Hepatitis Delta Virus Genomic RNA Research Articles

Analysis of Hepatitis D Virus in Minor Salivary Gland of Sjögren’s Disease

Hepatitis delta virus (HDV) has been detected in the minor salivary gland (MSG) tissue of Sjögren’s disease (SjD) patients in the absence of a hepatitis B virus (HBV) coinfection. Previous research has shown that HDV antigen (HDAg) expression can trigger an SjD-like phenotype in vivo, demonstrating a potential cause-and-effect relationship. We hypothesize that if HDV plays a role in the development of SjD, then HDV profiles may be correlated with disease manifestations. This retrospective study characterized HDV in a cohort of 48 SjD MSG samples collected between 2014 and 2021. Analyses of HDAg expression, including cell type and subcellular localization, in situ hybridization of HDV RNA, and comparative analyses with associated SjD and viral hepatitis clinical features, were conducted. HDAg was detected in MSG acinar, ductal, myoepithelial, and adipose cells and localized with the nuclei, cytoplasm, and mitochondria. In situ hybridization detected HDV genomic RNA localization in the MSG nuclei. A significant negative correlation was found between HDAg intensity and focal lymphocytic inflammation and in patients with both anti-SSA/Ro-52 and anti-SSA/Ro-60. In analyzing autoimmune disease comorbidities with SjD, it was found that SjD patients diagnosed with autoimmune thyroiditis and/or hypothyroidism were significantly more represented in the high HDAg intensity group compared to the negative and moderate HDAg intensity groups. No significant associations were detected between MSG-localized HDAg and liver enzymes or an evident HBV coinfection. This study has further confirmed that there is a nonhepatic reservoir for chronic HDV persistence in SjD-affected salivary gland tissue in a third independent SjD patient cohort. In addition, this study describes the unique colocalization of HDAg with mitochondria. The detection of HDV antigen and sequence within SjD-affected salivary gland tissue, and in the absence of an evident current or past HBV coinfection, warrants further investigation.

Role of RNA Polymerase II Promoter-Proximal Pausing in Viral Transcription.

The promoter-proximal pause induced by the binding of the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) to RNAP II is a key step in the regulation of metazoan gene expression. It helps maintain a permissive chromatin landscape and ensures a quick transcriptional response from stimulus-responsive pathways such as the innate immune response. It is also involved in the biology of several RNA viruses such as the human immunodeficiency virus (HIV), the influenza A virus (IAV) and the hepatitis delta virus (HDV). HIV uses the pause as one of its mechanisms to enter and maintain latency, leading to the creation of viral reservoirs resistant to antiretrovirals. IAV, on the other hand, uses the pause to acquire the capped primers necessary to initiate viral transcription through cap-snatching. Finally, the HDV RNA genome is transcribed directly by RNAP II and requires the small hepatitis delta antigen to displace NELF from the polymerase and overcome the transcriptional block caused by RNAP II promoter-proximal pausing. In this review, we will discuss the RNAP II promoter-proximal pause and the roles it plays in the life cycle of RNA viruses such as HIV, IAV and HDV.

Treatment of hepatitis delta and HIV infection.

Hepatitis delta virus (HDV) is a defective agent that only infects individuals with hepatitis B virus (HBV). Around 5-10% of chronic hepatitis B patients worldwide are superinfected with HDV, which means 15-25 million people. Hepatitis delta is the most severe of all chronic viral hepatitis, leading to cirrhosis, liver cancer and/or transplantation in most patients. Despite it, many HDV patients remain undiagnosed. The only treatment available until recently was peginterferon alfa, with poor results and significant side effects. The recent approval of bulevirtide, a lipopeptide that blocks HBV/HDV entry, has revolutionized the field. Another drug, lonafarnib, already approved to treat progeria, is expected to be available soon as HDV therapy. Since there is no cell reservoir for the HDV RNA genome, hypothetically viral clearance could be achieved if complete blocking of viral replication occurs for a minimum time frame. This is what happens in hepatitis C using direct-acting antivirals, with the achievement of cure in nearly all treated patients. We envision the cure of hepatitis delta using combination antiviral therapy. Given that sexual and parenteral transmission routes are the most frequent for the acquisition of HBV and HDV, shared with HIV infection and HBV/HDV and HIV coinfection. The clinical outcome of hepatitis delta is worst in the HIV setting, with more frequent liver complications. Since most persons infected with HIV are on regular health care follow-up, we propose that HIV-HDV patients should be prioritized for moving forward new and potentially curative treatments for hepatitis delta.

