Hepatitis Delta Antigen Mediates the Nuclear Import of Hepatitis Delta Virus RNA

Hepatitis delta virus (HDV) RNA replicates in the nuclei of virus-infected cells. The mechanism of nuclear import of HDV RNA is so far unknown. Using a fluorescein-labeled HDV RNA introduced into partially permeabilized HeLa cells, we found that HDV RNA accumulated only in the cytoplasm. However, in the presence of hepatitis delta antigen (HDAg), which is the only protein encoded by HDV RNA, the HDV RNA was translocated into the nucleus, suggesting that nuclear import of HDV RNA is mediated by HDAg. Deletion of the nuclear localization signal (NLS) or RNA-binding motifs of HDAg resulted in the failure of nuclear import of HDV RNA, indicating that both the NLS and an RNA-binding motif of HDAg are required for the RNA-transporting activity of HDAg. Surprisingly, any one of the three previously identified RNA-binding motifs was sufficient to confer the RNA-transporting activity. We have further shown that HDAg, via its NLS, interacts with karyopherin α2 in vitro, suggesting that nuclear import of the HDAg-HDV RNA complex is mediated by the karyopherin α2β heterodimer. The nuclear import of HDV RNA may be the first biological function of HDAg in the HDV life cycle.     

Genotype-Specific Complementation of Hepatitis Delta Virus RNA Replication by Hepatitis Delta Antigen

Characterizations of genetic variations among hepatitis delta virus (HDV) isolates have focused principally on phylogenetic analysis of sequences, which vary by 30 to 40% among three genotypes and about 10 to 15% among isolates of the same genotype. The significance of the sequence differences has been unclear but could be responsible for pathogenic variations associated with the different genotypes. Studies of the mechanisms of HDV replication have been limited to cDNA clones from HDV genotype I, which is the most common. To perform a comparative analysis of HDV RNA replication in genotypes I and III, we have obtained a full-length cDNA clone from an HDV genotype III isolate. In transfected Huh-7 cells, the functional roles of the two forms of the viral protein, hepatitis delta antigen (HDAg), in HDV RNA replication are similar for both genotypes I and III; the short form is required for RNA replication, while the long form inhibits replication. For both genotypes, HDAg was able to support replication of RNAs of the same genotype that were mutated so as to be defective for HDAg production. Surprisingly, however, neither genotype I nor genotype III HDAg was able to support replication of such mutated RNAs of the other genotype. The inability of genotype III HDAg to support replication of genotype I RNA could have been due to a weak interaction between the RNA and HDAg. The clear genotype-specific activity of HDAg in supporting HDV RNA replication confirms the original categorization of HDV sequences in three genotypes and further suggests that these should be referred to as types (i.e., HDV-I and HDV-III) rather than genotypes.     

Arginine-Rich Motifs Are Not Required for Hepatitis Delta Virus RNA Binding Activity of the Hepatitis Delta Antigen

Hepatitis delta virus (HDV) replication and packaging require interactions between the unbranched rodlike structure of HDV RNA and hepatitis delta antigen (HDAg), a basic, disordered, oligomeric protein. The tendency of the protein to bind nonspecifically to nucleic acids has impeded analysis of HDV RNA protein complexes and conclusive determination of the regions of HDAg involved in RNA binding. The most widely cited model suggests that RNA binding involves two proposed arginine-rich motifs (ARMs I and II) in the middle of HDAg. However, other studies have questioned the roles of the ARMs. Here, binding activity was analyzed in vitro using HDAg-160, a C-terminal truncation that binds with high affinity and specificity to HDV RNA segments in vitro. Mutation of the core arginines of ARM I or ARM II in HDAg-160 did not diminish binding to HDV unbranched rodlike RNA. These same mutations did not abolish the ability of full-length HDAg to inhibit HDV RNA editing in cells, an activity that involves RNA binding. Moreover, only the N-terminal region of the protein, which does not contain the ARMs, was cross-linked to a bound HDV RNA segment in vitro. These results indicate that the amino-terminal region of HDAg is in close contact with the RNA and that the proposed ARMs are not required for binding HDV RNA. Binding was not reduced by mutation of additional clusters of basic amino acids. This result is consistent with an RNA-protein complex that is formed via numerous contacts between the RNA and each HDAg monomer.     

