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A number of research studies, including ours, have spotlighted exosomes as critical facilitators of viral dissemination. While hepatitis B virus (HBV) transmission through exosomes has been studied, the focus on its satellite virus, the hepatitis delta virus (HDV), has been unexplored in this context. HDV, although being a defective virus, can replicate its genome autonomously within hepatocytes, independently of HBV. Investigations on Huh7 cells revealed an intriguing phenomenon: the HDV proteins, S-HDAg and L-HDAg, are transmitted between cells without a complete viral structure. Detailed analysis further revealed that the expression of these proteins not only bolstered exosome secretion but also ensured their enrichment within these vesicles. Our experimental approach utilized transfection of various plasmids to examine the role of HDV RNA and proteins in the process. One salient finding was the differential propagation of the HDV proteins S-HDAg and L-HDAg, suggesting intricate molecular mechanisms behind their transmission. Notably, the purity of our exosome preparations was monitored using markers such as TSG101 and CD81. Importantly, these exosomes were found to carry both HDV RNA and proteins, highlighting their role in HDV dissemination. This novel study underscores the role of exosomes in mediating the transmission of HDV components between hepatocytes independent of HBV. These revelations about the exosomal pathway of HDV transmission provide a foundation for the development of innovative therapeutic strategies against HDV infections.

Hepatitis D virus (HDV) infection represents the most severe form of chronic viral hepatitis. We have shown that the delivery of HDV replication-competent genomes to the hepatocytes using adeno-associated virus (AAV-HDV) as gene delivery vehicles offers a unique platform to investigate the molecular aspects of HDV and associated liver damage. For the purpose of this study, we generated HDV genomes modified by site-directed mutagenesis aimed to (i) prevent some post-translational modifications of HDV antigens (HDAgs) such as large-HDAg (L-HDAg) isoprenylation or short-HDAg (S-HDAg) phosphorylation; (ii) alter the localization of HDAgs within the subcellular compartments; and (iii) inhibit the right conformation of the delta ribozyme. First, the different HDV mutants were tested in vitro using plasmid-transfected Huh-7 cells and then in vivo in C57BL/6 mice using AAV vectors. We found that Ser177 phosphorylation and ribozymal activity are essential for HDV replication and HDAg expression. Mutations of the isoprenylation domain prevented the formation of infectious particles and increased cellular toxicity and liver damage. Furthermore, altering HDAg intracellular localization notably decreased viral replication, though liver damage remained unchanged versus normal HDAg distribution. In addition, a mutation in the nuclear export signal impaired the formation of infectious viral particles. These findings contribute valuable insights into the intricate mechanisms of HDV biology and have implications for therapeutic considerations.

RNA editing of the hepatitis delta virus (HDV) is essential for generating the large delta antigen, which is crucial for virion assembly. In HDV genotype 1 (HDV-1), editing occurs within the context of the unbranched rod-like structure characteristic of HDV RNA, while RNA editing in HDV-3 requires a branched double-hairpin structure. The regulation of RNA editing in HDV-2 and HDV-4 remains uncertain. Based on predictions of the unbranched rod-like RNA structures of HDV-2 and HDV-4, the editing site occurs as an A.C mismatch pair, surrounded by four base pairs upstream and two base pairs downstream of the editing site, respectively. To investigate HDV-2 and HDV-4 RNA editing, cultured cells were transfected with non-replicating editing reporters carrying wild-type sequences or specific mutations. The results revealed that the editing rates observed for wild-type HDV-2 and HDV-4 were fairly similar, albeit lower than that of HDV-1. Like HDV-1, both HDV-2 and HDV-4 showed a reduction in editing rate when the A.C mismatch pair and the immediately upstream base-paired region were disturbed. Notably, extending the downstream base-paired region from two to three or four (forming a structure identical to that of HDV-1) base pairs increased editing rate. Furthermore, we presented novel evidence that indicates the importance of the first bulge's size, located upstream of the editing site, and the base-pairing length within 7-13 and 28-39 nucleotides downstream of the editing site in influencing the HDV-4 editing rate. To summarize, our analyses suggest that the unbranched rod-like structures surrounding the editing site of HDV-2 and HDV-4 play a crucial role in regulating their RNA editing rates., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background & Aims: Chronic coinfection with HBV and HDV leads to the most aggressive form of chronic viral hepatitis. Herein, we aimed to elucidate the molecular mechanisms underlying the widely reported observation that HDV interferes with HBV in most coinfected patients., Methods: Patient liver tissues, primary human hepatocytes, HepaRG cells and human liver chimeric mice were used to analyze the effect of HDV on HBV using virological and RNA-sequencing analyses, as well as RNA synthesis, stability and association assays., Results: Transcriptomic analyses in cell culture and mouse models of coinfection enabled us to define an HDV-induced signature, mainly composed of interferon (IFN)-stimulated genes (ISGs). We also provide evidence that ISGs are upregulated in chronically HDV/HBV-coinfected patients but not in cells that only express HDV antigen (HDAg). Inhibition of the hepatocyte IFN response partially rescued the levels of HBV parameters. We observed less HBV RNA synthesis upon HDV infection or HDV protein expression. Additionally, HDV infection or expression of HDAg alone specifically accelerated the decay of HBV RNA, and HDAg was associated with HBV RNAs. On the contrary, HDAg expression did not affect other viruses such as HCV or SARS-CoV-2., Conclusions: Our data indicate that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms. Specifically, we uncover a new viral interference mechanism in which proteins of a satellite virus affect the RNA production of its helper virus. Exploiting these findings could pave the way to the development of new therapeutic strategies against HBV., Impact and Implications: Although the molecular mechanisms remained unexplored, it has long been known that despite its dependency, HDV decreases HBV viremia in patients. Herein, using in vitro and in vivo models, we showed that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms affecting HBV RNA metabolism, and we defined the HDV-induced modulation signature. The mechanisms we uncovered could pave the way for the development of new therapeutic strategies against HBV by mimicking and/or increasing the effect of HDAg on HBV RNA. Additionally, the HDV-induced modulation signature could potentially be correlated with responsiveness to IFN-α treatment, thereby helping to guide management of HBV/HDV-coinfected patients., (Copyright © 2023 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Hepatitis delta virus (HDV), a satellite virus of hepatitis B virus (HBV), is a small, defective RNA virus strongly associated with the most severe form of hepatitis and progressive chronic liver disease and cirrhosis. Chronic hepatitis D, resulting from HBV/HDV coinfection, is considered to be the most severe form of viral hepatitis and affects 12-20 million people worldwide. Involved in the endocytosis and exocytosis of cellular and viral proteins, clathrin contributes to the pathogenesis and morphogenesis of HDV. Previously, we demonstrated that HDV-I and -II large hepatitis delta antigens (HDAg-L) possess a putative clathrin box that interacts with clathrin heavy chain (CHC) and supports HDV assembly., Methods: Virus assembly and vesicular trafficking of HDV virus-like particles (VLPs) were evaluated in Huh7 cells expressing HDV-I, -II and -III HDAg-L and hepatitis B surface antigen (HBsAg). To elucidate the interaction motif between HDAg-L and CHC, site-directed mutagenesis was performed to introduce mutations into HDAg-L and CHC and analyzed using coimmunoprecipitation or pull-down assays., Results: Comparable to HDV-I virus-like particles (VLPs), HDV-III VLPs were produced at a similar level and secreted into the medium via clathrin-mediated post-Golgi vesicular trafficking. Mutation at F27 or E33 of CHC abolished the binding of CHC to the C-terminus of HDV-III HDAg-L. Mutation at W207 of HDV-III HDAg-L inhibited its association with CHC and interfered with HDV-III VLP formation. We elucidated mechanism of the binding of HDV-III HDAg-L to CHC and confirmed the pivotal role of clathrin binding in the assembly of genotype III HDV., Conclusions: A novel W box which was identified at the C terminus of HDV-III HDAg-L is known to differ from the conventional clathrin box but also interacts with CHC. The novel W box of HDAg-L constitutes a new molecular target for anti-HDV-III therapeutics., (© 2022. The Author(s).)