Hepatitis Delta Virus (HDV) and Delta-Like Agents: Insights Into Their Origin.

Hepatitis delta virus (HDV) is a human pathogen, and the only known species in the genus Deltavirus. HDV is a satellite virus and depends on the hepatitis B virus (HBV) for packaging, release, and transmission. Extracellular HDV virions contain the genomic HDV RNA, a single-stranded negative-sense and covalently closed circular RNA molecule, which is associated with the HDV-encoded delta antigen forming a ribonucleoprotein complex, and enveloped by the HBV surface antigens. Replication occurs in the nucleus and is mediated by host enzymes and assisted by cis-acting ribozymes allowing the formation of monomer length molecules which are ligated by host ligases to form unbranched rod-like circles. Recently, meta-transcriptomic studies investigating various vertebrate and invertebrate samples identified RNA species with similarities to HDV RNA. The delta-like agents may be representatives of novel subviral agents or satellite viruses which share with HDV, the self-complementarity of the circular RNA genome, the ability to encode a protein, and the presence of ribozyme sequences. The widespread distribution of delta-like agents across different taxa with considerable phylogenetic distances may be instrumental in comprehending their evolutionary history by elucidating the transition from transcriptome to cellular circular RNAs to infectious subviral agents.

Hepatitis delta virus interacts with splicing factor SF3B155 and alters pre-mRNA splicing of cell cycle control genes.

Hepatitis delta virus (HDV) is the agent responsible for the most severe form of human viral hepatitis. The HDV genome consists of a single-stranded circular RNA molecule that encodes for one single protein, the delta antigen. Given its simplicity, HDV must make use of several host cellular proteins to accomplish its life cycle processes, including transcription, replication, post-transcriptional, and post-translational modifications. Consequently, identification of the interactions established between HDV components and host proteins assumes a pivotal interest in the search of novel therapeutic targets. Here, we used the yeast three-hybrid system to screen a human liver cDNA library to identify host proteins that interact with the HDV genomic RNA. One of the identified proteins corresponded to the splicing factor SF3B155, a component of the U2snRNP complex that is essential for the early recognition of 3' splice sites in the pre-mRNAs of human genes. We show that the interaction between the HDV genomic RNA and SF3B155 occurs invivo and that the expression of HDV promotes changes in splicing of human genes whose alternative splicing is SF3B155-dependent. We further show that expression of HDV triggers alterations in several constitutive and alternative splicing events in the tumor suppressor RBM5 transcript, with consequent reduction of its protein levels. This is the first description that HDV expression promotes changes in the splicing of human genes, and we suggest that the HDV-induced alternative splicing changes, through SF3B155 sequester, may contribute for the early progression to hepatocellular carcinoma characteristic of HDV-infected patients.

