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.     

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.     

Interaction and replication activation of genotype I and II hepatitis delta antigens

The nucleotide sequences of hepatitis D viruses (HDV) vary 5 to 14% among isolates of the same genotype and 23 to 34% among different genotypes. The only viral-genome-encoded antigen, hepatitis delta antigen (HDAg), has two forms that differ in size. The small HDAg (HDAg-S) trans-activates viral replication, while the large form (HDAg-L) is essential for viral assembly. Previously, it has been shown that the packaging efficiency of HDAg-L is higher for genotype I than for genotype II. In this study, the question of whether other functional properties of the HDAgs are affected by genotype differences is addressed. By coexpression of the two antigens in HuH-7 cells followed by specific antibody precipitation, it was found that HDAgs of different origins interacted without genotypic discrimination. Moreover, in the presence of hepatitis B virus surface antigen, HDAg-S was incorporated into virion-like particles through interaction with HDAg-L without genotype restriction. As to the differences in replication activation of genotype I HDV RNA, all HDAg-S clones tested had some trans-activation activity, and this activity varied greatly among isolates. As to the support of HDV genotype II replication, only clones of HDAg-S from genotype II showed trans-activation activity, and this activity also varied among isolates. In conclusion, genotype has no effect on HDAg interaction and genotype per se only partly predicts how much the HDAg-S of an HDV isolate affects the replication of a second HDV isolate.     

The nucleolar phosphoprotein B23 interacts with hepatitis delta antigens and modulates the hepatitis delta virus RNA replication

Hepatitis delta virus (HDV) encodes two isoforms of delta antigens (HDAgs). The small form of HDAg is required for HDV RNA replication, while the large form of HDAg inhibits the viral replication and is required for virion assembly. In this study, we found that the expression of B23, a nucleolar phosphoprotein involved in disparate functions including nuclear transport, cellular proliferation, and ribosome biogenesis, is up-regulated by these two HDAgs. Using in vivo and in vitro experimental approaches, we have demonstrated that both isoforms of HDAg can interact with B23 and their interaction domains were identified as the NH(2)-terminal fragment of each molecule encompassing the nuclear localization signal but not the coiled-coil region of HDAg. Sucrose gradient centrifugation analysis indicated that the majority of small HDAg, but a lesser amount of the large HDAg, co-sedimented with B23 and nucleolin in the large nuclear complex. Transient transfection experiments also indicated that introducing exogenous full-length B23, but not a mutated B23 defective in HDAg binding, enhanced HDV RNA replication. All together, our results reveal that HDAg has two distinct effects on nucleolar B23, up-regulation of its gene expression and the complex formation, which in turn regulates HDV RNA replication. Therefore, this work demonstrates the important role of nucleolar protein in regulating the HDV RNA replication through the complex formation with the key positive regulator being small HDAg.     

Casein kinase II and protein kinase C modulate hepatitis delta virus RNA replication but not empty viral particle assembly.

Hepatitis delta virus (HDV) contains two virus-specific delta antigens (HDAgs), large and small forms, which are identical in sequence except that the large one contains 19 extra amino acids at the C terminus. HDAgs are nuclear phosphoproteins with distinct biological functions; the small form activates HDV RNA replication, whereas the large form suppresses this process but is required for viral particle assembly. In this study, we have characterized the phosphorylative property of HDAg in a human hepatoma cell line (HuH-7) and examined the role of phosphorylation in HDAg function. As demonstrated by in vivo labeling and kinase inhibitor experiments, the phosphorylation levels of both HDAgs were diminished by the inhibitor of casein kinase II (CKII). Nevertheless, phosphorylation of only the small form could be markedly reduced by the protein kinase C (PKC) inhibitor, suggesting different phosphorylation properties between the two HDAgs. When these two kinase inhibitors were added separately to the transient-expression system, HDV RNA replication was profoundly suppressed. In contrast, the inhibitors did not affect the assembly of empty HDAg particle from HDAgs and hepatitis B virus surface antigen. To further examine the role of phosphorylation in HDAg function, two conservative CKII recognition sites at Ser-2 and Ser-123 of both HDAgs and one potential PKC recognition site at Ser-210 of the large HDAg were altered to alanine by site-directed mutagenesis. Transfection experiments indicated that mutation at Ser-2, but not Ser-123, significantly impaired the activity of the small HDAg in assisting HDV RNA replication. This property is in accordance with our observation that Ser-2, not Ser-123, was the predominant CKII phosphorylation site in the small HDAg. Our studies also excluded the possibility that the phosphorylation of Ser-2, Ser-123, or Ser-210, had roles in the trans-suppression activity of the large HDAg, in the assembly of empty virus-like HDAg particle, and in the nuclear transport of HDAgs. In conclusion, our results indicate that both CKII and PKC positively modulate HDV RNA replication but not the assembly of empty HDAg particle. The role of CKII in HDV replication may at least in part be accounted for by the phosphorylation of Ser-2 in the small HDAg. The effect of PKC on HDV RNA replication is, however, not to mediate the phosphorylation of the conservative Ser-210 in the large HDAg but rather to act on as-yet-unidentified Ser or Thr residues in the small HDAg or cellular factors. These findings provide the first insight into the roles of phosphorylation of the two HDAgs in the HDV replication cycle.     