Multimerization of Hepatitis Delta Antigen Is a Critical Determinant of RNA Binding Specificity

Hepatitis delta virus (HDV) RNA forms an unbranched rod structure that is associated with hepatitis delta antigen (HDAg) in cells replicating HDV. Previous in vitro binding experiments using bacterially expressed HDAg showed that the formation of a minimal ribonucleoprotein complex requires an HDV unbranched rod RNA of at least about 300 nucleotides (nt) and suggested that HDAg binds the RNA as a multimer of fixed size. The present study specifically examines the role of HDAg multimerization in the formation of the HDV ribonucleoprotein complex (RNP). Disruption of HDAg multimerization by site-directed mutagenesis was found to profoundly alter the nature of RNP formation. Mutant HDAg proteins defective for multimerization exhibited neither the 300-nt RNA size requirement for binding nor specificity for the unbranched rod structure. The results unambiguously demonstrate that HDAg binds HDV RNA as a multimer and that the HDAg multimer is formed prior to binding the RNA. RNP formation was found to be temperature dependent, which is consistent with conformational changes occurring on binding. Finally, analysis of RNPs constructed with unbranched rod RNAs successively longer than the minimum length indicated that multimeric binding is not limited to the first HDAg bound and that a minimum RNA length of between 604 and 714 nt is required for binding of a second multimer. The results confirm the previous proposal that HDAg binds as a large multimer and demonstrate that the multimer is a critical determinant of the structure of the HDV RNP.Human hepatitis delta virus (HDV) is an unusual subviral agent that increases the severity of acute and chronic liver disease in those infected with its helper, hepatitis B virus (23). The HDV genome is a 1,680-nucleotide (nt) single-stranded circular RNA that is replicated by a double-rolling-circle mechanism (reviewed in references 15 and 28). Both the genome and antigenome RNAs form a characteristic unbranched rod structure due to 70% sequence complementarity between the noncoding and coding regions of the RNA (10, 11, 31). HDV encodes just one protein, hepatitis delta antigen (HDAg), which forms ribonucleoprotein (RNP) complexes with both the genome and the antigenome in cells replicating HDV (3, 5, 30). These complexes play fundamental roles in viral RNA replication and packaging and their characterization is essential for understanding these processes, which are not well characterized.HDAg has been shown to form dimers and higher order multimers, even in the absence of HDV RNA (25, 30, 32). The multimerization activity has been localized to the amino-terminal third of the 195-amino-acid (aa) protein (12, 24, 30, 32). X-ray crystallographic analysis of a peptide comprised of aa 12 to 60 indicated that antiparallel dimers are stabilized by a coiled coil (aa 16 to 48), as well as a hydrophobic core region (aa 50 to 60) that also stabilizes interactions between dimers such that an octameric structure may form (35). Zuccola et al. found that bacterially expressed HDAg could be cross-linked in an octameric structure, and Cornillez-Ty et al. obtained evidence supporting such a structure in cells replicating HDV (7, 35). Site-directed mutations of HDAg amino acids critical for dimerization and/or multimerization abolish the ability of HDAg to support RNA replication (18, 32), indicating that the formation of HDAg multimers is essential for this process.We recently showed that bacterially expressed, C-terminally truncated HDAg forms stable RNP complexes in vitro with segments of HDV RNA that form unbranched rod structures (8). No particular sequences or structures in the RNA, other than the HDV unbranched rod, were essential for complex formation, but, remarkably, binding required that the RNA have a minimum length of at least about 300 nt. Overall, the results were consistent with the formation of a large RNP containing multiple copies of the 19-kDa protein that bound to the RNA either in a highly cooperative manner or as a preformed multimer. On the other hand, based on indirect measures of the RNA-binding activity of site-directed HDAg mutations in cells, others have found that HDAg multimerization might not be required for RNA-binding activity (18).Here, we directly analyze the role of HDAg multimerization in the formation of the HDV RNP complex. We find that HDAg binds to HDV unbranched rod RNA as a preformed multimer. Site-directed mutations that disrupted protein multimerization did not abolish binding but profoundly altered the nature of the RNA-protein complex. In particular, we found that multimerization is associated with RNA-binding specificity, including the RNA length requirement for binding. For the wild-type protein, RNP formation was found to be strongly temperature dependent, suggesting that conformational changes occur on binding, and providing a plausible explanation of the RNA length requirement for binding. Furthermore, we show that the protein binds as multiple multimeric units on longer RNAs, provided the length of the RNA is sufficient. We conclude that the HDAg multimer plays a critical role in the formation of properly structured HDV RNPs.     

Hepatitis Delta Virus RNA Editing Is Highly Specific for the Amber/W Site and Is Suppressed by Hepatitis Delta Antigen

RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication.     

Delta Hepatitis in Denver

The prevalence of hepatitis D virus (HDV) infection in patients with hepatitis B virus (HBV) infection in the mid-United States is not well defined. We tested 65 patients seen between 1983 and 1986 with HBV infection in Denver for evidence of coexisting HDV infection. Five patients had anti-delta (δ) antibody. The prevalence of HDV infection was higher in patients with chronic hepatitis B (4/37) than in patients with acute hepatitis B (1/28). The prevalence of HDV infection in male homosexuals (3/32) was similar to reported figures, but the incidence of δ-infection in intravenous drug users in Denver was usually low (1/16). In comparison to Los Angeles, New York, southern Italy, and Sweden, Denver appears to have a low incidence of HDV infection, which probably reflects its low prevalence in the drug-using population.     