Hepatitis Delta Virus histone mimicry drives the recruitment of chromatin remodelers for viral RNA replication

Hepatitis Delta virus (HDV) is a satellite of Hepatitis B virus with a single-stranded circular RNA genome. HDV RNA genome synthesis is carried out in infected cells by cellular RNA polymerases with the assistance of the small hepatitis delta antigen (S-HDAg). Here we show that S-HDAg binds the bromodomain (BRD) adjacent to zinc finger domain 2B (BAZ2B) protein, a regulatory subunit of BAZ2B-associated remodeling factor (BRF) ISWI chromatin remodeling complexes. shRNA-mediated silencing of BAZ2B or its inactivation with the BAZ2B BRD inhibitor GSK2801 impairs HDV replication in HDV-infected human hepatocytes. S-HDAg contains a short linear interacting motif (SLiM) KacXXR, similar to the one recognized by BAZ2B BRD in histone H3. We found that the integrity of the S-HDAg SLiM sequence is required for S-HDAg interaction with BAZ2B BRD and for HDV RNA replication. Our results suggest that S-HDAg uses a histone mimicry strategy to co-activate the RNA polymerase II-dependent synthesis of HDV RNA and sustain HDV replication.

The Hepatitis Delta Virus accumulation requires paraspeckle components and affects NEAT1 level and PSP1 localization

The Hepatitis Delta Virus (HDV) relies mainly on host proteins for its replication. We previously identified that PSF and p54nrb associate with the HDV RNA genome during viral replication. Together with PSP1, these proteins are part of paraspeckles, which are subnuclear bodies nucleated by the long non-coding RNA NEAT1. In this work, we established the requirement for PSF, p54nrb and PSP1 in HDV replication using RNAi-mediated knockdown in HEK-293 cells replicating the HDV RNA genome. We determined that HDV replication induces the delocalization of PSP1 to cytoplasmic foci containing PABP and increases NEAT1 level causing an enlargement of NEAT1 foci. Overall, our data support a role for the main paraspeckles proteins in HDV life cycle and indicate that HDV replication causes a cellular stress and induces both a delocalization of the PSP1 to the cytoplasm and a disruption of paraspeckles.

Reduced genetic distance and high replication levels increase the RNA recombination rate of hepatitis delta virus

Hepatitis delta virus (HDV) replication is carried out by host RNA polymerases. Since homologous inter-genotypic RNA recombination is known to occur in HDV, possibly via a replication-dependent process, we hypothesized that the degree of sequence homology and the replication level should be related to the recombination frequency in cells co-expressing two HDV sequences. To confirm this, we separately co-transfected cells with three different pairs of HDV genomic RNAs and analyzed the obtained recombinants by RT-PCR followed by restriction fragment length polymorphism and sequencing analyses. The sequence divergence between the clones ranged from 24% to less than 0.1%, and the difference in replication levels was as high as 100-fold. As expected, significant differences were observed in the recombination frequencies, which ranged from 0.5% to 47.5%. Furthermore, varying the relative amounts of parental RNA altered the dominant recombinant species produced, suggesting that template switching occurs frequently during the synthesis of genomic HDV RNA. Taken together, these data suggest that during the host RNA polymerase-driven RNA recombination of HDV, both inter- and intra-genotypic recombination events are important in shaping the genetic diversity of HDV.

Conserved features of an RNA promoter for RNA polymerase II determined from sequence heterogeneity of a hepatitis delta virus population

The right terminal domain of genomic hepatitis delta virus (HDV) RNA is involved in viral replication by recruiting host RNA polymerase II. To identify conserved features of this region, we performed high-throughput 454 sequencing of an HDV population actively replicating in cells. We generated 473,139 sequences representing 2351 new HDV variants of this region and investigated nucleotide conservation and positions of covariation in the population. We found that the sequence is heterogeneous and the rod-like conformation is conserved for both polarities of the HDV RNA genome at this location. Additionally, we identified conserved nucleotides near the previously reported initiation site of transcription, and corroborated our finding with sequences from HDV variants isolated in various hosts. Our analysis highlights the importance of both a conserved sequence at the tip of the rod-like structure and the RNA secondary structure upstream of the tip, which are likely important for HDV replication.