Functional motifs of delta antigen essential for RNA binding and replication of hepatitis delta virus.

The functions of delta antigens (HDAgs) in the replication of hepatitis delta virus (HDV) have been identified previously. The small HDAg acts as a transactivator, whereas the large HDAg has a negative effect on replication. To understand the molecular mechanisms involved in the control of HDV replication, we have established a replication system in Huh-7 cells by cotransfecting a monomeric cDNA genome of HDV and a plasmid encoding the small HDAg. We demonstrate that a leucine repeat in the middle domain of the small HDAg is involved in binding to the HDV genome and transactivation of HDV replication. When the leucine repeat was disrupted by a substitution of valine for leucine at position 115, both RNA-binding and transactivation activity of the small HDAg were abolished. In contrast, the binding and transactivation activities were not affected when Leu-37 and Leu-44 of the small HDAg were replaced by valines. In addition, small and large HDAgs can interact with each other to form protein complexes in vitro. The complex formation that may lead to the trans-dominant negative regulation of large HDAg in HDV replication is mediated by a cryptic signal located between amino acid residues 35 and 65 other than the putative N-terminal leucine zipper motif. Furthermore, an extra 21-amino-acid extension near the N terminus converts the small HDAg into a pseudo-large HDAg with negative regulation activity of HDV replication even though the extreme C-terminal residue is unchanged.     

Isoprenylation mediates direct protein-protein interactions between hepatitis large delta antigen and hepatitis B virus surface antigen.

Hepatitis delta antigen (HDAg) consists of two protein species of 195 and 214 amino acids, respectively, which are identical in sequence except that the large HDAg has additional 19 amino acids at its C terminus and is prenylated. Previous studies have shown that the large HDAg and the surface antigen of hepatitis B virus (HBsAg) together can form empty hepatitis delta virus (HDV) particles. To understand the molecular mechanism of HDV virion morphogenesis, we investigated the possible direct protein-protein interaction between HDAg and HBsAg. We constructed recombinant baculoviruses expressing the major form of HBsAg and various mutant HDAgs and used these proteins for far-Western protein binding assays. We demonstrated that HBsAg interacted specifically with the large HDAg but not with the small HDAg. Using mutant HDAgs which have defective or aberrant prenylation, we showed that this interaction required isoprenylates on the cysteine residue of the C terminus of the large HDAg. Isoprenylation alone, without the remainder of the C-terminal amino acids of the large HDAg, was insufficient to mediate interaction with HBsAg. This study demonstrates a novel role of prenylates in HDV virion assembly.     

Polyadenylation of the mRNA of hepatitis delta virus is dependent on the structure of the nascent RNA and regulated by the small or large delta antigen.

