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.     

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.     

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.     

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.     

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.     

Tissue culture system for infection with human hepatitis delta virus.

An in vitro culture system was developed for assaying the infectivity of the human hepatitis delta virus (HDV). Hepatocytes were isolated from chimpanzee liver and grown in a serum-free medium. Cells were shown to be infectible by HDV and to remain susceptible to infection for at least 3 weeks in culture, as evidenced by the appearance of RNA species characteristic of HDV replication as early as 6 days postinfection. When repeated experiments were carried out on cells derived from an animal free of hepatitis B virus (HBV), HDV infection occurred in a consistent fashion but there was no indication of infection with the HBV that was present in the inoculum. Despite numerous attempts with different sources of HBV inocula free of HDV, there was no evidence that indicated susceptibility of these cells to HBV infection. This observation may indicate that HBV and HDV use different modes of entry into hepatocytes. When cells derived from an HBV-infected animal were exposed to HDV, synthesis and release of progeny HDV particles were obtained in addition to HBV replication and production of Dane particles. Although not infectible with HBV, primary cultures of chimpanzee hepatocytes are capable of supporting part of the life cycle of HBV and the entire life cycle of HDV.     

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.     

Primary Biliary Acids Inhibit Hepatitis D Virus (HDV) Entry into Human Hepatoma Cells Expressing the Sodium-Taurocholate Cotransporting Polypeptide (NTCP)

BackgroundThe sodium-taurocholate cotransporting polypeptide (NTCP) is both a key bile acid (BA) transporter mediating uptake of BA into hepatocytes and an essential receptor for hepatitis B virus (HBV) and hepatitis D virus (HDV). In this study we aimed to characterize to what extent and through what mechanism BA affect HDV cell entry.MethodsHuH-7 cells stably expressing NTCP (HuH-7/NTCP) and primary human hepatocytes (PHH) were infected with in vitro generated HDV particles. Infectivity in the absence or presence of compounds was assessed using immunofluorescence staining for HDV antigen, standard 50% tissue culture infectious dose (TCID50) assays and quantitative PCR.ResultsAddition of primary conjugated and unconjugated BA resulted in a dose dependent reduction in the number of infected cells while secondary, tertiary and synthetic BA had a lesser effect. This effect was observed both in HuH-7/NTCP and in PHH. Other replication cycle steps such as replication and particle assembly and release were unaffected. Moreover, inhibitory BA competed with a fragment from the large HBV envelope protein for binding to NTCP-expressing cells. Conversely, the sodium/BA-cotransporter function of NTCP seemed not to be required for HDV infection since infection was similar in the presence or absence of a sodium gradient across the plasma membrane. When chenodeoxycolic acid (15 mg per kg body weight) was administered to three chronically HDV infected individuals over a period of up to 16 days there was no change in serum HDV RNA.ConclusionsPrimary BA inhibit NTCP-mediated HDV entry into hepatocytes suggesting that modulation of the BA pool may affect HDV infection of hepatocytes.     

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.     

Proteoglycans Act as Cellular Hepatitis Delta Virus Attachment Receptors

The hepatitis delta virus (HDV) is a small, defective RNA virus that requires the presence of the hepatitis B virus (HBV) for its life cycle. Worldwide more than 15 million people are co-infected with HBV and HDV. Although much effort has been made, the early steps of the HBV/HDV entry process, including hepatocyte attachment and receptor interaction are still not fully understood. Numerous possible cellular HBV/HDV binding partners have been described over the last years; however, so far only heparan sulfate proteoglycans have been functionally confirmed as cell-associated HBV attachment factors. Recently, it has been suggested that ionotrophic purinergic receptors (P2XR) participate as receptors in HBV/HDV entry. Using the HBV/HDV susceptible HepaRG cell line and primary human hepatocytes (PHH), we here demonstrate that HDV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cellular heparan sulfate proteoglycans. We furthermore provide evidence that P2XR are not involved in HBV/HDV entry and that effects observed with inhibitors for these receptors are a consequence of their negative charge. HDV infection was abrogated by soluble GAGs and other highly sulfated compounds. Enzymatic removal of defined carbohydrate structures from the cell surface using heparinase III or the obstruction of GAG synthesis by sodium chlorate inhibited HDV infection of HepaRG cells. Highly sulfated P2XR antagonists blocked HBV/HDV infection of HepaRG cells and PHH. In contrast, no effect on HBV/HDV infection was found when uncharged P2XR antagonists or agonists were applied. In summary, HDV infection, comparable to HBV infection, requires binding to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors, while P2XR are not actively involved.     

