An extracellular humanized IFNAR immunocompetent mouse model for analyses of human interferon alpha and subtypes.

Type I interferons (IFN-Is) have key roles in immune defense and treatments for various diseases, including chronic hepatitis B virus (HBV) infection. All IFN-Is signal through a shared IFN-I heterodimeric receptor complex comprising IFN-α receptor 1 (IFNAR1) and IFNAR2 subunits, but differences in antiviral and immunomodulatory responses among IFN-I subtypes remain largely unknown. Because the IFN-IFNAR interactions are species-specific, mice exhibit weak responses to human IFN-I. To more fully characterize the actions of human IFN-α and its subtypes in vivo, a gene targeting strategy was employed to generate gene knock-in mice with extracellular-humanized IFNAR1/2 (IFNAR-hEC) in the C57BL/6N strain. IFNAR-hEC mice actively responded to human IFN-I, and endogenous mouse IFN-I signalling remained active in heterozygous mice (IfnarhEC/+). Analyses of IFNAR-hEC mice and isolated cells showed that human IFN-α2 and α14 subtypes exerted differential effect on the activation of JAK-STAT signalling and immune responses. Compared with IFN-α2, IFN-α14 induced greater activation of STAT1/2 and IFN-stimulated genes, synergistically elicited IFN-α and -γ signalling, and induced higher numbers of antigen-specific CD8+ T cells. Moreover, IFNAR-hEC mice with HBV replication displayed long-term viral suppression upon treatment with the clinically-used PEGylated hIFN-α2. These results indicate that IFNAR-hEC mice may be useful for elucidating antiviral and immunomodulatory functions of human IFN-Is and for conducting preclinical studies. A better understanding of the distinct activities of IFN-α subtypes can provide insights concerning the development of improved IFN-based therapy.

Genome-Wide Extrachromosomal Circular DNA Profiling of Paired Hepatocellular Carcinoma and Adjacent Liver Tissues.

Hepatocellular carcinoma (HCC) develops through multiple mechanisms. While recent studies have shown the presence of extrachromosomal circular DNA (eccDNA) in most cancer types, the eccDNA expression pattern and its association with HCC remain obscure. We aimed to investigate this problem. The genome-wide eccDNA profiles of eight paired HCC and adjacent non-tumor tissue samples were comprehensively elucidated based on Circle-seq, and they were further cross-analyzed with the RNA sequencing data to determine the association between eccDNA expression and transcriptome dysregulation. A total of 60,423 unique eccDNA types were identified. Most of the detected eccDNAs were smaller than 1 kb, with a length up to 182,363 bp and a mean sizes of 674 bp (non-tumor) and 813 bp (tumor), showing a greater association with gene-rich rather than with gene-poor regions. Although there was no statistical difference in length and chromosome distribution, the eccDNA patterns between HCC and adjacent non-tumor tissues showed significant differences at both the chromosomal and single gene levels. Five of the eight HCC tissues showed significantly higher amounts of chromosome 22-derived eccDNA expression compared to the non-tumor tissue. Furthermore, two genes, SLC16A3 and BAIAP2L2, with a higher transcription level in tumor tissues, were related to eccDNAs exclusively detected in three HCC samples and were negatively associated with survival rates in HCC cohorts from public databases. These results indicate the existence and massive heterogeneity of eccDNAs in HCC and adjacent liver tissues, and suggest their potential association with dysregulated gene expression.

HBV induced the discharge of intrinsic antiviral miRNAs in HBV-replicating hepatocytes via extracellular vesicles to facilitate its replication.