During the hepatitis delta virus (HDV) RNA replication, synthesis of either the mRNA for the delta antigen (HDAg) or the full-length antigenomic RNA is determined by selective usage of the potent poly(A) signal on the antigenome. To elucidate the regulatory mechanism, HDV cDNA cotransfection system was used to examine the potential effect of the secondary structure of the nascent RNA and that of the HDAg on HDV polyadenylation in transfected cells. We found that when the nascent RNA species could fold itself to form the rodlike structure, the HDV polyadenylation was suppressed 3 to 5 fold by the HDAg. In addition, we observed that the small and the large HDAg exerted a similar suppressive effect on the HDV polyadenylation, though they played different roles in HDV replication. We concluded that the HDV polyadenylation could be regulated by the structure of the nascent antigenomic RNA and by either the small or large HDAg.     

Expression of the hepatitis delta virus large and small antigens in transgenic mice.

Simultaneous infection with hepatitis delta virus (HDV) and hepatitis B virus (HBV) in humans is often associated with severe viral liver disease including fulminant hepatitis. Since HBV is thought to be noncytopathic to the hepatocyte, the enhanced disease severity observed during dual infection has been attributed to either simultaneous immune responses against the two viruses or direct cytotoxic effects of HDV products on the hepatocyte or both. To examine these alternate possibilities, we produced transgenic mice that express the small and large delta antigens (HDAg) in hepatocyte nuclei at levels equal to those observed during natural HDV infection. No biological or histopathological evidence of liver disease was detectable during 18 months of observation, suggesting that neither the large nor small form of HDAg is directly cytopathic to the hepatocyte in vivo.     

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.     

Mutational analysis of delta antigen: effect on assembly and replication of hepatitis delta virus.

Hepatitis delta virus requires a helper function from hepatitis B virus for packaging, release, and infection of hepatocytes. The assembly of large delta antigen (HDAg) is mediated by copackaging with the small surface antigen of hepatitis B virus (HBsAg), and the assembly of small HDAg requires interactions with large HDAg. To examine the molecular mechanisms by which small HBsAg, large HDAg, and small HDAg interact, we have established a virion assembly system in COS7 cells by cotransfecting plasmids encoding the small HBsAg, the small HDAg, and large HDAg mutants. Results indicate that sequences within the C-terminal 19-amino-acid domain flanking the Cxxx isoprenylation motif are important for the assembly of large HDAg. In addition, a large HDAg mutant bearing extra sequences separating the C-terminal 19-amino-acid domain from the common regions of the small and large HDAgs is capable, like the wild-type large HDAg, of copackaging with small HBsAg. The ability of assembly is also demonstrated for a large HDAg mutant from which nuclear localization signals have been removed. Furthermore, a cryptic signal within the N-terminal 50 amino acid residues other than the putative N-terminal coiled-coil structure and a subdomain between amino acid residues 50 and 65 of the large HDAg are important for the assembly of small HDAg as well as the trans-dominant negative regulation of large HDAg in hepatitis delta virus replication.     

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.     

RNA-binding activity of hepatitis delta antigen involves two arginine-rich motifs and is required for hepatitis delta virus RNA replication.

Hepatitis delta antigen (HDAg) is an RNA-binding protein with binding specificity for hepatitis delta virus (HDV) RNA (J. H. Lin, M. F. Chang, S. C. Baker, S. Govindarajan, and M. M. C. Lai, J. Virol. 64:4051-4058, 1990). By amino acid sequence homology search, we have identified within its RNA-binding domain two stretches of an arginine-rich motif (ARM), which is present in many prokaryotic and eukaryotic RNA-binding proteins. The first one is KERQDHRRRKA and the second is EDEKRERRIAG, and they are separated by 29 amino acids. Deletion of either one of these ARM sequences resulted in the total loss of the in vitro RNA-binding activity of HDAg. Thus, HDAg is different from other RNA-binding proteins in that it requires two ARM-like sequences for its RNA-binding activity. Replacement of the spacer sequence between the two ARMs with a shorter stretch of sequence also reduced RNA binding in vitro. Furthermore, site-specific mutations of the basic amino acid residues in both ARMs resulted in the total loss or reduction of RNA-binding activity. The biological significance of the RNA-binding activity was studied by examining the trans-activating activity of the RNA-binding mutants. The plasmids expressing HDAgs with various mutations in the RNA-binding motifs were cotransfected with a replication-defective HDV dimer cDNA construct into COS cells. It was found that all the HDAg mutants which had lost the in vitro RNA-binding activity also lost the ability to complement the defect of HDV RNA replication. We conclude that the trans-activating function of HDAg requires its binding to HDV RNA.     