Hepatitis delta virus (HDV) is a defective satellite virus that uses hepatitis B virus (HBV) envelope proteins to form its virions and infect hepatocytes via the HBV receptors. Concomitant HDV/HBV infection continues to be a major health problem, with at least 25 million people chronically infected worldwide. N 6 -methyladenine (m6A) modification of cellular and viral RNAs is the most prevalent internal modification that occurs cotranscriptionally, and this modification regulates various biological processes. We have previously described a wider range of functional roles of m6A methylation of HBV RNAs, including its imminent regulatory role in the encapsidation of pregenomic RNA. In this study, we present evidence that m6A methylation also plays an important role in the HDV life cycle. Using the methylated RNA immunoprecipitation (MeRIP) assay, we identified that the intracellular HDV genome and antigenome are m6A methylated in HDV- and HBV-coinfected primary human hepatocytes and HepG2 cell expressing sodium taurocholate cotransporting polypeptide (NTCP), while the extracellular HDV genome is not m6A methylated. We observed that HDV genome and delta antigen levels are significantly decreased in the absence of METTL3/14, while the extracellular HDV genome levels are increased by depletion of METTL3/14. Importantly, YTHDF1, an m6A reader protein, interacts with the m6A-methylated HDV genome and inhibits the interaction between the HDV genome and antigens. Thus, m6A of the HDV genome negatively regulates virion production by inhibiting the interaction of the HDV genome with delta antigens through the recruitment of YTHDF1. This is the first study that provides insight into the functional roles of m6A in the HDV life cycle. IMPORTANCE The functional roles of N 6 -methyladenine (m6A) modifications in the HBV life cycle have been recently highlighted. Here, we investigated the functional role of m6A modification in the HDV life cycle. HDV is a subviral agent of HBV, as it uses HBV envelope proteins to form its virions. We found that m6A methylation also occurs in the intracellular HDV genome and antigenome but not in the extracellular HDV genome. The m6A modification of the HDV genome recruits m6A reader protein (YTHDF1) onto the viral genome. The association of YTHDF1 with the HDV genome abrogates the interaction of delta antigens with the HDV genome and inhibits virion assembly. This study describes the unique effects of m6A on regulation of the HDV life cycle.

Hepatitis D virus (HDV) is a defective virus that relies on hepatitis B virus envelope proteins to complete its replication cycle. The HDV genome contains two isoforms of hepatitis delta antigen: the small and the large hepatitis delta antigens (S- and L-HDAg). Here we report the 1 H, 13 C and 15  N backbone and side chain resonance assignments of an N-terminally truncated form of S-HDAg (S Δ60 ), which lacks the 1-60 oligomerization domain. We derived secondary structures based on NMR chemical shifts, which will be used in further structural and functional studies. We show that S Δ60 is partially disordered, and that the central structured part contains two well-defined α-helices of 22 and 17 residues, respectively. A temperature titration allowed to identify the residues involved in hydrogen bonds., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Chronic hepatitis D is the most severe form of chronic viral hepatitis and to date, efficient therapeutic approaches against hepatitis D virus (HDV) are limited. Among the antiviral molecules currently tested in clinical trials, the farnesyl transferase inhibitor (FTI) Lonafarnib inhibits the prenylation of the large delta antigen (L-HDAg), blocking virus assembly. Given the importance of L-HDAg in the virus life cycle, we hypothesized that Lonafarnib treatment may have side effects on virus replication. Here, we setup an innovative method for the quantification of HDV RNA allowing the independent quantification of edited and non-edited versions of the HDV genome upon infection. We demonstrated that FTI treatment of HBV/HDV co-infected dHepaRG or primary human hepatocytes leads to an accumulation of intracellular HDV RNAs and a marked increase in the levels of edited RNAs non only within the infected cells but also in the viral particles that are produced. Interestingly, these viral particles were less infectious, probably due to an enrichment in edited genomes that are packaged, leading to unproductive infection given the absence of S-HDAg synthesis after viral entry. Taken together, we setup an innovative quantification method allowing the investigation of RNA editing during HDV infection in a simple, fast, clinically-relevant assay and demonstrated for the first time the dual antiviral activity of FTI on HDV infection., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Hepatitis D is the most severe form of human viral hepatitis and currently lacks an efficient therapy. Dendritic cell-derived exosomes (Dexs) have been found to induce immune responses capable of eliminating viruses. However, the therapeutic potential of antigen-loaded exosomes in hepatitis D is still unknown. Recently, we designed exosomes loaded with ubiquitinated hepatitis delta virus (HDV) small delta antigen (Ub-S-HDAg) and then treated mice bearing replicating HDV with these exosomes to explore their antiviral effect and mechanism. Mature dendritic cell-derived exosomes (mDexs) were loaded with Ub-S-HDAg and their antivirus function was evaluated in mice with HDV viremia. Furthermore, the proportion of CD8 + cells, the ratio of Th1/Th2 cells, the postimmunization levels of cytokines were explored, and the Janus kinases (JAK)/signal transducer and activator of transcription (STAT) pathway was evaluated with a JAK2 inhibitor AG490. In Ub-S-HDAg-Dexs group, the HDV RNA viral load was significantly decreased compared with other groups by CD8 + cell enrichment and an increase Th1/Th2 cell ratio. Furthermore, lymphocyte infiltration was increased, while the HDAg level was decreased in mouse liver tissue. However, there were no significant differences in HBV surface antigen (HBsAg), alanine aminotransferase (ALT), or aspartate aminotransferase (AST) levels among the groups. Moreover, p-JAK2, p-STAT1, p-STAT4, STAT1, and STAT4 expression was increased in Ub-S-HDAg-Dexs group. In conclusion, Ub-S-HDAg-Dexs might be a potential immunotherapeutic agent for eradicating HDV by inducing specific cellular immune response via the JAK/STAT pathway. IMPORTANCE Hepatitis D is the most severe viral hepatitis with accelerating the process of liver cirrhosis and increasing the risk of hepatocellular carcinoma. However, there are no effective antiviral drugs. Exosomes derived from mature dendritic cells are used not only as immunomodulators, but also as biological carriers to deliver antigens to induce robust immune response. Based on these properties, exosomes could be used as a biological immunotherapy by enhancing adaptive immune response to inhibit hepatitis D virus replication. Our research may provide a new therapeutic strategy to eradicate HDV in the future.

Hepatitis D virus (HDV) is a global health threat with more than 15 million humans affected. Current treatment options are largely unsatisfactory leaving chronically infected humans at high risk to develop liver cirrhosis and hepatocellular carcinoma. HDV is the only human satellite virus known. It encodes only two proteins, and requires Hepatitis B virus (HBV) envelope protein expression for productive virion release and spread of the infection. How HDV could evolve and why HBV was selected as a helper virus remains unknown. Since the discovery of Na + -taurocholate co-transporting polypeptide as the essential uptake receptor for HBV and HDV, we are beginning to understand the interactions of HDV and the immune system. While HBV is mostly regarded a stealth virus, that escapes innate immune recognition, HBV-HDV coinfection is characterized by a strong innate immune response. Cytoplasmic RNA sensor melanoma differentiation antigen 5 has been reported to recognize HDV RNA replication and activate innate immunity. Innate immunity, however, seems not to impair HDV replication while it inhibits HBV. In this review, we describe what is known up-to-date about the interplay between HBV as a helper and HDV's immune evasion strategy and identify where additional research is required., Competing Interests: Conflict-of-interest statement: The authors declare no conflict of interests concerning the content of this article., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