Intrinsic disorder and oligomerization of the hepatitis delta virus antigen

The 195 amino acid basic protein (δAg) of hepatitis delta virus (HDV) is essential for replication of the HDV RNA genome. Numerous properties have been mapped to full-length δAg and attempts made to link these to secondary, tertiary and quaternary structures. Here, for the full-size δAg, extensive intrinsic disorder was predicted using PONDR-FIT, a meta-predictor of intrinsic disorder, and evidenced by circular dichroism measurements. Most δAg amino acids are in disordered configurations with no more than 30% adopting an α-helical structure. In addition, dynamic light scattering studies indicated that purified δAg assembled into structures of as large as dodecamers. Cross-linking followed by denaturing polyacrylamide gel electrophoresis revealed hexamers to octamers for this purified δAg and at least this size for δAg found in virus-like particles. Oligomers of purified δAg were resistant to elevated NaCl and urea concentrations, and bound without specificity to RNA and single- and double-stranded DNAs.

Modification of Small Hepatitis Delta Virus Antigen by SUMO Protein

Hepatitis delta antigen (HDAg) is a nuclear protein that is intimately involved in hepatitis delta virus (HDV) RNA replication. HDAg consists of two protein species, the small form (S-HDAg) and the large form (L-HDAg). Previous studies have shown that posttranslational modifications of S-HDAg, such as phosphorylation, acetylation, and methylation, can modulate HDV RNA replication. In this study, we show that S-HDAg is a small ubiquitin-like modifier 1 (SUMO1) target protein. Mapping data showed that multiple lysine residues are SUMO1 acceptors within S-HDAg. Using a genetic fusion strategy, we found that conjugation of SUMO1 to S-HDAg selectively enhanced HDV genomic RNA and mRNA synthesis but not antigenomic RNA synthesis. This result supports our previous proposition that the cellular machinery involved in the synthesis of HDV antigenomic RNA is different from that for genomic RNA synthesis and mRNA transcription, requiring different modified forms of S-HDAg. Sumoylation represents a new type of modification for HDAg.

The hepatitis delta virus RNA genome interacts with eEF1A1, p54nrb, hnRNP-L, GAPDH and ASF/SF2

Because of its extremely limited coding capacity, the hepatitis delta virus (HDV) takes over cellular machineries for its replication and propagation. Despite the functional importance of host factors in both HDV biology and pathogenicity, little is known about proteins that associate with its RNA genome. Here, we report the identification of several host proteins interacting with an RNA corresponding to the right terminal stem-loop domain of HDV genomic RNA, using mass spectrometry on a UV crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation was used to confirm the interactions of eEF1A1, p54(nrb), hnRNP-L, GAPDH and ASF/SF2 with the right terminal stem-loop domain of HDV genomic RNA in vitro, and with both polarities of HDV RNA within HeLa cells. Our discovery that HDV RNA associates with RNA-processing pathways and translation machinery during its replication provides new insights into HDV biology and its pathogenicity.

The hepatitis delta virus RNA genome interacts with the human RNA polymerases I and III

The hepatitis delta virus (HDV) relies on human transcriptional machinery for its replication and transcription. Although the involvement of RNA polymerase II in HDV RNA biosynthesis is established, the contribution of additional polymerases remains uncertain. Here, we demonstrate the interaction of both RNA polymerase I and III with HDV RNA, both in vitro and in human cells. Binding of these polymerases occurs near the terminal stem-loop domains of both polarities of the HDV RNA genome. Based on interactions of HDV RNA with numerous host polymerases, our results suggest a higher level of complexity of HDV biology than previously envisioned.

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.

