Deciphering the Role of Post-Translational Modifications and Cellular Location of Hepatitis Delta Virus (HDV) Antigens in HDV-Mediated Liver Damage in Mice.

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.

Unbranched rod-like RNA is required for RNA editing of hepatitis delta virus genotype 2 and genotype 4.

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.

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.

Hepatitis D virus interferes with hepatitis B virus RNA production via interferon-dependent and -independent mechanisms.

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.

Identification of a novel interaction site between the large hepatitis delta antigen and clathrin that regulates the assembly of genotype III hepatitis delta virus.

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.

N6-Methyladenine Modification of Hepatitis Delta Virus Regulates Its Virion Assembly by Recruiting YTHDF1.

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. N6-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 N6-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.

1H, 15N and 13C backbone and side chain solution NMR assignments of the truncated small hepatitis delta antigen Δ60-S-HDAg.

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 1H, 13C and 15 N backbone and side chain resonance assignments of an N-terminally truncated form of S-HDAg (SΔ60), 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 SΔ60 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.

Loss of hepatitis D virus infectivity upon farnesyl transferase inhibitor treatment associates with increasing RNA editing rates revealed by a new RT-ddPCR method.

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.

Ubiquitinated Hepatitis D Antigen-Loaded Microvesicles Induce a Potent Specific Cellular Immune Response to Inhibit HDV Replication in Vivo.

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.

Innate immune recognition and modulation in hepatitis D virus infection.

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.

Statin inhibits large hepatitis delta antigen-Smad3 -twist-mediated epithelial-to-mesenchymal transition and hepatitis D virus secretion.

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-ß) 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-ß secretion, expression of EMT markers, and to evaluate efficacy of statins for HDV treatment. RESULTS: We found that L-HDAg activated Twist expression, TGF-ß 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-ß 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-ß activation. Treatment with statins suppressed Twist expression, and TGF-ß 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.

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.

Hepatitis Delta Virus Alters the Autophagy Process To Promote Its Genome Replication.

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.

Structural Pattern Differences in Unbranched Rod-like RNA of Hepatitis Delta Virus affect RNA Editing.

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.

Recapitulation of HDV infection in a fully permissive hepatoma cell line allows efficient drug evaluation.

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 (IC50: 1.4 nM) and interferon-α (IC50: 28 IU/ml, but max. 60-80% inhibition) interfere with primary infection. Lonafarnib inhibits virus secretion (IC50: 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 Antigen Regulates mRNA and Antigenome RNA Levels during Hepatitis Delta Virus Replication.

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.

Small hepatitis delta antigen selectively binds to target mRNA in hepatic cells: a potential mechanism by which hepatitis D virus downregulates glutathione S-transferase P1 and induces liver injury and hepatocarcinogenesis.

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.

Amino Acid Substitutions within HLA-B*27-Restricted T Cell Epitopes Prevent Recognition by Hepatitis Delta Virus-Specific CD8+ T Cells.

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.

Tat-enhanced delivery of the C terminus of HDAg-L inhibits assembly and secretion of hepatitis D virus.

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.

A new HDV mouse model identifies mitochondrial antiviral signaling protein (MAVS) as a key player in IFN-ß induction.

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.