Hepatitis B virus (HBV), which can cause chronic hepatitis B, has sophisticated machinery to establish persistent infection. Here, we report a novel mechanism whereby HBV changed miRNA packaging into extracellular vesicles (EVs) to facilitate replication. Disruption of the miRNA machinery in hepatocytes enhanced HBV replication, indicating an intrinsic miRNA-mediated antiviral state. Interference with EV release only decreased HBV replication if there was normal miRNA biogenesis, suggesting a possible link between HBV replication and EV-associated miRNAs. Microarray and qPCR analyses revealed that HBV replication changed miRNA expression in EVs. EV incubation, transfection of miRNA mimics and inhibitors, and functional pathway and network analyses showed that EV miRNAs are associated with antiviral function, suggesting that to promote survival HBV coopts EVs to excrete anti-HBV intracellular miRNAs. These data suggest a novel mechanism by which HBV maintains its replication, which has therapeutic implications.

Monocytic MDSCs homing to thymus contribute to age-related CD8+ T cell tolerance of HBV.

Hepatitis B virus exposure in children usually develops into chronic hepatitis B (CHB). Although hepatitis B surface antigen (HBsAg)-specific CD8+ T cells contribute to resolve HBV infection, they are preferentially undetected in CHB patients. Moreover, the mechanism for this rarely detected HBsAg-specific CD8+ T cells remains unexplored. We herein found that the frequency of HBsAg-specific CD8+ T cells was inversely correlated with expansion of monocytic myeloid-derived suppressor cells (mMDSCs) in young rather than in adult CHB patients, and CCR9 was upregulated by HBsAg on mMDSCs via activation of ERK1/2 and IL-6. Sequentially, the interaction between CCL25 and CCR9 mediated thymic homing of mMDSCs, which caused the cross-presentation, transferring of peripheral HBsAg into the thymic medulla, and then promoted death of HBsAg-specific CD8+ thymocytes. In mice, adoptive transfer of mMDSCs selectively obliterated HBsAg-specific CD8+ T cells and facilitated persistence of HBV in a CCR9-dependent manner. Taken together, our results uncovered a novel mechanism for establishing specific CD8+ tolerance to HBsAg in chronic HBV infection.

Human hepatocyte-enriched miRNA-192-3p promotes HBV replication through inhibiting Akt/mTOR signalling by targeting ZNF143 in hepatic cell lines.

Previous studies have revealed multiple tissue- or cell-specific or enriched miRNA profiles. However, miRNA profiles enriched in hepatic cell types and their effect on HBV replication have not been well elucidated. In this study, primary human hepatocytes (PHHs), Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs) were prepared from liver specimens of non-HBV-infected patients. Four hepatic cell type-enriched miRNA profiles were identified from purified liver cells miRNA microarray assay. The results revealed that 12 miRNAs, including miR-122-5p and miR-192-3p were PHH-enriched; 9 miRNAs, including miR-142-5p and miR-155-5p were KC-enriched; 6 miRNAs, including miR-126-3p and miR-222-3p were LSEC-enriched; and 14 miRNAs, including miR-214-3p and miR-199a-3p were HSC-enriched. By testing the effect of 11 PHH-enriched miRNAs on HBV production, we observed that miR-192-3p had the greatest pro-virus effect in hepatic cell lines. Moreover, we further found that miR-192-3p promoted HBV replication and gene expression through inhibiting Akt/mTOR signalling by direct targeting of ZNF143 in HepG2.2.15 cells. Additionally, the serum and hepatic miR-192-3p expression levels were significantly higher in chronic hepatitis B patients than in healthy controls and serum miR-192-3p positively correlated with the serum levels of HBV DNA and HBsAg. Collectively, we identified miRNA profiles enriched in four hepatic cell types and revealed that PHH-enriched miR-192-3p promoted HBV replication through inhibiting Akt/mTOR signalling by direct targeting of ZNF143 in hepatic cell lines. Our study provides a specific perspective for the role of hepatic cell type-enriched miRNA in interaction with viral replication and various liver pathogenesis.

Interferon Alpha Induces Cellular Autophagy and Modulates Hepatitis B Virus Replication.

