Expression of hepatitis delta virus RNA deletions: cis and trans requirements for self-cleavage, ligation, and RNA packaging.

The hepatitis delta virus (HDV) genome is a circular, single-stranded, rod-shaped, 1.7-kb RNA that replicates via a rolling-circle mechanism. Viral ribozymes function to cleave replication intermediates which are then ligated to generate the circular product. HDV expresses two forms of a single protein, the small and large delta antigens (delta Ag-S and delta Ag-L), which associate with viral RNA in a ribonucleoprotein (RNP) structure. While delta Ag-S is required for RNA replication, delta Ag-L inhibits this process but promotes the assembly of the RNP into mature virions. In this study, we have expressed full-length and deleted HDV RNA inside cells to determine the minimal RNA sequences required for self-cleavage, ligation, RNP packaging, and virion assembly and to assess the role of either delta antigen in each of these processes. We report the following findings. (i) The cleavage and ligation reactions did not require either delta antigen and were not inhibited in their presence. (ii) delta Ag-L, in the absence of delta Ag-S, formed an RNP with HDV RNA which could be assembled into secreted virus-like particles. (iii) Full-length HDV RNAs were stabilized in the presence of either delta antigen and accumulated to much higher levels than in their absence. (iv) As few as 348 nucleotides of HDV RNA were competent for circle formation, RNP assembly, and incorporation into virus-like particles. (v) An HDV RNA incapable of folding into the rod-like structure was not packaged by delta Ag-L.     

Hepatitis delta virus ribonucleoproteins shuttle between the nucleus and the cytoplasm

Hepatitis delta virus (HDV) infection of individuals infected with hepatitis B virus (HBV) is associated with more severe liver damage and an increased risk of fulminant disease. HDV is a single-stranded RNA virus that encodes a single protein, the delta antigen, which is expressed in two forms, small (S-HDAg) and large (L-HDAg). Here we show that although HDV ribonucleoproteins are mainly detected in the nucleus, they are also present in the cytoplasm of cells infected with HDV or transfected with HDV cDNA. Making use of an heterokaryon assay, we demonstrate that HDV ribonucleoproteins shuttle continuously between the nucleus and the cytoplasm. In the absence of HDV RNA, both forms of the delta antigen are retained in the nucleus, whereas in the absence of the delta antigen, HDV RNA is predominantly detected in the cytoplasm. Coexpression of HDV RNA and S-HDAg (which binds to the viral RNA and contains a nuclear localization signal) results in nuclear accumulation of the viral RNA. This suggests that HDV RNA mediates export of viral particles to the cytoplasm whereas the delta antigen triggers their reimport into the nucleus.     

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.     

Epitopes exposed on hepatitis delta virus ribonucleoproteins.

A total of 17 antibodies, raised in several nonhuman species and specific for different regions on the delta antigen (delta Ag), were used to map, via immunoprecipitation, those domains exposed on the surface of the viral ribonucleoprotein (RNP). These studies showed that the domains for the nuclear localization signal and the C-terminal extension, unique to the large form of delta Ag, are exposed. Also exposed is the C-terminal region of the small form of delta Ag. In contrast, reactivity was not found with the coiled-coil domain needed for protein dimerization. When the hepatitis delta virus (HDV) RNA was released by treatment of viral RNP with vanadyl ribonucleoside complexes, no change in the pattern of delta Ag epitope presentation was detected, consistent with the interpretation that a multimeric protein structure persists in the absence of RNA. These RNP studies have implications not only for understanding of the process of HDV assembly but also for evaluation of the immune responses of an infected host to HDV replication.     

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.     

Functional domains of delta antigens and viral RNA required for RNA packaging of hepatitis delta virus.

