an additional 19 residues present at the C terminus of delta Ag-L. While delta Ag-S is required to promote genome replication, delta Ag-L potently inhibits this process and also facilitates packaging of the viral genome by envelope proteins of the helper virus (hepatitis B virus). Regions within the antigens responsible for nuclear localization, RNA binding, and dimerization have been identified, yet it is not clear how these particular activities contribute to the ultimate replication and packaging phenotypes. Here we report the following findings. (i) Although the removal of the nuclear localization signal from either antigen resulted in significant cytoplasmic accumulation, both proteins still had access to the nucleus. As a consequence, no functional defect was observed with either mutant. (ii) The RNA-binding domain, although necessary for delta Ag-S function, could be deleted from delta Ag-L without compromising its ability to either inhibit replication or promote packaging. (iii) In contrast, the coiled-coil dimerization domain was required for both the activation of replication by delta Ag-S and the inhibition of replication by delta Ag-L. This region, with an additional 20 amino acids C-terminal to it, was necessary and sufficient to potently inhibit replication by interacting with the small antigen. (iv) The packaging property of delta Ag-L required a C-terminal Pro/Gly-rich region which is hypothesized to interact with the hepatitis B virus envelope proteins during the assembly process.     

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.     

Heterogeneity and evolution rates of delta virus RNA sequences.

To investigate the geographical divergence of delta virus RNA sequences, 868 nucleotides (nt), including the delta antigen-coding region, were determined in isolates from two Japanese patients, M and S, by polymerase chain reaction and direct sequencing and compared with three previously reported nucleotide sequences. The sequence obtained for hepatitis delta virus RNA from patient M was approximately 92% identical to sequences previously obtained for two other strains of hepatitis delta virus, whereas the sequence of hepatitis delta virus RNA obtained from patient S was approximately 81% identical to the previously sequenced strains. This suggests that delta agent in Japan has a heterogeneous origin and the delta virus RNA sequence from Japanese patient S is the most divergent delta virus isolate yet analyzed. To study the evolution rate of delta virus RNA, viral isolates obtained 3 and 4 years apart from each of two patients were also sequenced. It was estimated that the substitution rate of viral RNA was 0.57 x 10(-3) nt per site per year in patient M and 0.64 x 10(-3) nt per site per year in patient S for the delta antigen gene.     

Solution structure of an N-capping peptide from the N-terminal leucine-repeat region of hepatitis delta antigen

Hepatitis delta virus (HDV) is a satellite virus of the hepatitis B virus (HBV) which provides the surface antigen for the viral coat. The RNA genome of HDV encodes two proteins, the small delta antigen and the large delta antigen, which differ only with the latter having an additional 19 amino acids at the C-terminus. Previously, we have shown that dAg24-50, a synthetic peptide corresponding to residues 24-50 of the N-terminal leucine-repeat region of hepatitis delta antigen, binds to the viral RNA and forms an alpha-helical conformation in TFE-containing solution. However, it exhibited low alpha-helicity (less than 5%) in the absence of TFE. In order to obtain biologically active delta antigen peptides with higher structural stability in solution, an N-capping 21-residue polypeptide corresponding to residues 24-38 of hepatitis delta antigen (dAg(Cap24-38am)) was synthesized and, surprisingly, its solution structure was found to be a stable alpha-helix (64%) by circular dichroism and 1H NMR techniques. Moreover, the structure of the capping box shows the characteristic L-shaped bend perpendicular to the helix axis. This structural knowledge provides a molecular basis for understanding the role of the N-terminal leucine-repeat region of hepatitis delta antigen and has a significant potential for the development of diagnostic and therapeutic methods for HDV.     

Limitations to replication of hepatitis delta virus in avian cells

Human hepatitis delta virus (HDV) is a natural subviral agent that uses hepatitis B virus as a helper. Experimentally, HDV can be made to replicate in woodchucks, using woodchuck hepatitis B virus as a helper virus. Also, independent of such helper activity, replication of the HDV RNA genome can be achieved in many mammalian cells. In this study we examined whether such replication could also be achieved in avian cells. We used cotransfection strategies and initially found no detectable genome replication in chicken LMH cells relative to the mammalian cell line Huh7, used as a positive control. We also found that, in contrast to transfected Huh7 cells, the avian cell line was readily and efficiently killed by expression of the delta protein. Three strategies were used to reduce such killing: (i) the delta protein was expressed from a separate expression vector, the amount of which was then reduced as much as 33-fold; (ii) the protein was expressed transiently, using a promoter under tetracycline control; and (iii) the transfected cells were treated with Z-VAD-fmk, a broad-spectrum caspase inhibitor, which reduced cell killing. This last result indicated that cell killing occurred via an apoptotic pathway. After application of these three strategies to reduce cell killing, together with a novel procedure to improve the signal-to-noise ratio in Northern analyses, replication of the HDV genome was then detected in LMH cells. However, even after removal of obvious signs of toxicity, the amount was still >50 times lower than in the Huh7 cells. Our findings explain previous unsuccessful attempts to demonstrate replication of the HDV genome in avian cells and establish the precedent that in certain situations HDV replication can be cytotoxic.     