Hepatitis B virus (HBV) infection causes acute and chronic liver diseases, including severe hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Interferon alpha 2a (IFNα-2a) is commonly used for treating chronic HBV infection. However, its efficacy remains relatively low. Yet, the immunological and molecular mechanisms for successful IFNα-2a treatment remain elusive. One issue is whether the application of increasing IFNα doses may modulate cellular processes and HBV replication in hepatic cells. In the present study, we focused on the interaction of IFNα signaling with other cellular signaling pathways and the consequence for HBV replication. The results showed that with the concentration of 6000 U/ml IFNα-2a treatment downregulated the activity of not only the Akt/mTOR signaling but also the AMPK signaling. Additionally, IFNα-2a treatment increased the formation of the autophagosomes by blocking autophagic degradation. Furthermore, IFNα-2a treatment inhibited the Akt/mTOR signaling and initiated autophagy under low and high glucose concentrations. In reverse, inhibition of autophagy using 3-methyladenine (3-MA) and glucose concentrations influenced the expression of IFNα-2a-induced ISG15 and IFITM1. Despite of ISGs induction, HBV replication and gene expression in HepG2.2.15 cells, a cell model with continuous HBV replication, were slightly increased at high doses of IFNα-2a. In conclusion, our study indicates that IFNα-2a treatment may interfere with multiple intracellular signaling pathways, facilitate autophagy initiation, and block autophagic degradation, thereby resulting in slightly enhanced HBV replication.

Long-Term Hepatitis B Virus Infection Induces Cytopathic Effects in Primary Human Hepatocytes, and Can Be Partially Reversed by Antiviral Therapy.

Chronic infection of hepatitis B virus (HBV) remains a major health burden worldwide. While the immune response has been recognized to play crucial roles in HBV pathogenesis, the direct cytopathic effects of HBV infection and replication on host hepatocytes and the HBV-host interactions are only partially defined due to limited culture systems. Here, based on our recently developed 5 chemical-cultured primary human hepatocytes (5C-PHHs) model that supports long-term HBV infection, we performed multiplexed quantitative analysis of temporal changes of host proteome and transcriptome on PHHs infected by HBV for up to 4 weeks. We showed that metabolic-, complement-, cytoskeleton-, mitochondrial-, and oxidation-related pathways were modulated at transcriptional or posttranscriptional levels during long-term HBV infection, which led to cytopathic effects and could be partially rescued by early, rather than late, nucleot(s)ide analog (NA) administration and could be significantly relieved by blocking viral antigens with RNA interference (RNAi). Overexpression screening of the dysregulated proteins identified a series of host factors that may contribute to pro- or anti-HBV responses of the infected hepatocytes. In conclusion, our results suggest that long-term HBV infection in primary human hepatocytes leads to cytopathic effects through remodeling the proteome and transcriptome and early antiviral treatment may reduce the extent of such effects, indicating a role of virological factors in HBV pathogenesis and a potential benefit of early administration of antiviral treatment. IMPORTANCE Global temporal quantitative proteomic and transcriptomic analysis using long-term hepatitis B virus (HBV)-infected primary human hepatocytes uncovered extensive remodeling of the host proteome and transcriptome and revealed cytopathic effects of long-term viral replication. Metabolic-, complement-, cytoskeleton-, mitochondrial-, and oxidation-related pathways were modulated at transcriptional or posttranscriptional levels, which could be partially rescued by early, rather than late, NA therapy and could be relieved by blocking viral antigens with RNAi. Overexpression screening identified a series of pro- or anti-HBV host factors. These data have deepened the understanding of the mechanisms of viral pathogenesis and HBV-host interactions in hepatocytes, with implications for therapeutic intervention.

Differential interferon-α subtype induced immune signatures are associated with suppression of SARS-CoV-2 infection.

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2-infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2.

HBV covalently closed circular DNA minichromosomes in distinct epigenetic transcriptional states differ in their vulnerability to damage.