The functions of delta antigens (HDAgs) in the morphogenesis of hepatitis delta virus (HDV) have been studied previously. The C terminus of large HDAg has been shown to complex with the small surface antigen (HBsAg) of helper hepatitis B virus, whereas the assembly of small HDAg requires interaction with the N terminus of large HDAg (M.-F. Chang, C.-J. Chen, and S. C. Chang, J. Virol. 68:646-653, 1994). To further examine the molecular mechanisms by which HDAgs are involved in the assembly of HDV RNA, we have cotransfected Huh-7 cells with plasmids representing a longer than unit-length HDV and the small HBsAg cDNAs. We found that HDAg mRNA could be generated from an endogenous promoter within the HDV cDNA that was translated into large HDAg. Large HDAg is capable of complexing with monomeric HDV genomic RNA to form ribonucleoprotein particles (RNPs) and is capable of forming enveloped HDV-like particles in the presence of small HBsAg without undergoing HDV replication. In addition, the middle region from amino acid residues 89 to 145 of large HDAg is required for assembly of the RNPs but is dispensable for assembly of the enveloped particles. RNA assembly is also demonstrated with small HDAg when it is cotransfected with a packaging-defective large HDAg mutant and small HBsAg. Leu-115 within the putative helix-loop-helix structure of the small HDAg is important for the replication of HDV but is not essential for RNA assembly, suggesting that conformational requirements of small HDAg for replication and assembly of viral RNA may be different. Further studies indicate that a 312-nucleotide linear HDV RNA from one end of the HDV and structure is sufficient to form RNP complexes competent for assembly of virus-like particles with large HDAg and small HBsAg.     

Production of hepatitis delta virus and suppression of helper hepatitis B virus in a human hepatoma cell line.

The hepatitis delta virus (HDV) is a defective virus with a coat composing of the surface antigen of its helper virus, hepatitis B virus (HBV). Replication of HDV in the absence of HBV has been shown in cell cultures by transient transfection of the HDV plasmid. However, the formation and release of HDV virions have not been observed. In this report, a human hepatoma cell line HuH-7 was transiently cotransfected with HDV and HBV plasmids. The production of monomeric and multimeric antigenomic RNAs of HDV in the transfected cells indicated replication of the HDV genome. The major 3.5- and 2.1-kb RNAs of HBV were also expressed. Virions of both HDV and HBV were released from the cotransfected cells, as shown by the detection of monomeric genomic HDV RNA and partially double-stranded HBV DNA in the culture medium. Thus, this is the first report that describes the assembly and the release of HDV viral particles in an in vitro cell culture. The HDV virions released possessed physicochemical properties identical to those of the HDV virions found in infected human serum. Furthermore, expression of both the 3.5- and 2.1-kb RNAs of HBV was shown to be dramatically decreased by the presence of HDV, indicating suppression of the expression of HBV genes by HDV. The amount of HBV virions released was similarly suppressed by HDV. Cotransfection of HBV with an expression plasmid of the HDV delta antigen remarkably reduced the levels of the 3.5- and 2.1-kb HBV RNAs, indicating that suppression of the expression of HBV RNAs by HDV occurs via the action of the delta antigen. This HBV- and HDV-cotransfected human hepatoma cell line should provide an excellent system for the study of the function of the delta antigen and the interaction between HDV and its helper, HBV.     

Solution structure and RNA-binding activity of 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. The two proteins resemble each other except for the presence of an additional 19 amino acids at the C terminus of the latter species. We have found that the N-terminal leucine-repeat region of hepatitis delta antigen (HDAg) binds to the autolytic domain of HDV genomic RNA and attenuates its autolytic activity. A 27-residue polypeptide corresponding to residues 24-50 of HDAg, designated dAg(24-50), was synthesized, and its solution structure was found to be an alpha-helix by circular dichroism and (1)H-nuclear magnetic resonance (NMR) techniques. Binding affinity of dAg(24-50) with HDV genomic RNA was found to increase with its alpha-helical content, and it was further confirmed by modifying its N- and C-terminal groups. Furthermore, the absence of RNA binding activity in the mutant peptides, dAgM(24-50am) and dAgM(Ac24-50am), in which Lys38, Lys39, and Lys40 were changed to Glu, indicates a possible involvement of these residues in their binding activity. Structural knowledge of the N-terminal leucine-repeat region of HDAg thus provides a molecular basis for the understanding of its role in the interaction with RNA. Proteins 1999;37:121-129.     

Editing on the genomic RNA of human hepatitis delta virus.