The human RNA polymerase II interacts with the terminal stem–loop regions of the hepatitis delta virus RNA genome

The hepatitis delta virus (HDV) is an RNA virus that depends on DNA-dependent RNA polymerase (RNAP) for its transcription and replication. While it is generally accepted that RNAP II is involved in HDV replication, its interaction with HDV RNA requires confirmation. A monoclonal antibody specific to the carboxy terminal domain of the largest subunit of RNAP II was used to establish the association of RNAP II with both polarities of HDV RNA in HeLa cells. Co-immunoprecipitations using HeLa nuclear extract revealed that RNAP II interacts with HDV-derived RNAs at sites located within the terminal stem–loop domains of both polarities of HDV RNA. Analysis of these regions revealed a strong selection to maintain a rod-like conformation and demonstrated several conserved features. These results provide the first direct evidence of an association between human RNAP II and HDV RNA and suggest two transcription start sites on both polarities of HDV RNA.

Binding of the polypyrimidine tract-binding protein-associated splicing factor (PSF) to the hepatitis delta virus RNA

The hepatitis delta virus (HDV) has a very limited protein coding capacity and must rely on host proteins for its replication. A ribonucleoprotein complex was detected following UV cross-linking between HeLa nuclear proteins and an RNA corresponding to the right terminal stem–loop domain of HDV genomic RNA. Mass spectrometric analysis of the complex revealed the polypyrimidine tract-binding protein-associated splicing factor (PSF) as a novel HDV RNA-interacting protein. Co-immunoprecipitation demonstrated the interaction between HDV RNA and PSF both in vitro in HeLa nuclear extract and in vivo within HeLa cells containing both polarities of the HDV genome. Analysis of the binding of various HDV-derived RNAs to purified, recombinant PSF further confirmed the specificity of the interaction and revealed that PSF directly binds to the terminal stem–loop domains of both polarities of HDV RNA. Our findings provide evidence of the involvement of a host mRNA processing protein in the HDV life cycle.

Evolution of Hepatitis Delta Virus RNA Genome following Long-Term Replication in Cell Culture

Previous studies have defined a novel cell culture system in which a modified RNA genome of hepatitis delta virus (HDV) is able to maintain a low level of continuous replication for at least 1 year, using a separate and limited DNA-directed source of mRNA for the essential small delta protein. This mode of replication is analogous to that used by plant viroids. An examination was made of the nucleotide changes that accumulated on the HDV RNA during 1 year of replication. The length of the RNA genome was maintained, except for some single-nucleotide deletions and insertions. There was an abundance of single-nucleotide substitutions, with a 22-fold excess of these being base transitions rather than transversions. Of the detected transitions, at least 70% were consistent with being the consequences of posttranscriptional RNA editing by an adenosine deaminase acting on RNA. The remainder of the changes, including the single-nucleotide insertions and deletions, are likely to be the consequence of misincorporation during transcription. In addition, an intermolecular competition assay was used to show that the majority of the genomes present after 1 year of replication were essentially as competent in replication as the original single HDV RNA sequence that was used to initiate the genome replication. A model is provided to explain how, in this experimental system, the observed single-nucleotide changes were essentially neutral in terms of their effect on the ability of the HDV genome to carry out continued rounds of replication.

Novel Nuclear Export Signal-Interacting Protein, NESI, Critical for the Assembly of Hepatitis Delta Virus

The process of host factor-mediated nucleocytoplasmic transport is critical for diverse cellular events in eukaryotes and the life cycle of viruses. We have previously identified a chromosome region maintenance 1-independent nuclear export signal (NES) at the C terminus of the large form of hepatitis delta antigen (HDAg), designated NES(HDAg-L) that is required for the assembly of hepatitis delta virus (HDV) (C.-H. Lee et al., J. Biol. Chem. 276:8142-8148, 2001). To look for interacting proteins of the NES(HDAg-L), yeast two-hybrid screening was applied using the GAL4-binding domain fused to the NES(HDAg-L) as bait. Among the positive clones, one encodes a protein, designated NESI [NES(HDAg-L) interacting protein] that specifically interacted with the wild-type NES(HDAg-L) but not with the export/package-defective HDAg-L mutant, NES*(HDAg-L), in which Pro-205 has been replaced by Ala. Northern blot analysis revealed NESI as the gene product of a 1.9-kb endogenous mRNA transcript that is present predominantly in human liver tissue. NESI consists of 467 amino acid residues and bears a putative actin-binding site and a bipartite nuclear localization signal. Specific interaction between HDAg-L and NESI was further confirmed by coimmunoprecipitation and immunofluorescence staining. Overexpression of antisense NESI RNAs inhibited the expression of NESI and abolished HDAg-L-mediated nuclear export and assembly of HDV genomic RNA. These data indicate a critical role of NESI in the assembly of HDV through interaction with HDAg-L.