Properties of Antigenomic Hepatitis Delta Virus Ribozyme That Consists of Three RNA Oligomer Strands

A three-strand ribozyme, a derivative of antigenomic hepatitis delta virus (HDV) ribozyme, which consists of subfragments of 16 (L), 17 (S), and 33 nucleotides (B), has been constructed. The ternary B-L-S complex formed by the subfragments in stoichiometric ratio was able to catalyze a self-cleavage reaction. Kinetics of this reaction exhibited biphasic behavior and the same parameters as in the case of natural cis-ribozyme. Study of kinetics of reaction initiated by adding various reaction components and the study of binary complex formation between subfragments B and L, B and S, and also ternary B-L-S complex formation revealed that: 1) in the presence of Mg2+, B and S form a stoichiometric complex, L and S do not form complex at all, while B and L form 2 types of complexes, probably B-L and 2B-L; and addition of S subfragment prevented the formation of the latter complex; 2) the reaction initiated by S subfragment proceeds much slower than that initiated by other components pointing to the possibility that in the absence of S L may form a nonproductive complex with B, which is slowly displaced by S followed by productive ternary complex formation. Dissociation constants for binary B-L, B-S and ternary B-L-S complexes have been estimated.     

Efficient Hepatitis Delta Virus RNA Replication in Avian Cells Requires a Permissive Factor(s) from Mammalian Cells

Hepatitis delta virus (HDV) is a highly pathogenic human RNA virus whose genome is structurally related to those of plant viroids. Although its spread from cell to cell requires helper functions supplied by hepatitis B virus (HBV), intracellular HDV RNA replication can proceed in the absence of HBV proteins. As HDV encodes no RNA-dependent RNA polymerase, the identity of the (presumably cellular) enzyme responsible for this reaction remains unknown. Here we show that, in contrast to mammalian cells, avian cells do not support efficient HDV RNA replication and that this defect cannot be rescued by provision of HDV gene products in trans. Contrary to earlier assertions, this defect is not due to enhanced apoptosis triggered in avian cells by HDV. Fusion of avian cells to mammalian cells rescues HDV replication in avian nuclei, indicating that the nonpermissive phenotype of avian cells is not due to the presence of dominantly acting inhibitors of replication. Rather, avian cells lack one or more essential permissive factors present in mammalian cells. These results set the stage for the identification of such factors and also explain the failure of earlier efforts to transmit HDV infection to avian hosts harboring indigenous hepadnaviruses.     

Anti-HDV IgM as a Marker of Disease Activity in Hepatitis Delta

Background

Hepatitis delta frequently leads to liver cirrhosis and hepatic decompensation. As treatment options are limited, there is a need for biomarkers to determine disease activity and to predict the risk of disease progression. We hypothesized that anti-HDV IgM could represent such a marker.

Methods

Samples of 120 HDV-infected patients recruited in an international multicenter treatment trial (HIDIT-2) were studied. Anti-HDV IgM testing was performed using ETI-DELTA-IGMK-2-assay (DiaSorin). In addition, fifty cytokines, chemokines and angiogenetic factors were measured using multiplex technology (Bio-Plex System). A second independent cohort of 78 patients was studied for the development of liver-related clinical endpoints (decompensation, HCC, liver transplantation or death; median follow up of 3.0 years, range 0.6–12).

Results

Anti-HDV IgM serum levels were negative in 18 (15%), low (OD<0.5) in 76 (63%), and high in 26 (22%) patients of the HIDIT-2 cohort. Anti-HDV IgM were significantly associated with histological inflammatory (p<0.01) and biochemical disease activity (ALT, AST p<0.01). HDV replication was independent from anti-HDV IgM, however, low HBV-DNA levels were observed in groups with higher anti-HDV IgM levels (p<0.01). While high IP-10 (CXCL10) levels were seen in greater groups of anti-HDV IgM levels, various other antiviral cytokines were negatively associated with anti-HDV IgM. Associations between anti-HDV IgM and ALT, AST, HBV-DNA were confirmed in the independent cohort. Clinical endpoints occurred in 26 anti-HDV IgM positive patients (39%) but in only one anti-HDV IgM negative individual (9%; p = 0.05).

Conclusions

Serum anti-HDV IgM is a robust, easy-to-apply and relatively cheap marker to determine disease activity in hepatitis delta which has prognostic implications. High anti-HDV IgM levels may indicate an activated interferon system but exhausted antiviral immunity.     

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.     