It has been shown previously that during replication of the genome of human hepatitis delta virus (HDV), a specific nucleotide change occurs to eliminate the termination codon for the small delta antigen (G. Luo, M. Chao, S.-Y. Hsieh, C. Sureau, K. Nishikura, and J. Taylor, J. Virol. 64:1021-1027, 1990). This change creates an extension in the length of the open reading frame for the delta antigen from 195 to 214 amino acids. These two proteins, the small and large delta antigens, have important and distinct roles in the life cycle of HDV. To further investigate the mechanism of this specific nucleotide alteration, we developed a sensitive assay involving the polymerase chain reaction to monitor changes on HDV RNA sequences as they occurred in transfected cells. We found that the substrate for the sequence change was the viral genomic RNA rather than the antigenomic RNA. This sequence change occurred independently of genome replication or the presence of the delta antigen. Less than full-length genomic RNA could act as a substrate, but only if it also contained a corresponding RNA sequences from the other side of the rodlike structure, which is characteristic of HDV. We were also able to reproduce the HDV base change in vitro, by addition of purified viral RNA to nuclear extracts of cells from a variety of species.     

Hepatitis D Virus Infection of Mice Expressing Human Sodium Taurocholate Co-transporting Polypeptide

Hepatitis D virus (HDV) is the smallest virus known to infect human. About 15 million people worldwide are infected by HDV among those 240 million infected by its helper hepatitis B virus (HBV). Viral hepatitis D is considered as one of the most severe forms of human viral hepatitis. No specific antivirals are currently available to treat HDV infection and antivirals against HBV do not ameliorate hepatitis D. Liver sodium taurocholate co-transporting polypeptide (NTCP) was recently identified as a common entry receptor for HDV and HBV in cell cultures. Here we show HDV can infect mice expressing human NTCP (hNTCP-Tg). Antibodies against critical regions of HBV envelope proteins blocked HDV infection in the hNTCP-Tg mice. The infection was acute yet HDV genome replication occurred efficiently, evident by the presence of antigenome RNA and edited RNA species specifying large delta antigen in the livers of infected mice. The resolution of HDV infection appears not dependent on adaptive immune response, but might be facilitated by innate immunity. Liver RNA-seq analyses of HDV infected hNTCP-Tg and type I interferon receptor 1 (IFNα/βR1) null hNTCP-Tg mice indicated that in addition to induction of type I IFN response, HDV infection was also associated with up-regulation of novel cellular genes that may modulate HDV infection. Our work has thus proved the concept that NTCP is a functional receptor for HDV infection in vivo and established a convenient small animal model for investigation of HDV pathogenesis and evaluation of antiviral therapeutics against the early steps of infection for this important human pathogen.     

Assembly of hepatitis delta virus: particle characterization, including the ability to infect primary human hepatocytes

Efficient assembly of hepatitis delta virus (HDV) was achieved by cotransfection of Huh7 cells with two plasmids: one to provide expression of the large, middle, and small envelope proteins of hepatitis B virus (HBV), the natural helper of HDV, and another to initiate replication of the HDV RNA genome. HDV released into the media was assayed for HDV RNA and HBV envelope proteins and characterized by rate-zonal sedimentation, immunoaffinity purification, electron microscopy, and the ability to infect primary human hepatocytes. Among the novel findings were that (i) immunostaining for delta antigen 6 days after infection with 300 genome equivalents (GE) per cell showed only 1% of cells as infected, but this was increased to 16% when 5% polyethylene glycol was present during infection; (ii) uninfected cells did not differ from infected cells in terms of albumin accumulation or the presence of E-cadherin at cell junctions; and (iii) sensitive quantitative real-time PCR assays detected HDV replication even when the multiplicity of infection was 0.2 GE/cell. In the future, this HDV assembly and infection system can be further developed to better understand the mechanisms shared by HBV and HDV for attachment and entry into host cells.     

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.     

Characterization of hepatitis delta antigen: specific binding to hepatitis delta virus RNA.