Background: Hepatitis D virus (HDV) infection may induce fulminant hepatitis in chronic hepatitis B patients (CHB) or rapid progression of CHB to cirrhosis or hepatocellular carcinoma. There is no effective treatment for HDV infection. HDV encodes small delta antigens (S-HDAg) and large delta antigens (L-HDAg). S-HDAg is essential for HDV replication. Prenylated L-HDAg plays a key role in HDV assembly. Previous studies indicate that L-HDAg transactivates transforming growth factor beta (TGF-β) and induces epithelial-mesenchymal transition (EMT), possibly leading to liver fibrosis. However, the mechanism is unclear., Methods: The mechanisms of the activation of Twist promoter by L-HDAg were investigated by luciferase reporter assay, chromatin immunoprecipitation, and co-immunoprecipitation analysis. ELISA and Western blotting were used to analyze L-HDAg prenylation, TGF-β secretion, expression of EMT markers, and to evaluate efficacy of statins for HDV treatment., Results: We found that L-HDAg activated Twist expression, TGF-β expression and consequently induced EMT, based on its interaction with Smad3 on Twist promoter. The treatment of statin, a prenylation inhibitor, resulted in reduction of Twist promoter activity, TGF-β expression, and EMT, and reduces the release of HDV virions into the culture medium., Conclusions: We demonstrate that L-HDAg activates EMT via Twist and TGF-β activation. Treatment with statins suppressed Twist expression, and TGF-β secretion, leading to downregulation of EMT. Our findings clarify the mechanism of HDV-induced EMT, and provide a basis for possible novel therapeutic strategies against HDV infection.

A substantial number of viruses have been demonstrated to subvert autophagy to promote their own replication. Recent publications have reported the proviral effect of autophagy induction on hepatitis B virus (HBV) replication. Hepatitis delta virus (HDV) is a defective virus and an occasional obligate satellite of HBV. However, no previous work has studied the relationship between autophagy and HDV. In this article, we analyze the impact of HBV and HDV replication on autophagy as well as the involvement of the autophagy machinery in the HDV life cycle when produced alone and in combination with HBV. We prove that HBxAg and HBsAg can induce early steps of autophagy but ultimately block flux. It is worth noting that the two isoforms of the HDV protein, the small HDAg (S-HDAg) and large HDAg (L-HDAg) isoforms, can also efficiently promote autophagosome accumulation and disturb autophagic flux. Using CRISPR-Cas9 technology to generate specific knockouts, we demonstrate that the autophagy machinery, specifically the proteins implicated in the elongation step (ATG7, ATG5, and LC3), is important for the release of HBV without affecting the level of intracellular HBV genomes. Surprisingly, the knockout of ATG5 and ATG7 decreased the intracellular HDV RNA level in both Huh7 and HepG2.2.15 cells without an additional effect on HDV secretion. Therefore, we conclude that HBV and HDV have evolved to utilize the autophagy machinery so as to assist at different steps of their life cycle. IMPORTANCE Hepatitis delta virus is a defective RNA virus that requires hepatitis B virus envelope proteins (HBsAg) to fulfill its life cycle. Thus, HDV can only infect individuals at the same time as HBV (coinfection) or superinfect individuals who are already chronic carriers of HBV. The presence of HDV in the liver accelerates the progression of infection to fibrosis and to hepatic cancer. Since current treatments against HBV are ineffective against HDV, it is of paramount importance to study the interaction between HBV, HDV, and host factors. This will help unravel new targets whereby a therapy that is capable of simultaneously impeding both viruses could be developed. In this research paper, we evidence that the autophagy machinery promotes the replication of HBV and HDV at different steps of their life cycle. Notwithstanding their contribution to HBV release, autophagy proteins seem to assist HDV intracellular replication but not its secretion., (Copyright © 2020 American Society for Microbiology.)

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.

Hepatitis delta virus (HDV) RNA forms an unbranched rod-like structure and complexes with the delta antigen (HDAg). Host ADAR1-catalyzed RNA editing at the amber/W site of the small HDAg leads to the production of the large HDAg, which inhibits replication and is required for virion assembly. For HDV genotype 1, amber/W editing is controlled by HDAg and the RNA structure immediate vicinity and downstream of the editing site. Here, the effects of 20 mutants carrying an increased length of consecutive base-pairing at various sites in HDV RNA on amber/W site editing were examined. All nine mutants carrying genomic regions that formed up to 15 consecutive base pairs, which is also the maximum length observed in 41 naturally occurring HDV genomes, showed normal editing rate. However, mutants carrying a 16 or 17 consecutive base-paired antigenomic segment located as far as 114 nt upstream could increase editing efficiency, possibly by interfering with HDAg binding. These data show for the first time that extended base-pairing upstream of the amber/W site could increase HDV RNA editing efficiency. Furthermore, it appears that the naturally occurring HDV RNA structures have been selected for suboptimal amber/W RNA editing, which favors the HDV replication cycle., Competing Interests: The authors declare no conflict of interest.

Hepatitis delta virus (HDV) depends on the helper function of hepatitis B virus (HBV), which provides the envelope proteins for progeny virus secretion. Current infection-competent cell culture models do not support assembly and secretion of HDV. By stably transducing HepG2 cells with genes encoding the NTCP-receptor and the HBV envelope proteins we produce a cell line (HepNB2.7) that allows continuous secretion of infectious progeny HDV following primary infection. Evaluation of antiviral drugs shows that the entry inhibitor Myrcludex B (IC 50 : 1.4 nM) and interferon-α (IC 50 : 28 IU/ml, but max. 60-80% inhibition) interfere with primary infection. Lonafarnib inhibits virus secretion (IC 50 : 36 nM) but leads to a substantial intracellular accumulation of large hepatitis delta antigen and replicative intermediates, accompanied by the induction of innate immune responses. This work provides a cell line that supports the complete HDV replication cycle and presents a convenient tool for antiviral drug evaluation.

Hepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of acute and chronic liver disease. HDV produces three processed RNAs that accumulate in infected cells: the circular genome; the circular antigenome, which serves as a replication intermediate; and lesser amounts of the mRNA, which encodes the sole viral protein, hepatitis delta antigen (HDAg). The HDV genome and antigenome RNAs form ribonucleoprotein complexes with HDAg. Although HDAg is required for HDV replication, it is not known how the relative amounts of HDAg and HDV RNA affect replication, or whether HDAg synthesis is regulated by the virus. Using a novel transfection system in which HDV replication is initiated using in vitro -synthesized circular HDV RNAs, HDV replication was found to depend strongly on the relative amounts of HDV RNA and HDAg. HDV controls these relative amounts via differential effects of HDAg on the production of HDV mRNA and antigenome RNA, both of which are synthesized from the genome RNA template. mRNA synthesis is favored at low HDAg levels but becomes saturated at high HDAg concentrations. Antigenome RNA accumulation increases linearly with HDAg and dominates at high HDAg levels. These results provide a conceptual model for how HDV antigenome RNA production and mRNA transcription are controlled from the earliest stage of infection onward and also demonstrate that, in this control, HDV behaves similarly to other negative-strand RNA viruses, even though there is no genetic similarity between them. IMPORTANCE Hepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of liver disease; approximately 15 million people are chronically infected worldwide. There are no licensed therapies available. HDV is not related to any known virus, and few details regarding its replication cycle are known. One key question is whether and how HDV regulates the relative amounts of viral RNA and protein in infected cells. Such regulation might be important because the HDV RNA and protein form complexes that are essential for HDV replication, and the proper stoichiometry of these complexes could be critical for their function. Our results show that the relative amounts of HDV RNA and protein in cells are indeed important for HDV replication and that the virus does control them. These observations indicate that further study of these regulatory mechanisms is required to better understand replication of this serious human pathogen., (Copyright © 2019 American Society for Microbiology.)