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.     

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.     

Small-form hepatitis B surface antigen is sufficient to help in the assembly of hepatitis delta virus-like particles.

Hepatitis delta virus (HDV) has an envelope composed of large-, middle-, and small-form hepatitis B surface antigens (HBsAgs) provided by the helper hepatitis B virus (HBV). In order to examine the roles of individual HBsAgs in HDV assembly, we constructed plasmids containing each specific HBsAg gene and then cotransfected each plasmid with HDV cDNA into a permissive human hepatoma cell line (HuH-7) to examine the effects on HDV production. Results indicated that the plasmids containing only the HBsAg genes were able to complement HDV cDNA as efficiently as the plasmid containing the complete HBV genome in generating HDV-like particles. Moreover, the small-form HBsAg alone was sufficient for HDV packaging. The particles produced from the cotransfection experiments have density and protein composition characteristics similar to those of naturally occurring HDV. With the electron microscope, they were identified as 36- to 38-nm-diameter particles. It was concluded that only the HBsAgs were able to help in the assembly of HDV-like particles.     

Human hepatitis delta antigen is a nuclear phosphoprotein with RNA-binding activity.

The genetic origin, structure, and biochemical properties of the delta antigen (HDAg) of a human hepatitis delta virus (HDV) were investigated. A cDNA fragment containing the open reading frame encoding the HDAg was transcribed into RNA and used for in vitro translation in rabbit reticulocyte lysates. The HDAg open reading frame was also inserted into an expression vector containing a simian virus 40 T-antigen promoter and expressed into COS 7 cells. In both systems, a protein species of 26 kilodaltons was synthesized from this open reading frame and could be specifically immunoprecipitated with antisera obtained from patients with delta hepatitis. A similar protein was also synthesized from antigenomic-sense monomeric HDV RNA in both systems, although the efficiency of translation was lower than that of the isolated open reading frame. This protein was found to be phosphorylated at the serine residues. Immunoperoxidase studies with anti-HDV sera demonstrated that the HDAg was expressed mainly in the nuclei of the transfected COS 7 cells. Moreover, the HDAg was shown to bind the genomic RNA of HDV. These studies indicate that HDAg is encoded by the antigenomic-sense RNA of HDV and is a nuclear phosphoprotein associated with an RNA-binding activity.     

Roles of carboxyl-terminal and farnesylated residues in the functions of the large hepatitis delta antigen

The large hepatitis delta antigen (HDAg-L) mediates hepatitis delta virus (HDV) assembly and inhibits HDV RNA replication. Farnesylation of the cysteine residue within the HDAg-L carboxyl terminus is required for both functions. Here, HDAg-L proteins from different HDV genotypes and genotype chimeric proteins were analyzed for their ability to incorporate into virus-like particles (VLPs). Observed differences in efficiency of VLP incorporation could be attributed to genotype-specific differences within the HDAg-L carboxyl terminus. Using a novel assay to quantify the extent of HDAg-L farnesylation, we found that genotype 3 HDAg-L was inefficiently farnesylated when expressed in the absence of the small hepatitis delta antigen (HDAg-S). However, as the intracellular ratio of HDAg-S to HDAg-L was increased, so too was the extent of HDAg-L farnesylation for all three genotypes. Single point mutations within the carboxyl terminus of HDAg-L were screened, and three mutants that severely inhibited assembly without affecting farnesylation were identified. The observed assembly defects persisted under conditions where the mutants were known to have access to the site of VLP assembly. Therefore, the corresponding residues within the wild-type protein are likely required for direct interaction with viral envelope proteins. Finally, it was observed that when HDAg-S was artificially myristoylated, it could efficiently inhibit HDV RNA replication. Hence, a general association with membranes enables HDAg to inhibit replication. In contrast, although myristoylated HDAg-S was incorporated into VLPs far more efficiently than HDAg-S or nonfarnesylated HDAg-L, it was incorporated far less efficiently than wild-type HDAg-L; thus, farnesylation was required for efficient assembly.