BACKGROUND AND AIMS: HBV covalently closed circular DNA (cccDNA) is a major obstacle for a cure of chronic hepatitis B. Accumulating evidence suggests that epigenetic modifications regulate the transcriptional activity of cccDNA minichromosomes. However, it remains unclear how the epigenetic state of cccDNA affects its stability. APPROACHES AND RESULTS: By using HBV infection cell models and in vitro and in vivo recombinant cccDNA (rcccDNA) and HBVcircle models, the reduction rate of HBV cccDNA and the efficacy of apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A)-mediated and CRISPR/CRISPR-associated 9 (Cas9)-mediated cccDNA targeting were compared between cccDNAs with distinct transcriptional activities. Interferon-α treatment and hepatitis B x protein (HBx) deletion were applied as two strategies for cccDNA repression. Chromatin immunoprecipitation and micrococcal nuclease assays were performed to determine the epigenetic pattern of cccDNA. HBV cccDNA levels remained stable in nondividing hepatocytes; however, they were significantly reduced during cell division, and the reduction rate was similar between cccDNAs in transcriptionally active and transcriptionally repressed states. Strikingly, HBV rcccDNA without HBx expression exhibited a significantly longer persistence in mice. The cccDNA with low transcriptional activity exhibited an epigenetically inactive pattern and was more difficult to access by APOBEC3A and engineered CRISPR-Cas9. The epigenetic regulator activating cccDNA increased its vulnerability to APOBEC3A. CONCLUSIONS: HBV cccDNA minichromosomes in distinct epigenetic transcriptional states showed a similar reduction rate during cell division but significantly differed in their accessibility and vulnerability to targeted nucleases and antiviral agents. Epigenetic sensitization of cccDNA makes it more susceptible to damage and may potentially contribute to an HBV cure.

Interferon and Hepatitis B: Current and Future Perspectives.

Chronic hepatitis B virus (HBV) infection remains a major health burden worldwide for which there is still no effective curative treatment. Interferon (IFN) consists of a group of cytokines with antiviral activity and immunoregulatory and antitumor effects, that play crucial roles in both innate and adaptive immune responses. IFN-α and its pegylated form have been used for over thirty years to treat chronic hepatitis B (CHB) with advantages of finite treatment duration and sustained virologic response, however, the efficacy is limited and side effects are common. Here, we summarize the status and unique advantages of IFN therapy against CHB, review the mechanisms of IFN-α action and factors affecting IFN response, and discuss the possible improvement of IFN-based therapy and the rationale of combinations with other antiviral agents in seeking an HBV cure.

Functional Comparison of Interferon-α Subtypes Reveals Potent Hepatitis B Virus Suppression by a Concerted Action of Interferon-α and Interferon-γ Signaling.

BACKGROUND AND AIMS: Interferon (IFN)-α, composed of numerous subtypes, plays a crucial role in immune defense. As the most studied subtype, IFN-α2 has been used for treating chronic hepatitis B virus (HBV) infection, with advantages of finite treatment duration and sustained virologic response, but its efficacy remains relatively low. This study aimed to screen for IFN-α subtypes with the highest anti-HBV potency and to characterize mechanisms of IFN-α-mediated HBV restriction. APPROACH AND RESULTS: Using cell culture-based HBV infection systems and a human-liver chimeric mouse model, IFN-α subtype-mediated antiviral response and signaling activation were comprehensively analyzed. IFN-α14 was identified as the most effective subtype in suppression of HBV covalently closed circular DNA transcription and HBV e antigen/HBV surface antigen production, with median inhibitory concentration values approximately 100-fold lower than those of the conventional IFN-α2. IFN-α14 alone elicited IFN-α and IFN-γ signaling crosstalk in a manner similar to the combined use of IFN-α2 and IFN-γ, inducing multiple potent antiviral effectors, which synergistically restricted HBV replication. Guanylate binding protein 5, one of the most differentially expressed genes between IFN-α14-treated and IFN-α2-treated liver cells, was identified as an HBV restriction factor. A strong IFN-α-IFN-α receptor subunit 1 interaction determines the anti-HBV activity of IFN-α. The in vivo anti-HBV activity of IFN-α14 and treatment-related transcriptional patterns were further confirmed, and few adverse effects were observed. CONCLUSIONS: A concerted IFN-α and IFN-γ response in liver, which could be efficiently elicited by IFN-α subtype 14, is associated with potent HBV suppression. These data deepen the understanding of the divergent activities of IFN-α subtypes and the mechanism underlying the synergism between IFN-α and IFN-γ signaling, with implications for improved IFN therapy and HBV curative strategies.