Liver coinfection by hepatitis B virus (HBV) and hepatitis D virus (HDV) can result in a severe form of hepatocellular carcinoma with poor prognosis. Coinfection with HDV and HBV causes more deleterious effects than infection with HBV alone. Clinical research has shown that glutathione S-transferase P1 (GSTP1), a tumor suppressor gene, is typically downregulated in liver samples from hepatitis-infected patients. In the present study, our data indicated that small HDV antigen (s-HDAg) could specifically bind to GSTP1 mRNA and significantly downregulate GSTP1 protein expression. For the human fetal hepatocyte cell line L-02, cells transfected with s-HDAg, along with decreased GSTP1 expression, there was a significant accumulation of reactive oxygen species (ROS) and increased apoptotic ratios. Restoring GSTP1 expression through silencing s-HDAg via RNAi or overexpressing exogenous GSTP1 could largely recover the abnormal cell status. Our results revealed a novel potential mechanism of HDV-induced liver injury and hepatocarcinogenesis: s-HDAg can inhibit GSTP1 expression by directly binding to GSTP1 mRNA, which leads to accumulation of cellular ROS, resulting in high cellular apoptotic ratios and increased selective pressure for malignant transformation. To our knowledge, this is the first study to examine s-HDAg-specific pathogenic mechanisms through potential protein-RNA interactions.

Virus-specific CD8 T cell response seems to play a significant role in the outcome of hepatitis delta virus (HDV) infection. However, the HDV-specific T cell epitope repertoire and mechanisms of CD8 T cell failure in HDV infection have been poorly characterized. We therefore aimed to characterize HDV-specific CD8 T cell epitopes and the impacts of viral mutations on immune escape. In this study, we predicted peptide epitopes binding the most frequent human leukocyte antigen (HLA) types and assessed their HLA binding capacities. These epitopes were characterized in HDV-infected patients by intracellular gamma interferon (IFN-γ) staining. Sequence analysis of large hepatitis delta antigen (L-HDAg) and HLA typing were performed in 104 patients. The impacts of substitutions within epitopes on the CD8 T cell response were evaluated experimentally and by in silico studies. We identified two HLA-B*27-restricted CD8 T cell epitopes within L-HDAg. These novel epitopes are located in a relatively conserved region of L-HDAg. However, we detected molecular footprints within the epitopes in HLA-B*27-positive patients with chronic HDV infections. The variant peptides were not cross-recognized in HLA-B*27-positive patients with resolved HDV infections, indicating that the substitutions represent viral escape mutations. Molecular modeling of HLA-B*27 complexes with the L-HDAg epitope and its potential viral escape mutations indicated that the structural and electrostatic properties of the bound peptides differ considerably at the T cell receptor interface, which provides a possible molecular explanation for the escape mechanism. This viral escape from the HLA-B*27-restricted CD8 T cell response correlates with a chronic outcome of hepatitis D infection. T cell failure resulting from immune escape may contribute to the high chronicity rate in HDV infection. IMPORTANCE Hepatitis delta virus (HDV) causes severe chronic hepatitis, which affects 20 million people worldwide. Only a small number of patients are able to clear the virus, possibly mediated by a virus-specific T cell response. Here, we performed a systematic screen to define CD8 epitopes and investigated the role of CD8 T cells in the outcome of hepatitis delta and how they fail to eliminate HDV. Overall the number of epitopes identified was very low compared to other hepatotropic viruses. We identified, two HLA-B*27-restricted epitopes in patients with resolved infections. In HLA-B*27-positive patients with chronic HDV infections, however, we detected escape mutations within these identified epitopes that could lead to viral evasion of immune responses. These findings support evidence showing that HLA-B*27 is important for virus-specific CD8 T cell responses, similar to other viral infections. These results have implications for the clinical prognosis of HDV infection and for vaccine development., (Copyright © 2018 American Society for Microbiology.)

Hepatitis D virus (HDV) contains a single-stranded circular RNA genome that encodes two forms of hepatitis delta antigen (HDAg), the small delta antigen (HDAg-S) and the large delta antigen (HDAg-L). The two proteins have an identical amino acid sequence, except that HDAg-L has a 19-amino-acid extension at the C terminus. The domain spanning amino acid residues 198-210 of the HDAg-L (HDAg-L(198-210)) contains a nuclear export signal (NES), which is important for the nuclear export of HDV ribonucleoprotein to the cytoplasm. In this study, we established a cell permeable TAT-HA-HDAg-L(198-210) fusion protein using an E. coli protein expression system, to determine its function during HDV infection. The cytotoxicity of the TAT-HA-HDAg-L(198-210) fusion protein was investigated using an MTT assay, while a GST pull-down assay revealed that the TAT-HA-HDAg-L(198-210) fusion protein interfered with the interaction between HDAg-L and clathrin heavy chain (CHC). In addition, the cellular distribution of HDAg-L, in the presence of HBsAg, was observed by immunofluorescence staining and the TAT-HA-HDAg-L(198-210) fusion protein was found to impede the nuclear export of HDAg-L. Furthermore, assembly of HDV virus-like particles (VLPs) was decreased by the expression of the TAT-HDAg-L(198-210) fusion protein. The TAT-HA-HDAg-L(198-210) fusion protein also inhibited virus particle assembly and HDV secretion in a mouse model. These results suggest that the TAT-HA-HDAg-L(198-210) fusion protein inhibits the nuclear export of HDAg-L and competes with the C terminus of HDAg-L for interaction with CHC, and may have potential as a therapeutic agent for HDV infection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background & Aims: Studying hepatitis delta virus (HDV) and developing new treatments is hampered by the limited availability of small animal models. Herein, a description of a robust mouse model of HDV infection that mimics several important characteristics of the human disease is presented., Methods: HDV and hepatitis B virus (HBV) replication competent genomes were delivered to the mouse liver using adeno-associated viruses (AAV; AAV-HDV and AAV-HBV). Viral load, antigen expression and genomes were quantified at different time points after AAV injection. Furthermore, liver pathology, genome editing, and the activation of the innate immune response were evaluated., Results: AAV-HDV infection initiated HDV replication in mouse hepatocytes. Genome editing was confirmed by the presence of small and large HDV antigens and sequencing. Viral replication was detected for 45days, even after the AAV-HDV vector had almost disappeared. In the presence of HBV, HDV infectious particles were detected in serum. Furthermore, as observed in patients, co-infection was associated with the reduction of HBV antigen expression and the onset of liver damage that included the alteration of genes involved in the development of liver pathologies. HDV replication induced a sustained type I interferon response, which was significantly reduced in immunodeficient mice and almost absent in mitochondrial antiviral signaling protein (MAVS)-deficient mice., Conclusion: The animal model described here reproduces important characteristics of human HDV infection and provides a valuable tool for characterizing the viral infection and for developing new treatments. Furthermore, MAVS was identified as a main player in HDV detection and adaptive immunity was found to be involved in the amplification of the innate immune response. Lay summary: Co-infection with hepatitis B and D virus (HBV and HDV, respectively) often causes a more severe disease condition than HBV alone. Gaining more insight into HDV and developing new treatments is hampered by limited availability of adequate immune competent small animal models and new ones are needed. Here, a mouse model of HDV infection is described, which mimics several important characteristics of the human disease, such as the initiation and maintenance of replication in murine hepatocytes, genome editing and, in the presence of HBV, generation of infectious particles. Lastly, the involvement of an adaptive immunity and the intracellular signaling molecule MAVS in mounting a strong and lasting innate response was shown. Thus, our model serves as a useful tool for the investigation of HDV biology and new treatments., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Short peptide motifs in unstructured regions of clathrin-adaptor proteins recruit clathrin to membranes to facilitate post-Golgi membrane transport. Three consensus clathrin-binding peptide sequences have been identified and structural studies show that each binds distinct sites on the clathrin heavy chain N-terminal domain (NTD). A fourth binding site for adaptors on NTD has been functionally identified but not structurally characterised. We have solved high resolution structures of NTD bound to peptide motifs from the cellular clathrin adaptors β2 adaptin and amphiphysin plus a putative viral clathrin adaptor, hepatitis D virus large antigen (HDAg-L). Surprisingly, with each peptide we observe simultaneous peptide binding at multiple sites on NTD and viral peptides binding to the same sites as cellular peptides. Peptides containing clathrin-box motifs (CBMs) with the consensus sequence LΦxΦ[DE] bind at the 'arrestin box' on NTD, between β-propeller blades 4 and 5, which had previously been thought to bind a distinct consensus sequence. Further, we structurally define the fourth peptide binding site on NTD, which we term the Royle box. In vitro binding assays show that clathrin is more readily captured by cellular CBMs than by HDAg-L, and site-directed mutagenesis confirms that multiple binding sites on NTD contribute to efficient capture by CBM peptides., (© 2016 The Authors. Traffic Published by John Wiley & Sons Ltd.)

Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus (HBV). HDV genome encodes two forms of hepatitis delta antigen (HDAg), small HDAg (HDAg-S), which is required for viral replication, and large HDAg (HDAg-L), which is essential for viral assembly. HDAg-L is identical to HDAg-S except that it bears a 19-amino acid extension at the C terminus. Both HDAgs contain a nuclear localization signal (NLS), but only HDAg-L contains a CRM1-independent nuclear export signal at its C terminus. The nuclear export activity of HDAg-L is important for HDV particle formation. However, the mechanisms of HDAg-L-mediated nuclear export of HDV ribonucleoprotein are not clear. In this study, the host cellular RNA export complex TAP-Aly was found to form a complex with HDAg-L, but not with an export-defective HDAg-L mutant, in which Pro 205 was replaced by Ala. HDAg-L was found to colocalize with TAP and Aly in the nucleus. The C-terminal domain of HDAg-L was shown to directly interact with the N terminus of TAP, whereas an HDAg-L mutant lacking the NLS failed to interact with full-length TAP. In addition, small hairpin RNA-mediated down-regulation of TAP or Aly reduced nuclear export of HDAg-L and assembly of HDV virions. Furthermore, a peptide, TAT-HDAg-L(198-210), containing the 10-amino acid TAT peptide and HDAg-L(198-210), inhibited the interaction between HDAg-L and TAP and blocked HDV virion assembly and secretion. These data demonstrate that formation and release of HDV particles are mediated by TAP and Aly., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Viral hepatitis remains a worldwide public health problem. The hepatitis D virus (HDV) must either coinfect or superinfect with the hepatitis B virus (HBV). HDV contains a small RNA genome (approximately 1.7 kb) with a single open reading frame (ORF) and requires HBV supplying surface antigens (HBsAgs) to assemble a new HDV virion. During HDV replication, two isoforms of a delta antigen, a small delta antigen (SDAg) and a large delta antigen (LDAg), are produced from the same ORF of the HDV genome. The SDAg is required for HDV replication, whereas the interaction of LDAg with HBsAgs is crucial for packaging of HDV RNA. Various clinical outcomes of HBV/HDV dual infection have been reported, but the molecular interaction between HBV and HDV is poorly understood, especially regarding how HBV and HDV compete with HBsAgs for assembling virions. In this paper, we review the role of endoplasmic reticulum stress induced by HBsAgs and the molecular pathway involved in their promotion of LDAg nuclear export. Because the nuclear sublocalization and export of LDAg is regulated by posttranslational modifications (PTMs), including acetylation, phosphorylation, and isoprenylation, we also summarize the relationship among HBsAg-induced endoplasmic reticulum stress signaling, LDAg PTMs, and nuclear export mechanisms in this review.

Background & Aims: Immunohistochemical assessment of liver tissue in chronic delta hepatitis (CDH) is underinvestigated. Aim of the study was (i) to assess variables associated with hepatitis D antigen (HDAg), hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) staining in the liver., Methods: Demographic, biochemical and virologic data collected from the HIDIT 1 study were used. HBsAg, HBcAg and HDAg immunohistochemical (IHC) staining was semiquantitatively assessed., Results: Hepatitis D antigen immunohistochemical staining displayed positive correlations with age and alanine aminotransferase (ALT) and negative correlations with serum HBsAg (P = 0.01 for all). HBsAg IHC displayed a negative correlation with gamma glutamyl transferase and positive correlations with serum HBV DNA, serum HBsAg levels and HBeAg serology (P < 0.001, P = 0.02 and P = 0.007 respectively). HBcAg staining was mainly nuclear and displayed negative correlations with serum HBsAg and histologic activity (P = 0.002 and P = 0.02 respectively). Pegylated IFN based treatment led to a decline of all IHC markers, however, these markers had no impact on treatment outcome., Conclusions: These data suggest an association of liver injury with HDAg expression in CDH whereas the negative correlation between HBcAg expression and liver injury and the overall nuclear localization of HBcAg suggest that HBcAg does not contribute to liver injury in CDH. HDV cases with high level of HBV replication, high serum HBsAg levels, HBeAg positivity, that are probably in the earlier stages of disease (low gamma-glutamyl transferase), had a more intense HBsAg staining profile. Overall, the data enforce the importance of HDAg and HBsAg in different phases of CDH infection., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Unlabelled: The circular genome and antigenome RNAs of hepatitis delta virus (HDV) form characteristic unbranched, quasi-double-stranded RNA secondary structures in which short double-stranded helical segments are interspersed with internal loops and bulges. The ribonucleoprotein complexes (RNPs) formed by these RNAs with the virus-encoded protein hepatitis delta antigen (HDAg) perform essential roles in the viral life cycle, including viral replication and virion formation. Little is understood about the formation and structure of these complexes and how they function in these key processes. Here, the specific RNA features required for HDAg binding and the topology of the complexes formed were investigated. Selective 2'OH acylation analyzed by primer extension (SHAPE) applied to free and HDAg-bound HDV RNAs indicated that the characteristic secondary structure of the RNA is preserved when bound to HDAg. Notably, the analysis indicated that predicted unpaired positions in the RNA remained dynamic in the RNP. Analysis of the in vitro binding activity of RNAs in which internal loops and bulges were mutated and of synthetically designed RNAs demonstrated that the distinctive secondary structure, not the primary RNA sequence, is the major determinant of HDAg RNA binding specificity. Atomic force microscopy analysis of RNPs formed in vitro revealed complexes in which the HDV RNA is substantially condensed by bending or wrapping. Our results support a model in which the internal loops and bulges in HDV RNA contribute flexibility to the quasi-double-stranded structure that allows RNA bending and condensing by HDAg., Importance: RNA-protein complexes (RNPs) formed by the hepatitis delta virus RNAs and protein, HDAg, perform critical roles in virus replication. Neither the structures of these RNPs nor the RNA features required to form them have been characterized. HDV RNA is unusual in that it forms an unbranched quasi-double-stranded structure in which short base-paired segments are interspersed with internal loops and bulges. We analyzed the role of the HDV RNA sequence and secondary structure in the formation of a minimal RNP and visualized the structure of this RNP using atomic force microscopy. Our results indicate that HDAg does not recognize the primary sequence of the RNA; rather, the principle contribution of unpaired bases in HDV RNA to HDAg binding is to allow flexibility in the unbranched quasi-double-stranded RNA structure. Visualization of RNPs by atomic force microscopy indicated that the RNA is significantly bent or condensed in the complex., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Recent reports show that many cellular RNAs are processed to form circular species that are relatively abundant and resistant to host nucleases. In some cases, such circles actually bind host microRNAs. Such depletion of available microRNAs appears to have biological roles; for instance, in homeostasis and disease. These findings regarding host RNA circles support a speculative reappraisal of the origin and mode of replication of hepatitis delta virus, hepatitis delta virus (HDV), an agent with a small circular RNA genome; specifically, it is proposed that in hepatocytes infected with hepatitis B virus (HBV), some viral RNA species are processed to circular forms, which by a series of chance events lead to an RNA that can be both replicated by host enzymes and assembled, using HBV envelope proteins, to form particles some of which are infectious. Such a model also may provide some new insights into the potential pathogenic potential of HDV infections. In return, new insights into HDV might provide information leading to a better understanding of the roles of the host RNA circles.