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.     

Hepatitis B surface antigen levels and sequences of natural hepatitis B virus variants influence the assembly and secretion of hepatitis d virus

Various domains of hepatitis B surface antigen (HBsAg) are essential for the assembly and secretion of hepatitis D virus (HDV). This study investigated the influences of the levels and sequences of HBsAg of naturally occurring HBV variants on the assembly and secretion of HDV. Six hepatitis B virus (HBV)-producing plasmids (three genotype B and three genotype C) and six HBsAg expression plasmids that expressed various HBsAg levels were constructed from the sera of HDV-infected patients. These plasmids were cotransfected with six expression plasmids of HDV of genotype 1, 2, or 4 into the Huh-7 hepatoma cell line. Serum HBsAg and HBV DNA levels were correlated with HDV RNA levels and outcomes of chronic hepatitis D (CHD) patients. The secretion of genotype 1, 2, or 4 HDV generally correlated with HBsAg levels but not with HBV genotypes or HBV DNA levels. Swapping and residue mutagenesis experiments of HBsAg-coding sequences revealed that the residue Pro-62 in the cytosolic domain-I affects the assembly and secretion of genotype 2 and 4 HDV and not those of genotype 1. The pre-S2 N-terminal deletion HBV mutant adversely affects secretion of the three HDV genotypes. In patients, serum HDV RNA levels correlated with HBsAg levels but not with HBV DNA levels. Viremia of HDV or HBV correlated with poor outcomes. In conclusion, the assembly and secretion of HDV were influenced by the amounts and sequences of HBsAg. For an effective treatment of CHD, reduction of HBsAg production in addition to the suppression of HBV and HDV replication might be crucial.     

Introduction of hepatitis delta virus into animal cell lines via cationic liposomes.

Cationic liposomes are known to facilitate efficient transfection of animal cells with DNA and even some viruses. As reported here, we have been able to use such a commercially available formulation (Lipofectamine) and introduce human hepatitis delta virus (HDV) into lines of cultured cells and demonstrate replication of the HDV genome both by immunofluorescence and by Northern (RNA) analysis. As much as 10% of the human hepatoma cell line Huh7 was transfected with HDV. Also transfected were the baby hamster kidney cell line BHK-21 and the Morris rat hepatoma line 7777. Two initial applications of HDV transfection have been made. (i) The ribonucleoprotein structure of HDV was isolated from disrupted virions and demonstrated as being sufficient to transfect Huh7 cells. In contrast, naked HDV RNA was not sufficient. (ii) From a study of cells transfected with HDV particles, it was found that, even after as long as 7 weeks and the associated replication of the transfected cells, the HDV RNA genome was still replicating. Apparently, HDV, in the absence of helper virus and in the absence of virus assembly, can maintain persistent replication and expression of the HDV genome. Transfection was also achieved with woodchuck hepatitis virus introduced into Huh7 cells. In summary, this transfection procedure should be of use for the study of these and maybe other recalcitrant animal viruses.     