Functional mapping of B-cell linear epitopes of SARS-CoV-2 in COVID-19 convalescent population.

ABSTRACT Pandemic SARS-CoV-2 has caused unprecedented mortalities. Vaccine is in urgent need to stop the pandemic. Despite great progresses on SARS-CoV-2 vaccine development, the efficacy of the vaccines remains to be determined. Deciphering the interactions of the viral epitopes with the elicited neutralizing antibodies in convalescent population inspires the vaccine development. In this study, we devised a peptide array composed of 20-mer overlapped peptides of spike (S), membrane (M) and envelope (E) proteins, and performed a screening with 120 COVID-19 convalescent sera and 24 non-COVID-19 sera. We identified five SARS-CoV-2-specific dominant epitopes that reacted with above 40% COVID-19 convalescent sera. Of note, two peptides non-specifically interacted with most of the non-COVID-19 sera. Neutralization assay indicated that only five sera completely blocked viral infection at the dilution of 1:200. By using a peptide-compete neutralizing assay, we found that three dominant epitopes partially competed the neutralization activity of several convalescent sera, suggesting antibodies elicited by these epitopes played an important role in neutralizing viral infection. The epitopes we identified in this study may serve as vaccine candidates to elicit neutralizing antibodies in most vaccinated people or specific antigens for SARS-CoV-2 diagnosis.

Pathogenicity and transmissibility of 2019-nCoV-A quick overview and comparison with other emerging viruses.

A zoonotic coronavirus, tentatively labeled as 2019-nCoV by the World Health Organization (WHO), has been identified as the causative agent of the viral pneumonia outbreak in Wuhan, China, at the end of 2019. Although 2019-nCoV can cause a severe respiratory illness like SARS and MERS, evidence from clinics suggested that 2019-nCoV is generally less pathogenic than SARS-CoV, and much less than MERS-CoV. The transmissibility of 2019-nCoV is still debated and needs to be further assessed. To avoid the 2019-nCoV outbreak turning into an epidemic or even a pandemic and to minimize the mortality rate, China activated emergency response procedures, but much remains to be learned about the features of the virus to refine the risk assessment and response. Here, the current knowledge in 2019-nCoV pathogenicity and transmissibility is summarized in comparison with several commonly known emerging viruses, and information urgently needed for a better control of the disease is highlighted.

AMPK and Akt/mTOR signalling pathways participate in glucose-mediated regulation of hepatitis B virus replication and cellular autophagy.

A growing consensus indicates that host metabolism plays a vital role in viral infections. Hepatitis B virus (HBV) infection occurs in hepatocytes with active glucose metabolism and may be regulated by cellular metabolism. We addressed the question whether and how glucose regulates HBV replication in hepatocytes. The low glucose concentration at 5 mM significantly promoted HBV replication via enhanced transcription and autophagy when compared with higher glucose concentrations (10 and 25 mM). At low glucose concentration, AMPK activity was increased and led to ULK1 phosphorylation at Ser 555 and LC3-II accumulation. By contrast, the mTOR pathway was activated by high glucose concentrations, resulting in reduced HBV replication. mTOR inhibition by rapamycin reversed negative effects of high glucose concentrations on HBV replication, suggesting that low glucose concentration promotes HBV replication by stimulating the AMPK/mTOR-ULK1-autophagy axis. Consistently, we found that glucose transporters inhibition using phloretin also enhanced HBV replication via increased AMPK/mTOR-ULK1-induced autophagy. Surprisingly, the glucose analogue 2-deoxy-D-glucose reduced HBV replication through activating the Akt/mTOR signalling pathway also at the low glucose concentrations. Our study reveals that glucose is an important factor for the HBV life cycle by regulating HBV transcription and posttranscriptional steps of HBV replication via cellular autophagy.