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.

Nuclear export is an important process that not only regulates the functions of cellular factors but also facilitates the assembly of viral nucleoprotein complexes. Chromosome region maintenance 1 (CRM1) that mediates the transport of proteins bearing the classical leucine-rich nuclear export signal (NES) is the best-characterized nuclear export receptor. Recently, several CRM1-independent nuclear export pathways were also identified. The nuclear export of the large form of hepatitis delta antigen (HDAg-L), a nucleocapsid protein of hepatitis delta virus (HDV), which contains a CRM1-independent proline-rich NES, is mediated by the host NES-interacting protein (NESI). The mechanism of the NESI protein in mediating nuclear export is still unknown. In this study, NESI was characterized as a highly glycosylated membrane protein. It interacted and colocalized well in the nuclear envelope with lamin A/C and nucleoporins. Importantly, HDAg-L could be coimmunoprecipitated with lamin A/C and nucleoporins. In addition, binding of the cargo HDAg-L to the C terminus of NESI was detected for the wild-type protein but not for the nuclear export-defective HDAg-L carrying a P205A mutation [HDAg-L(P205A)]. Knockdown of lamin A/C effectively reduced the nuclear export of HDAg-L and the assembly of HDV. These data indicate that by forming complexes with lamin A/C and nucleoporins, NESI facilitates the CRM1-independent nuclear export of HDAg-L.

Hepatitis delta is an inflammatory liver disease caused by infection with HDV. HDV is a single-stranded circular RNA pathogen with a diameter of 36 nm. HDV is classified in the genus Deltavirus and is still awaiting a final taxonomic classification up to the family level. HDV shares similarities with satellite RNA and viroids including a small circular single-stranded RNA with secondary structure that replicates through the 'double rolling circle' mechanism. The HDV RNA genome is capable of self-cleavage through a ribozyme and encodes only one structural protein, the hepatitis delta antigen (HDAg), from the antigenomic RNA. There are two forms of HDAg, a shorter (S; 22 kDa) and a longer (L; 24 kDa) form, the latter generated from an RNA editing mechanism. The S form is essential for viral genomic replication. The L form participates in the assembly and formation of HDV. For complete replication and transmission, HDV requires the hepatitis B surface antigen (HBsAg). Thus, HDV infection only occurs in HBsAg-positive individuals, either as acute coinfection in treatment-naive HBV-infected persons, or as superinfection in patients with pre-existing chronic hepatitis B (CHB). HDV is found throughout the world, but its prevalence, incidence, clinical features and epidemiological characteristics vary by geographic region. There are eight genotypes (1 to 8) distributed over different geographic areas: HDV-1 is distributed worldwide, whereas HDV-2 to 8 are seen more regionally. Levels of HDV viraemia change over the course of HDV infection, being significantly higher in patients with early chronic hepatitis than in cirrhosis. Chronic HDV infection leads to more severe liver disease than chronic HBV monoinfection with an accelerated course of fibrosis progression, an increased risk of hepatocellular carcinoma and early decompensation in the setting of established cirrhosis. Current treatments include pegylated interferon-α and liver transplantation; the latter of which can be curative. Further studies are needed to develop better treatment strategies for this challenging disease.

Hepatitis delta virus (HDV) is an RNA virus and eight clades of HDV have been identified. HDV clade 3 (HDV-3) is isolated only in the northern area of South America. The outcome of HDV-3 infection is associated with severe fulminant hepatitis. Variations in the large delta antigen (LDAg) between HDV clade 1 (HDV-1) and HDV-3 have been proposed to contribute to differences in viral secretion efficiency, but which changes might be relevant remains unclear. The control of subcellular localization of LDAg has been reported to be associated with post-translational modifications, such as phosphorylation and isoprenylation. We have observed evidence for acetylation on the LDAg of HDV-3 (LDAg-3) and LDAg of HDV-1 (LDAg-1). Green fluorescent protein-fused LDAg-3 (GFP-LD3) was used to investigate the cellular distribution and secretion of the protein. Sequence alignment of LDAg amino acids suggested that lysine-71 of LDAg-3 could be an acetylation site. Expression of a mutant form of LDAg-3 with an arginine-substitution at lysine-71 (GFP-LD3K71R) showed a distribution of the protein predominantly in the cytoplasm instead of the nucleus. Western blot analyses of secreted empty viral particles (EVPs) revealed a higher amount of secreted GFP-LD3K71R compared to GFP-LD3. Furthermore, the ectopic expression of p300, a histone acetyltransferase, led to a reduction of GFP-LD3 in EVPs. By contrast, expression of three histone deacetylases (HDAC-4, -5, and -6) facilitated the secretion of GFP-LD3. Combined, our observations support the hypothesis that the acetylation status of LDAg-3 plays a role in regulating LDAg-3's localization inside the nucleus or cytoplasm, and its secretion., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Unlabelled: No specific drugs are currently available against hepatitis delta virus (HDV), a defective virus leading to the most severe form of chronic viral hepatitis in man. The lack of convenient HDV infection models has hampered the development of effective therapeutics. In this study, naïve and hepatitis B virus (HBV) chronically infected humanized uPA/SCID mice were employed to establish a small animal model of HBV/HDV coinfection and superinfection. For preclinical antiviral drug evaluation, the GMP version of the myristoylated preS-peptide (Myrcludex-B), a lipopeptide derived from the pre-S1 domain of the HBV envelope, was applied to prevent de novo HBV/HDV coinfection in vivo. Virological parameters were determined at serological and intrahepatic level both by real-time polymerase chain reaction (PCR) and by immunohistochemistry. Establishment of HDV infection was highly efficient in both HBV-infected and naïve chimeric mice with HDV titers rising up to 1 × 10E9 copies/mL. Notably, HDV superinfection led to a median 0.6log reduction of HBV viremia, which although not statistically significant suggests that HDV may hinder HBV replication. In the setting of HBV/HDV simultaneous infection, a majority of human hepatocytes stained HDAg-positive long before HBV spreading was completed, confirming that HDV can replicate intrahepatically also in the absence of HBV infection. Furthermore, the increase of HBV viremia and intrahepatic cccDNA loads was significantly slower than in HBV mono-infected mice. Treatment with the HBV entry inhibitor Myrcludex-B, efficiently hindered the establishment of HDV infection in vivo., Conclusion: We established an efficient model of HBV/HDV infection to exploit mechanisms of viral interference in human hepatocytes and to test the efficacy of an HDV-entry inhibitor in vivo., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Hepatitis B virus (HBV) produces high quantities of subviral surface antigen particles (HBsAg) which circulate in the blood outnumbering virions of about 1\10(3-6) times. In individuals coinfected with the defective hepatitis Delta virus (HDV) the small HDV-RNA-genome and Delta antigen circulate as ribonucleoprotein complexes within HBsAg subviral particles. We addressed the question whether subviral HBsAg particles may carry in the same way cellular microRNAs (miRNAs) which are released into the bloodstream within different subcellular forms such as exosomes and microvescicles. Circulating HBsAg particles were isolated from sera of 11 HBsAg carriers by selective immunoprecipitation with monoclonal anti-HBs-IgG, total RNA was extracted and human miRNAs were screened by TaqMan real-time quantitative PCR Arrays. Thirty-nine human miRNAs were found to be significantly associated with the immunoprecipitated HBsAg, as determined by both comparative DDCT analysis and non-parametric tests (Mann-Whitney, p<0.05) with respect to controls. Moreover immunoprecipitated HBsAg particles contained Ago2 protein that could be revealed in ELISA only after 0.5% NP40. HBsAg associated miRNAs were liver-specific (most frequent = miR-27a, miR-30b, miR-122, miR-126 and miR-145) as well as immune regulatory (most frequent = miR-106b and miR-223). Computationally predicted target genes of HBsAg-associated miRNAs highlighted molecular pathways dealing with host-pathogen. The finding that HBsAg particles carry selective pools of hepatocellular miRNAs opens new avenues of research to disentangle the complex interactions between host and HBV and provides a non invasive tool to study the physiopathology of liver epigenetics.

