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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Large hepatitis delta antigen in packaging and replication inhibition: role of the carboxyl-terminal 19 amino acids and amino-terminal sequences.

Hepatitis delta virus (HDV) encodes two proteins, the small delta antigen (SHDAg) and large delta antigen (LHDAg). The latter is identical to the former except for the presence of additional 19 amino acids at the C terminus. While SHDAg is required for HDV replication, LHDAg inhibits replication and, together with hepatitis B surface antigen (HBsAg), is required for the assembly of HDV. The last 19 C-terminal amino acids of LHDAg are essential for HDV assembly. Most of LHDAg (amino acids 19 to 146 and 163 to 195) had been shown to be dispensable for packaging with HBsAg. To discern whether the last 19 C-terminal amino acids solely constitute the signal for packaging with HBsAg, we constructed two LHDAg deletion mutants and tested their abilities to be packaged with HBsAg in cotransfection experiments. We found that deletion of amino acids 2 to 21 and 142 to 165 did not affect LHDAg packaging. This result suggested that only the last 19 C-terminal amino acids of LHDAg are required for packaging. We further constructed two plasmids which expressed c-H-ras with or without additional 19 C-terminal amino acids identical to those in LHDAg. Only c-H-ras with additional 19 amino acids could be cosecreted with HBsAg in the cotransfection experiment. This result confirmed that the C-terminal 19 amino acids are the packaging signal for HBsAg. We also tested the trans activation activity and trans-dominant inhibitory activity of the deletion mutants of SHDAg and LHDAg, respectively. In contrast to deletion of amino acids 142 to 165, deletion of amino acids 2 to 21 impaired the trans-dominant inhibitory activity of LHDAg. Deletion of amino acids 2 to 21 and 142 to 165 did not affect the trans activation activity of SHDAg. This result suggested that a functional domain which is important for the trans-dominant inhibitory activity of LHDAg exists in the amino terminus of HDAg.     

Two regions of an avian hepadnavirus RNA pregenome are required in cis for encapsidation.

We have constructed a series of deletion mutants spanning the genome of duck hepatitis B virus in order to determine which regions of the viral genome are required in cis for packaging of the pregenome into capsid particles. Deletion of sequences within either of two nonadjacent regions prevented replication of the mutant viral genomes expressed in a permissive avian hepatoma cell line in the presence of functionally active viral core and P proteins. Extraction of RNA from cells transfected with these replication-defective mutants showed that the mutants retained the capacity to be transcribed into a pregenomic-size viral RNA, but that these RNA species were not packaged into viral capsids. The two regions defined by these deletions are located 36 to 126 (region I) and 1046 to 1214 (region II) nucleotides downstream of the 5' end of the pregenome and contain sequences which are required in cis for encapsidation of the duck hepatitis B virus pregenome.     

In vitro-synthesized hepatitis delta virus RNA initiates genome replication in cultured cells.

Monomers of the genomic strand of hepatitis delta virus RNA were transcribed in vitro and then delivered to NIH 3T3 fibroblasts by using a liposome fusion technique. After 7 days, genome replication was detected, but only in fibroblasts that stably expressed the delta antigen. Sequence analysis of the replicated products identified them as faithful copies of the hepatitis delta virus genome found in virions.     

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.     

Role of the large hepatitis B virus envelope protein in infectivity of the hepatitis delta virion.

The hepatitis delta virus (HDV) is coated with large (L), middle (M), and small (S) envelope proteins encoded by coinfecting hepatitis B virus (HBV). To study the role of the HBV envelope proteins in the assembly and infectivity of HDV, we produced three types of recombinant particles in Huh7 cells by transfection with HBV DNA and HDV cDNA: (i) particles with an envelope containing the S HBV envelope protein only, (ii) particles with an envelope containing S and M proteins, and (iii) particles with an envelope containing S, M, and L proteins. Although the resulting S-, SM-, and SML-HDV particles contained both hepatitis delta antigen and HDV RNA, only particles coated with all three envelope proteins (SML) showed evidence of infectivity in an in vitro culture system susceptible to HDV infection. We concluded that the L HBV envelope protein, and more specifically the pre-S1 domain, is important for infectivity of HDV particles and that the M protein, which has been reported to bear a site for binding to polymerized albumin in the pre-S2 domain, is not sufficient for infectivity. Our data also show that the helper HBV is not required for initiation of HDV infection. The mechanism by which the L protein may affect HDV infectivity is discussed herein.     

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.     

Genetic organization and replication strategy of hepatitis delta virus

Hepatitis delta virus (HDV) is a subviral satellite of human hepatitis B virus (HBV). The discovery in 1977 and subsequent demonstration of HDV as an infectious agent was primarily due to the work of Rizzetto and co-workers. In nature, HDV infections occur only if HBV is present. This is because HDV is a subviral satellite of HBV; HBV provides the envelope, or surface antigens, needed for the assembly of HDV particles. Other than this dependence, HDV seems fundamentally different from HBV; it has a single-stranded RNA genome and replicates via RNA-directed RNA synthesis. Five years ago the first nucleotide sequence of the genome was obtained and as a consequence we have progressively gained a picture of the genetic organization of this unusual agent and of its replication strategy.