Residues Asn118 and Glu119 of hepatitis B virus X protein are critical for HBx-mediated inhibition of RIG-I-MAVS signaling.

Hepatitis B virus (HBV) X protein (HBx) has been reported to counteract the innate immune responses through interfering with the pattern recognition receptors signaling activated by retinoic acid-inducible gene-I (RIG-I)-mitochondrial antiviral signaling protein (MAVS). Here, we showed that, compared to the HBx derived from genotype (gt) A, C and D, HBx of gtB exhibited more potent inhibitory activity on the RIG-I-MAVS-mediated interferon-ß promoter activation. Functional analysis of the genotype-associated differences in amino acid sequence and the reciprocal mutation experiments in transient-transfection and infection cell models revealed that HBx with asparagine (N) and glutamic acid (E) at 118-119 positions inhibited RIG-I signaling and interacted with MAVS more efficiently than that with lysine (K) and aspartic acid (D). An impaired RIG-I-induced MAVS aggregation was observed in the presence of HBx-118N119E while MAVS-TRAF3 interaction was not affected. These results implicated that HBx gene heterogeneity may affect the innate immune responses to HBV infection.

Label-Free Proteomic Analysis of Exosomes Secreted from THP-1-Derived Macrophages Treated with IFN-α Identifies Antiviral Proteins Enriched in Exosomes.

Exosomes are extracellular vesicles that function in intercellular communication. We have previously reported that exosomes play an important role in the transmission of antiviral molecules during interferon-α (IFN-α) treatment. In this study, the protein profiles of THP-1-derived macrophages with or without interferon-α treatment and the exosomes secreted from these cells were analyzed by label-free liquid chromatography-tandem mass spectrometry quantitation technologies. A total of 1845 and 1550 protein groups were identified in the THP-1 macrophages and the corresponding exosomes, respectively. Treating the cells with IFN-α resulted in the differential abundance of 94 proteins in cells and 67 proteins in exosomes (greater than 2.0-fold), among which 23 proteins were up-regulated in both the IFN-α treated cells and corresponding exosomes, while 14 proteins were specifically up-regulated in exosomes but not in the donor cells. GO and KEGG analysis of the identified proteins suggested that IFN-α promoted the abundance of proteins involved in the "defense response to virus" and "type I interferon signaling pathway" in both exosomes and cells. Functional analysis further indicated that exosomes from IFN-α-treated cells exhibited potent antiviral activity that restored the impaired antiviral response of IFN-α in hepatitis B virus-replicating hepatocytes. These results have deepened the understanding of the exosome-mediated transfer of IFN-α-induced antiviral molecules and may provide a new basis for therapeutic strategies to control viral infection.

Exosomes Exploit the Virus Entry Machinery and Pathway To Transmit Alpha Interferon-Induced Antiviral Activity.