Background: Hepatitis delta virus (HDV) is considered to be a satellite virus of the Hepatitis B virus. The genome consists of a 1679 nt ssRNA molecule in which a single ORF was identified. This ORF codes for a unique protein, the Delta antigen (HDAg). During transcription, two forms, small (S-HDAg; p24) and large (L-HDAg; p27) of this antigen are derived as a result of an editing mechanism catalyzed by cellular adenosine deaminase 1. Despite its simplicity, little is still known about the host factors that interact with the virus RNA and antigens being to modulate virus replication., Methods: A yeast two-hybrid screening of a human liver cDNA library, using the hepatitis delta virus (HDV) small antigen (S-HDAg) as bait, was performed. Blot overlay and co-immunoprecipitation assays were used in an attempt to confirm the interaction of hnRNPC and S-HDAg. siRNA knockdown assays of hnRNPC were performed to assess the effect on HDV antigen expression., Results: Thirty known proteins were identified as S-HDAg interactors in the yeast two-hybrid screening. One of the identified proteins, hnRNPC, was found to interact with S-HDAg in vitro and in vivo in human liver cells. The interaction of the two proteins is mediated by the C-terminal half of the S-HDAg which contains a RNA-binding domain (aa 98-195). HDV RNA, S-HDAg, and hnRNPC, were also found to co-localize in the nucleus of human liver cells. Knockdown of hnRNPC mRNA using siRNAs resulted in a marked decreased expression of HDV antigens., Conclusions: S-HDAg was found to interact with human liver proteins previously assigned to different functional categories. Among those involved in nucleic acid metabolism, hnRNPC was found to interact in vitro and in vivo in human liver cells. Similar to other RNA viruses, it seems plausible that hnRNPC may also be involved in HDV replication. However, further investigation is mandatory to clarify this question.

Caenorhabditis elegans is a model organism that has been used to study human bacterial and viral pathogenesis. We report here the expression of human hepatitis delta viral antigens (HDAg) in C. elegans and measure the effect on the sterility, growth, and brood size in transgenic worms. Expression of HDAg under two different promoters, fib-1 (a ubiquitous promoter) and myo-2 (a pharynx-specific promoter), was achieved in C. elegans using dicistronic or tricistronic vectors derived from the operon CEOP5428. Transgenic worms expressing HDAg ubiquitously resulted in 20% to 70% sterility while those expressing HDAg in the pharynx displayed 70% sterility. Most of worms expressing HDAg in pharynx were arrested at larvae stage 2 or 3 and displayed a 70% reduction in brood size. Domain mapping experiments suggested that the nuclear localization signal of HDAg is required for the observed effect. Heat-shock induction of HDAg expression revealed that L4 larvae were the most sensitive to brood size reduction. These studies demonstrate that C. elegans can provide an additional model for studying HDAg interactions with host targets.

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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.

Recent studies revealed that posttranslational modifications (e.g., phosphorylation and methylation) of the small hepatitis delta antigen (SHDAg) are required for hepatitis delta virus (HDV) replication from antigenomic to genomic RNA. The phosphorylation of SHDAg at serine 177 (Ser(177)) is involved in this step, and this residue is crucial for interaction with RNA polymerase II (RNAP II), the enzyme assumed to be responsible for antigenomic RNA replication. This study demonstrated that SHDAg dephosphorylated at Ser(177) interacted preferentially with hypophosphorylated RNAP II (RNAP IIA), which generally binds at the transcription initiation sites. In contrast, the Ser(177)-phosphorylated counterpart (pSer(177)-SHDAg) exhibited preferential binding to hyperphosphorylated RNAP II (RNAP IIO). In addition, RNAP IIO associated with pSer(177)-SHDAg was hyperphosphorylated at both the Ser(2) and Ser(5) residues of its carboxyl-terminal domain (CTD), which is a hallmark of the transcription elongation isoform. Moreover, the RNAP II CTD kinase inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) not only blocked the interaction between pSer(177)-SHDAg and RNAP IIO but also inhibited HDV antigenomic RNA replication. Our results suggest that the phosphorylation of SHDAg at Ser177 shifted its affinitytoward the RNAP IIO isoform [corrected] and thus is a switch for HDV antigenomic RNA replication from the initiation to the elongation stage.

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.

Clathrin is involved in the endocytosis and exocytosis of cellular proteins and the process of virus infection. We have previously demonstrated that large hepatitis delta antigen (HDAg-L) functions as a clathrin adaptor, but the detailed mechanisms of clathrin involvement in the morphogenesis of hepatitis delta virus (HDV) are not clear. In this study, we found that clathrin heavy chain (CHC) is a key determinant in the morphogenesis of HDV. HDAg-L with a single amino acid substitution at the clathrin box retained nuclear export activity but failed to interact with CHC and to assemble into virus-like particles. Downregulation of CHC function by a dominant-negative mutant or by short hairpin RNA reduced the efficiency of HDV assembly, but not the secretion of hepatitis B virus subviral particles. In addition, the coexistence of a cell-permeable peptide derived from the C terminus of HDAg-L significantly interfered with the intracellular transport of HDAg-L. HDAg-L, small HBsAg, and CHC were found to colocalize with the trans-Golgi network and were highly enriched on clathrin-coated vesicles. Furthermore, genotype II HDV, which assembles less efficiently than genotype I HDV does, has a putative clathrin box in its HDAg-L but interacted only weakly with CHC. The assembly efficiency of the various HDV genotypes correlates well with the CHC-binding activity of their HDAg-Ls and coincides with the severity of disease outcome. Thus, the clathrin box and the nuclear export signal at the C terminus of HDAg-L are potential new molecular targets for HDV therapy.

Hepatitis delta virus (HDV) infection causes fulminant hepatitis and liver cirrhosis. To elucidate the molecular mechanism of HDV pathogenesis, we examined the effects of HDV viral proteins, the small hepatitis delta antigen (SHDAg) and the large hepatitis delta antigen (LHDAg), on NF-kappaB signaling pathway. In this study, we demonstrated that TNF-alpha-induced NF-kappaB transcriptional activation was increased by LHDAg but not by SHDAg in both HEK293 and Huh7 cells. Furthermore, LHDAg promoted TRAF2-induced NF-kappaB activation. Using coimmunoprecipitation assays, we demonstrated that both SHDAg and LHDAg interacted with TRAF2 protein. We showed that isoprenylation of LHDAg was not required for the increase of NF-kappaB activity. We further showed that only LHDAg but not SHDAg increased the TNF-alpha-mediated nuclear translocation of p65. This was accomplished by activation of IkappaBalpha degradation by LHDAg. Finally, we demonstrated that LHDAg augmented the COX-2 expression level in Huh7 cells. These data suggest that LHDAg modulates NF-kappaB signaling pathway and may contribute to HDV pathogenesis.