Replication of human hepatitis delta virus: recent developments

In a natural setting, hepatitis delta virus (HDV) is only found in patients that are also infected with hepatitis B virus (HBV). In hepatocytes infected with these two viruses, HDV RNA genomes are assembled using the envelope proteins of HBV. Since 1986, we have known that HDV has a small single-stranded RNA genome with a unique circular conformation that is replicated using a host RNA polymerase. These and other features make HDV and its replication unique, at least among agents that infect animals. This mini-review focuses on advances gained over the last 2-3 years, together with an evaluation of HDV questions that are either unsolved or not yet solved satisfactorily.     

Recombinant retroviral DNA yielding high expression of hepatitis B surface antigen

A genomic fragment of hepatitis B virus encoding the surface antigen (HBsAg) was inserted into the proviral genome of Moloney mouse sarcoma virus (MSV), obtained from the mouse cell line G8 -124. The recombinant DNA was introduced into NIH 3T3 mouse fibroblasts. Cells, morphologically transformed by the oncogene of MSV (v-mosM) were selected, established as cell lines and tested for expression of HBsAg. An expression level of up to 4.5 micrograms/10(7) cells/day was detected.     

Self-ligating RNA sequences on the antigenome of human hepatitis delta virus.

A 179-base fragment of RNA from the 1,679-base antigenome of hepatitis delta virus can not only self-cleave but, when the ends of the resultant fragments are brought into apposition by base pairing to another RNA, also self-ligate. Thus, processing events needed for genome replication in vivo may be strictly RNA mediated.     

A specific base transition occurs on replicating hepatitis delta virus RNA.

Three independent lines of evidence showed that when an infectious clone of hepatitis delta virus of known sequence was used to initiate genome replication, up to 41% of the genomes were specifically mutated in the amber termination codon (UAG to UGG) for the open reading frame of the delta antigen, thereby increasing the length of the predicted protein from 195 to 214 amino acids. This change was detected only on molecules that participated in RNA-directed RNA synthesis.     

Antigenomic RNA of human hepatitis delta virus can undergo self-cleavage.

The structure and replication of the single-stranded circular RNA genome of hepatitis delta virus (HDV) are unique relative to those of known animal viruses, and yet there are real similarities between HDV and certain infectious RNAs of plants. Therefore, since some of the latter RNAs have been shown to undergo in vitro site-specific cleavage and even ligation, we tested the hypothesis that similar events might also occur for HDV RNA. In partial confirmation of this hypothesis, we found that in vitro the RNA complementary to the HDV genome, the antigenomic RNA, could undergo a self-cleavage that was not only more than 90% efficient but also occurred only at a single location. This cleavage was found to produce junction fragments consistent with a 5'-hydroxyl and a cyclic 2',3'-monophosphate. Since the observed cleavage was both site-specific and occurred only once per genome length, we propose that the site may be relevant to the normal intracellular replication of the HDV genome. Because the site is located almost adjacent to the 3' end of the delta antigen-coding region, the only known functional open reading frame of HDV, we suggest that the cleavage may have a role not only in genome replication but also in RNA processing, helping to produce a functional mRNA for the translation of delta antigen.     

RNA editing in the replication cycle of human hepatitis delta virus

For some time it has been known that the RNA genome of human hepatitis delta virus (HDV) undergoes a specific RNA editing event. This review describes the editing phenomenon and its potential biological significance, and evaluates the data regarding the mechanism involved, including the possible relationship to other RNA editing phenomena.     

Molecular cloning of hepatitis delta virus RNA from an infected woodchuck liver: sequence, structure, and applications.

cDNA prepared from the single-stranded circular RNA genome of hepatitis delta virus was cloned in lambda gt11 by using RNA from the liver of an infected woodchuck. From the sequence of overlapping clones, we assembled the full sequence of 1,679 nucleotides. The sequence indicated an exceptional ability for intramolecular base pairing, yielding a rod structure with at least 70% of the bases paired and a predicted free energy of -805 kcal (-3,368 kJ)/mol. Three of the lambda clones contained sequences that were not only expressed as fusion proteins with beta-galactosidase but were recognized by human hepatitis delta virus-specific antibody. These clones were sequenced so as to establish the reading frame of the delta antigen on the antigenomic strand. The fusion protein produced by one clone was purified by immunoaffinity chromatography and then was used to raise rabbit antibodies specific for the delta antigen.