Alpha interferon (IFN-α) induces the transfer of resistance to hepatitis B virus (HBV) from liver nonparenchymal cells (LNPCs) to hepatocytes via exosomes. However, little is known about the entry machinery and pathway involved in the transmission of IFN-α-induced antiviral activity. In this study, we found that macrophage exosomes uniquely depend on T cell immunoglobulin and mucin receptor 1 (TIM-1), a hepatitis A virus (HAV) receptor, to enter hepatocytes for delivering IFN-α-induced anti-HBV activity. Moreover, two primary endocytic routes for virus infection, clathrin-mediated endocytosis (CME) and macropinocytosis, collaborate to permit exosome entry and anti-HBV activity transfer. Subsequently, lysobisphosphatidic acid (LBPA), an anionic lipid closely related to endosome penetration of virus, facilitates membrane fusion of exosomes in late endosomes/multivesicular bodies (LEs/MVBs) and the accompanying exosomal cargo uncoating. Together, our findings provide comprehensive insights into the transmission route of macrophage exosomes to efficiently deliver IFN-α-induced antiviral substances and highlight the similarities between the entry mechanisms of exosomes and virus.IMPORTANCE Our previous study showed that LNPC-derived exosomes could transmit IFN-α-induced antiviral activity to HBV replicating hepatocytes, but the concrete transmission mechanisms, which include exosome entry and exosomal cargo release, remain unclear. In this study, we found that virus entry machinery and pathway were also applied to exosome-mediated cell-to-cell antiviral activity transfer. Macrophage-derived exosomes distinctively exploit hepatitis A virus receptor for access to hepatocytes. Later, CME and macropinocytosis are utilized by exosomes, followed by exosome-endosome fusion for efficient transfer of IFN-α-induced anti-HBV activity. We believe that understanding the cellular entry pathway of exosomes will be beneficial to designing exosomes as efficient vehicles for antiviral therapy.

Identification of Retinoic Acid Receptor Agonists as Potent Hepatitis B Virus Inhibitors via a Drug Repurposing Screen.

Currently available therapies for chronic hepatitis B virus (HBV) infection can efficiently reduce viremia but induce hepatitis B surface antigen (HBsAg) loss in very few patients; also, these therapies do not greatly affect the viral covalently closed circular DNA (cccDNA). To discover new agents with complementary anti-HBV effects, we performed a drug repurposing screen of 1,018 Food and Drug Administration (FDA)-approved compounds using HBV-infected primary human hepatocytes (PHH). Several compounds belonging to the family of retinoic acid receptor (RAR) agonists were identified that reduced HBsAg levels in a dose-dependent manner without significant cytotoxicity. Among them, tazarotene exhibited the most potent anti-HBV effect, with a half-maximal inhibitory concentration (IC50) for HBsAg of less than 30 nM in PHH. The inhibitory effect was also observed in HBV-infected differentiated HepaRG (dHepaRG) models, but not in HepG2.215 cells, and HBV genotypes A to D were similarly inhibited. Tazarotene was further demonstrated to repress HBV cccDNA transcription, as determined by the levels of HBV cccDNA and RNAs and the activation of HBV promoters. Moreover, RNA sequence analysis showed that tazarotene did not induce an interferon response but altered the expression of a number of genes associated with RAR and metabolic pathways. Inhibition of RARß, but not RARα, by a specific antagonist significantly attenuated the anti-HBV activity of tazarotene, suggesting that tazarotene inhibits HBV in part through RARß. Finally, a synergistic effect of tazarotene and entecavir on HBV DNA levels was observed. Therefore, RAR agonists as represented by tazarotene were identified as potential novel anti-HBV agents.

In vitro studies identify a low replication phenotype for hepatitis B virus genotype H generally associated with occult HBV and less severe liver disease.

Hepatitis B virus (HBV) exists as 9 major genotypes and multiple subtypes, many of which exhibit differences in pathogenicity and treatment response. Genotype H identified in Central America is associated with low incidence of liver disease and HCC, but higher incidence of occult HBV (low level HBV DNA positivity, HBsAg negative). The replication phenotype of genotype H associated with less severe forms of liver disease is unknown. We hypothesized that the reduced pathogenesis associated with this genotype may be due to by lower rates of viral replication and/or secretion compared to other characterised strains. We used transient transfection and infection cell culture models to characterise the replication phenotype, compared to our D3 reference strain. Genotype H exhibited reduced viral replication and altered envelope protein expression compared to genotype D, with functional studies showing that low replication was in part likely due to sequence differences in the major transcriptional regulatory region.