Hepatitis delta virus (HDV) encodes one protein, hepatitis delta antigen (deltaAg), a 195-amino-acid RNA binding protein essential for the accumulation of HDV RNA-directed RNA transcripts. It has been accepted that deltaAg localizes predominantly to the nucleolus in the absence of HDV genome replication while in the presence of replication, deltaAg facilitates HDV RNA transport to the nucleoplasm and helps redirect host RNA polymerase II (Pol II) to achieve transcription and accumulation of processed HDV RNA species. This study used immunostaining and confocal microscopy to evaluate factors controlling the localization of deltaAg in the presence and absence of replicating and nonreplicating HDV RNAs. When deltaAg was expressed in the absence of full-length HDV RNAs, it colocalized with nucleolin, a predominant nucleolar protein. With time, or more quickly after induced cell stress, there was a redistribution of both deltaAg and nucleolin to the nucleoplasm. Following expression of nonreplicating HDV RNAs, deltaAg moved to the nucleoplasm, but nucleolin was unchanged. When deltaAg was expressed along with replicating HDV RNA, it was found predominantly in the nucleoplasm along with Pol II. This localization was insensitive to inhibitors of HDV replication, suggesting that the majority of deltaAg in the nucleoplasm reflects ribonucleoprotein accumulation rather than ongoing transcription. An additional approach was to reevaluate several forms of deltaAg altered at specific locations considered to be essential for protein function. These studies provide evidence that deltaAg does not interact directly with either Pol II or nucleolin and that forms of deltaAg which support replication are also capable of prior nucleolar transit.

Hepatitis delta virus (HDV) is a subviral pathogen that increases the severity of liver disease caused by hepatitis B virus. Both the small circular RNA genome and its complement, the antigenome, form a characteristic unbranched rod structure in which approximately 70% of the nucleotides are base paired. These RNAs are associated with the sole virally encoded protein, hepatitis delta antigen (HDAg), in infected cells; however, the nature of the ribonucleoprotein complexes (RNPs) is not well understood. Previous analyses of binding in vitro using native, bacterially expressed HDAg have been hampered by a lack of specificity for HDV RNA. Here, we show that removal of the C-terminal 35 amino acids of HDAg yields a native, bacterially expressed protein, HDAg-160, that specifically binds HDV unbranched rod RNA with high affinity. In an electrophoretic mobility shift assay, this protein produced a discrete, micrococcal nuclease-resistant complex with an approximately 400-nucleotide (nt) segment of HDV unbranched rod RNA. Binding occurred with several segments of HDV RNA, although with various affinities and efficiencies. Analysis of the effects of deleting segments of the unbranched rod indicated that binding did not require one or two specific binding sites within these RNA segments. Rather, a minimum-length HDV RNA unbranched rod approximately 311 nt was essential for RNP formation. The results are consistent with a model in which HDAg binds HDV unbranched rod RNA as multimers of fixed size rather than as individual subunits.

Background: Hepatitis delta virus (HDV) is a defected RNA virus and requires its encoded large antigen (LDAg) to interact with helper viral proteins (HBsAgs) during assembly. Recently, a study demonstrated a direct binding of the LDAg C-terminus from genotype I HDV to the clathrin heavy chain (CHC), which suggests that this interaction might facilitate HDV assembly. If LDAg binding to clathrin is essential to HDV life cycle, a clathrin box sequence at the C-terminus of LDAg should be conserved across all HDV. However, the C-terminal sequence of LDAg is variable among 43 HDV isolates., Results: Based on the presence and location of clathrin box at the C-terminus of LDAg from 43 isolates of HDV, we classified them into three groups. Group 1 (13 isolates) and 2 (26 isolates) contain a clathrin box located at amino acids 199-203 and 206-210, respectively, as found in genotype I and genotype II. Group 3 (4 isolates) contains no clathrin box as found in genotype III. CHC binding by three different LDAg (genotype I to III) was then tested by in vivo and in vitro experiments. Transfection of plasmids which encode fusion proteins of EGFP and full-length of LDAg from three genotypes into HuH-7 cells, a human hepatoma cell line, was performed. GFP-pull down assays showed that a full-length of CHC was co-precipitated by EGFP-LDI, -LDII and -LDIII but not by EGFP. Further in vitro studies showed a full-length or fragment (amino acids 1 to 107) of CHC can be pull-down by 13-amino-acid peptides of LDAg from three genotypes of HDV., Conclusion: Both in vivo and in vitro studies showed that CHC can bind to various sequences of LDAg from the three major genotypes of HDV. We therefore suggest that the clathrin-LDAg interaction is essential to the HDV life-cycle and that sequences binding to clathrin are evolutionarily selected, but nonetheless show the diversity across different HDV genotypes.

Hepatitis delta virus (HDV) is a subviral agent dependent upon hepatitis B virus (HBV). HDV uses the envelope proteins of HBV to achieve assembly and infection of target cells. Otherwise, the replication of the RNA genome of HDV is totally different from that of its helper virus, and involves redirection of host polymerase activity. This chapter is concerned with recent developments in our understanding of the genome replication process.

Hepatitis delta virus (HDV) is a pathogenic RNA virus with a plant viroid-like genome structure. HDV encodes two isoforms of delta antigen (HDAg), the small and large forms of HDAg (SHDAg and LHDAg), which are essential for HDV RNA replication and virion assembly, respectively. Replication of HDV RNA depends on host cellular transcription machinery, and the exact molecular mechanism for HDV RNA replication is still unclear. In this study, we demonstrated that both isoforms of HDAg interact with transcription factor YY1 (Yin Yang 1) in vivo and in vitro. Their interaction domains were identified as the middle region encompassing the RNA binding domain of HDAg and the middle GA/GK-rich region and the C-terminal zinc-finger region of YY1. Results of sucrose gradient centrifugation analysis indicated the cosedimentation of the majority of SHDAg and a portion of the LHDAg with YY1 and its associated acetyltransferases CBP (CREB-binding protein) and p300 as a large nuclear complex in vivo. Furthermore, exogenous expression of YY1 or CBP/p300 in HDV RNA replication system showed an enhancement of HDV RNA replication. Interestingly, the acetyltransferase activity of p300 is important for this enhancement. Moreover, SHDAg could be acetylated in vivo, and treatment with cellular deacetylase inhibitor elevated the replication of HDV RNA and acetylation of SHDAg. All together, our results reveal that HDAg interacts with cellular transcription factor YY1 and its associated acetyltransferases CBP and p300 in a large nuclear complex, which in turn modulates the replication of HDV RNA.

The evolutionary origin of human hepatitis delta virus (HDV) replication by RNA-directed transcription is unclear. Here we identify two species of 5'-capped, approximately 18-25-nucleotide small RNAs. One was of antigenomic polarity, corresponding to the 5' end of hepatitis delta antigen (HDAg) mRNA, and interacted with HDAg and RNA polymerase II (Pol II), whereas the other mapped to a structurally analogous region on the genomic RNA hairpin. An HDAg-interaction screen indicated that HDAg interacts with MOV10, the human homolog of the Arabidopsis thaliana RNA amplification factor gene SDE3 and Drosophila melanogaster RISC-maturation factor gene Armitage (armi). MOV10 knockdown inhibited HDV replication, but not HDAg mRNA translation, further supporting a role for MOV10 in RNA-directed transcription. Together, our studies define RNA hairpins as critical elements for the initiation of HDV-related, RNA-directed transcription. The identification of capped small RNAs and the involvement of MOV10 in HDV replication further suggest a conserved mechanism related to RNA-directed transcription in lower eukaryotes.

Hepatitis delta virus (HDV) encodes two isoforms of delta antigens (HDAgs). The small form of HDAg (SHDAg) is required for HDV RNA replication, while the large form of HDAg (LHDAg) is required for viral assembly. Using tandem affinity purification method combined with mass spectrometry, we found that linker histone H1e bound to SHDAg. The binding domain of SHDAg to histone H1e was mapped to the N-terminal 67 amino acids. Oligomerization of SHDAg was required for its interaction with histone H1e. LHDAg barely bound to histone H1e and was masked at N-terminus. The binding domain of histone H1e to SHDAg was mapped to its central globular domain. HDV replication was inhibited by N- or C-terminal deletion mutants of histone H1e and was rescued by wild-type histone H1e. We conclude that histone H1e plays a significant role in HDV replication through forming protein complex with SHDAg.

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.