Trans-acting hepatitis delta virus ribozyme: catalytic core and global structure are dependent on the 5' substrate sequence.

The hepatitis delta virus (HDV), an infectious human pathogen affecting millions of people worldwide, leads to intensified disease symptoms, including progression to liver cirrhosis upon coinfection with its helper virus, HBV. Both the circular RNA genome of HDV and its complementary antigenome contain a common cis-cleaving catalytic RNA motif, the HDV ribozyme, which plays a crucial role in viral replication. Previously, the crystal structure of the product form of the cis-acting genomic HDV ribozyme has been determined, and the precursor form has been suggested to be structurally similar. In contrast, solution studies by fluorescence resonance energy transfer (FRET) on a trans-cleaving form of the ribozyme have shown significant global conformational changes upon catalysis, while 2-aminopurine (AP) fluorescence assays have detected concomitant local conformational changes in the catalytic core. Here, we augment these studies by using terbium(III) to probe the structure of the trans-acting HDV ribozyme at nucleotide resolution. We observe significant structural differences between the precursor and product forms, especially in the P1.1 helix and the trefoil turn in the single-stranded region connecting P4 and P2 (termed J4/2) of the catalytic core. We show, using terbium(III) footprinting and sensitized luminescence spectroscopy as well as steady-state, time-resolved, and gel-mobility FRET assays on a systematic set of substrates, that the substrate sequence immediately 5' to the cleavage site significantly modulates these local as well as resultant global structural differences. Our results suggest a structural basis for the previously observed impact of the 5' substrate sequence on catalytic activity.

A mechanistic framework for co-transcriptional folding of the HDV genomic ribozyme in the presence of downstream sequence.

Hepatitis delta virus (HDV) is a circular pathogenic RNA that uses self-cleavage by closely related 84 nt genomic and antigenomic ribozymes to facilitate the replication of its genome. Downstream of each ribozyme is a stretch of nucleotides termed the attenuator that functions to base-pair with and unfold the ribozyme into a rod-like fold. The competing rates of RNA synthesis, ribozyme folding and cleavage, and rod folding are therefore likely to affect the efficiency of co-transcriptional self-cleavage. In these studies, co-transcriptional folding of the genomic ribozyme was assayed in vitro by monitoring co-transcriptional self-cleavage of transcripts having variable lengths of sequence downstream of the ribozyme. Co-transcriptional cleavage data were simulated successfully only with kinetic models in which cleavage-inactive channels were populated during transcription. Partitioning to and escape from these channels was influenced, in part, by whether the available attenuator sequence could form structures with the ribozyme, and by the stability of such structures. Surprisingly, only 23 nt of attenuator were needed for strong inactivation of cleavage. Self-cleavage of certain 3'-virus-containing sequences could be restored, partially, by renaturation; however, self-cleavage of transcripts with a full-length attenuator could not be restored efficiently by renaturation in vitro. This suggests that in the presence of the attenuator, the cleavage-active ribozyme fold is not the thermodynamically most stable species. In accordance with this model, the efficiency of self-cleavage of the ribozyme followed by a full-length attenuator was increased by decreasing the rate of transcription. These results suggest that, in the absence of additional factors, efficient co-transcriptional cleavage of the full-length genomic HDV RNA may require cleavage to occur prior to synthesis of the attenuator.