Sphingomyelin Activates Hepatitis C Virus RNA Polymerase in a Genotype-Specific Manner

Hepatitis C virus (HCV) replication and infection depend on the lipid components of the cell, and replication is inhibited by inhibitors of sphingomyelin biosynthesis. We found that sphingomyelin bound to and activated genotype 1b RNA-dependent RNA polymerase (RdRp) by enhancing its template binding activity. Sphingomyelin also bound to 1a and JFH1 (genotype 2a) RdRps but did not activate them. Sphingomyelin did not bind to or activate J6CF (2a) RdRp. The sphingomyelin binding domain (SBD) of HCV RdRp was mapped to the helix-turn-helix structure (residues 231 to 260), which was essential for sphingomyelin binding and activation. Helix structures (residues 231 to 241 and 247 to 260) are important for RdRp activation, and 238S and 248E are important for maintaining the helix structures for template binding and RdRp activation by sphingomyelin. 241Q in helix 1 and the negatively charged 244D at the apex of the turn are important for sphingomyelin binding. Both amino acids are on the surface of the RdRp molecule. The polarity of the phosphocholine of sphingomyelin is important for HCV RdRp activation. However, phosphocholine did not activate RdRp. Twenty sphingomyelin molecules activated one RdRp molecule. The biochemical effect of sphingomyelin on HCV RdRp activity was virologically confirmed by the HCV replicon system. We also found that the SBD was the lipid raft membrane localization domain of HCV NS5B because JFH1 (2a) replicon cells harboring NS5B with the mutation A242C/S244D moved to the lipid raft while the wild type did not localize there. This agreed with the myriocin sensitivity of the mutant replicon. This sphingomyelin interaction is a target for HCV infection because most HCV RdRps have 241Q.Hepatitis C virus (HCV) has a positive-stranded RNA genome and belongs to the family Flaviviridae (21). HCV chronically infects more than 130 million people worldwide (34), and HCV infection often induces liver cirrhosis and hepatocellular carcinoma (19, 28). To date, pegylated interferon (PEG-IFN) and ribavirin are the standard treatments for HCV infection. However, many patients cannot tolerate their serious side effects. Therefore, the development of new and safer therapeutic methods with better efficacy is urgently needed.Lipids play important roles in HCV infection and replication. For example, the HCV core associates with lipid droplets and recruits nonstructural proteins and replication complexes to lipid droplet-associated membranes which are involved in the production of infectious virus particles (24). HCV RNA replication depends on viral protein association with raft membranes (2, 30). The association of cholesterol and sphingolipid with HCV particles is also important for virion maturation and infectivity (3). The inhibitors of the sphingolipid biosynthetic pathway, ISP-1 and HPA-12, which specifically inhibit serine palmitoyltransferase (SPT) (23) and ceramide trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus (37), suppress HCV virus production in cell culture but not viral RNA replication by the JFH1 replicon (3). Other serine SPT inhibitors (myriocin and NA255) inhibit genotype 1b replication (4, 29, 33). Very-low-density lipoprotein (VLDL) also interacts with the HCV virion (15).Sakamoto et al. reported that sphingomyelin bound to HCV RNA-dependent polymerase (RdRp) at the sphingomyelin binding domain (SBD; amino acids 230 to 263 of RdRp) to recruit HCV RdRp on the lipid rafts, where the HCV complex assembles, and that NA255 suppressed HCV replication by releasing HCV RdRp from the lipid rafts (29). In the present study, we analyzed the effect of sphingomyelin on HCV RdRp activity in vitro and found that sphingomyelin activated HCV RdRp activity in a genotype-specific manner. We also determined the sphingomyelin activation domain and the activation mechanism. Finally, we confirmed our biochemical data by a HCV replicon system.     

RNA干扰对乙肝病毒基因的抑制作用

双链介导的遗传干涉的机制是1998年发现的。它通过双链RNA的介导特异性地降解相应序列从而导致转录后水平的基因沉默。RNA干扰作为后基因组时代的一种下调基因表达的工具已被广泛用于基因功能的研究以及疾病的治疗。利用小干扰RNA与乙肝病毒DNA通过共转染于HepG2肝癌细胞中使乙肝病毒基因沉默以达到抑制乙肝病毒复制作用。     

RNA干扰对乙肝病毒基因的抑制作用

双链介导的遗传干涉的机制是1998年发现的。它通过双链RNA的介导特异性地降解相应序列从而导致转录后水平的基因沉默。RNA干扰作为后基因组时代的一种下调基因表达的工具已被广泛用于基因功能的研究以及疾病的治疗。利用小干扰RNA与乙肝病毒DNA通过共传染于HepG2肝癌细胞中使乙肝病毒基因沉默以达到抑制乙肝病毒复制作用。