Changes in the proteome of Huh7 cells induced by transient expression of hepatitis D virus RNA and antigens

Hepatitis delta virus (HDV) infection of human hepatocytes infected with the hepatitis B virus (HBV) is associated with increased liver damage and risk of fulminant disease. Although considerable progress has been made towards the elucidation of the mechanisms of HDV replication and pathogenesis, little is still known about the host factors involved in the different steps of the replication cycle. Here, we made use of a proteomic approach to analyse the global alterations in protein expression that arise in human hepatocytes separately transfected with each of the HDV components. Huh7 cells were transiently transfected with plasmids that code for the small delta antigen (S-HDAg), large delta antigen (L-HDAg), genomic RNA (gRNA), and antigenomic RNA (agRNA), respectively. Total protein extracts were separated by 2-DE and differentially expressed spots were identified by MALDI-TOF followed by database searching. We identified 32 proteins known to be involved in different pathways namely nucleic acid metabolism, protein metabolism, transport, signal transduction, apoptosis, and cell growth. Moreover, the down regulation of hnRNP D, HSP105, and triosephosphate isomerase was further confirmed by Real time PCR.     

Effects of conserved RNA secondary structures on hepatitis delta virus genotype I RNA editing, replication, and virus production

RNA editing of the hepatitis delta virus (HDV) antigenome at the amber/W site by the host RNA adenosine deaminase ADAR1 is a critical step in the HDV replication cycle. Editing is required for production of the viral protein hepatitis delta antigen long form (HDAg-L), which is necessary for viral particle production but can inhibit HDV RNA replication. The RNA secondary structural features in ADAR1 substrates are not completely defined, but base pairing in the 20-nucleotide (nt) region 3' of editing sites is thought to be important. The 25-nt region 3' of the HDV amber/W site in HDV genotype I RNA consists of a conserved secondary structure that is mostly base paired but also has asymmetric internal loops and single-base bulges. To understand the effect of this 3' region on the HDV replication cycle, mutations that either increase or decrease base pairing in this region were created and the effects of these changes on amber/W site editing, RNA replication, and virus production were studied. Increased base pairing, particularly in the region 15 to 25 nt 3' of the editing site, significantly increased editing; disruption of base pairing in this region had little effect. Increased editing resulted in a dramatic inhibition of HDV RNA synthesis, mostly due to excess HDAg-L production. Although virus production at early times was unaffected by this reduced RNA replication, at later times it was significantly reduced. Therefore, it appears that the conserved RNA secondary structure around the HDV genotype I amber/W site has been selected not for the highest editing efficiency but for optimal viral replication and secretion.     

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.     

Hepatitis delta virus RNA encoding the large delta antigen cannot sustain replication due to rapid accumulation of mutations associated with RNA editing

Hepatitis delta virus (HDV) contains two RNA species (HDV-S and HDV-L), which encode the small and large forms of hepatitis delta antigens (S- and L-HDAg), respectively. HDV-L RNA is a result of an RNA editing event occurring at an amber/W site of HDV-S RNA. RNA editing must be regulated to prevent premature and excessive accumulation of HDV-L RNA in the viral life cycle. In this study, we used an RNA transfection procedure to study the replication abilities of HDV-L and HDV-S RNA. While HDV-S led to robust RNA replication, HDV-L could not replicate even after 6 days following transfection. The failure of HDV-L to replicate was not due to insufficient amounts of S-HDAg, as identical results were obtained in a cell line that stably overexpresses S-HDAg. Also, it was not due to possible inhibition by L-HDAg, as HDV-S RNA replication was not affected when both HDV-L and HDV-S RNA were cotransfected. Further, when L-HDAg expression from HDV-L RNA was abolished by site-directed mutagenesis, the mutant HDV-L RNA also failed to replicate. Unexpectedly, when the kinetics of RNA replication was examined daily, HDV-L was found to replicate at a low level at the early time points (1 to 2 days posttransfection) but then lose this capability at later time points. Sequence analysis of the replicated HDV-L RNA at day 1 posttransfection showed that it had undergone multiple nucleotide changes, particularly in the region near the putative promoter region of HDV RNA replication. In contrast, very few mutations were found in HDV-S RNA. These results suggest that the editing at the amber/W site triggers a series of additional mutations which rapidly reduce the replication efficiency of the resultant HDV genome and thus help regulate the amount of HDV-L RNA in infected cells. They also explain why L-HDAg is not produced early in HDV infection, despite the fact that HDV-L RNA is present in the virion.