It has previously been shown that human hepatitis virus delta antigen has an RNA-binding activity (Chang et al., J. Virol. 62:2403-2410, 1988). In the present study, the specificity of such an RNA-protein interaction was demonstrated by expressing various domains of the delta antigen in Escherichia coli as TrpE fusion proteins and testing their RNA-binding activities in a Northwestern protein-RNA immunoblot assay and RNA gel mobility shift assay. Hepatitis delta virus (HDV) RNA bound specifically to the delta antigen in the presence of an excess amount of unrelated RNAs and a relatively high salt concentration. Both genome- and antigenome-sense HDV RNAs and at least two different regions of HDV genomic RNA bound to the delta antigen. Surprisingly, these two different regions of HDV genomic RNA could compete with each other for delta antigen binding, although they do not have common nucleotide sequences. In contrast, this binding could not be competed with by other viral or cellular RNA. Since both the genomic and antigenomic HDV RNAs had strong intramolecular complementary sequences, these results suggest that the binding of delta antigen is probably specific for a secondary structure unique to the HDV RNA. By expressing different subdomains of the delta antigen, we found that the middle one-third of delta antigen was responsible for binding HDV RNA. Neither the N-terminal nor the C-terminal domain bound HDV RNA. Binding between the delta antigen and HDV RNA was also demonstrated within the HDV particles isolated from the plasma of a human delta hepatitis patient. This in vivo binding resisted treatment with 0.1% sodium dodecyl sulfate and 0.5% Nonidet P-40. In addition, we showed that the antiserum from a human patient with delta hepatitis reacted with all three subdomains of the delta antigen, indicating that all of the domains are immunogenic in vivo. These studies demonstrated the specific interaction between delta antigen and HDV RNA.     

Genetic changes in hepatitis delta virus from acutely and chronically infected woodchucks

A woodchuck-derived hepatitis delta virus (HDV) inoculum was created by transfection of a genotype I HDV cDNA clone directly into the liver of a woodchuck that was chronically infected with woodchuck hepatitis virus. All woodchucks receiving this inoculum became positive for HDV RNA in serum, and 67% became chronically infected, similar to the rate of chronic HDV infection in humans. Analysis of HDV sequences obtained at 73 weeks postinfection indicated that changes had occurred at a rate of 0.5% per year; many of these modifications were consistent with editing by host RNA adenosine deaminase. The appearance of sequence changes, which were not evenly distributed on the genome, was correlated with the course of HDV infection. A limited number of modifications occurred in the consensus sequence of the viral genome that altered the sequence of the hepatitis delta antigen (HDAg). All chronically infected animals examined exhibited these changes 73 weeks following infection, but at earlier times, only one of the HDV carriers exhibited consensus sequence substitutions. On the other hand, sequence modifications in animals that eventually recovered from HDV infection were apparent after 27 weeks. The data are consistent with a model in which HDV sequence changes are selected by host immune responses. Chronic HDV infection in woodchucks may result from a delayed and weak immune response that is limited to a small number of epitopes on HDAg.     

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.     

Deleterious effects of hepatitis delta virus replication on host cell proliferation

Hepatitis delta virus (HDV) infection and spread in vivo are dependent upon coinfection by hepatitis B virus (HBV), and dual HDV/HBV infection is frequently more severe than HBV infection alone, raising the possibility that HDV infection may be deleterious to cells. Here we have examined the effects of HDV replication on the long-term growth of cultured cells. Our results show that most cells transfected with HDV cDNA do not give rise to stable cell lines expressing viral antigens or replicative intermediates; in addition, cotransfection of HDV replicons with a plasmid vector expressing a hygromycin resistance marker results in a dose-dependent impairment of hygromycin-resistant colony formation. When cells transfected with replication-competent HDV cDNA are followed prospectively, a progressive decline in viral RNA replication and a steady decrease in the proportion of cells expressing delta antigen are observed. However, in transient transfection assays, no evidence was found to link HDV replication to apoptosis or to cell cycle arrest, nor did HDV replication confer on host cells enhanced sensitivity to inducers of apoptosis. Thus, HDV replication does not appear to be acutely cytotoxic. However, in dividing cells HDV replication is associated with a subtler growth disadvantage, leading to selection in culture for cells displaying diminished HDV expression. This effect would not be expected to cause hepatitis in vivo but might contribute to impaired liver regeneration in the setting of ongoing hepatocellular injury.