Jiangxia Liu

Exosomes mediate the cell-to-cell transmission of IFN-α-induced antiviral activity

The cell-to-cell transmission of viral resistance is a potential mechanism for amplifying the interferon-induced antiviral response. In this study, we report that interferon-α (IFN-α) induced the transfer of resistance to hepatitis B virus (HBV) from nonpermissive liver nonparenchymal cells (LNPCs) to permissive hepatocytes via exosomes. Exosomes from IFN-α-treated LNPCs were rich in molecules with antiviral activity. Moreover, exosomes from LNPCs were internalized by hepatocytes, which mediated the intercellular transfer of antiviral molecules. Finally, we found that exosomes also contributed to the antiviral response of IFN-α to mouse hepatitis virus A59 and adenovirus in mice. Thus, we propose an antiviral mechanism of IFN-α activity that involves the induction and intercellular transfer of antiviral molecules via exosomes.

Subversion of Cellular Autophagy Machinery by Hepatitis B Virus for Viral Envelopment

ABSTRACT Autophagy is a conserved eukaryotic mechanism that mediates the removal of long-lived cytoplasmic macromolecules and damaged organelles via a lysosomal degradative pathway. Recently, a multitude of studies have reported that viral infections may have complex interconnections with the autophagic process. The findings reported here demonstrate that hepatitis B virus (HBV) can enhance the autophagic process in hepatoma cells without promoting protein degradation by the lysosome. Mutation analysis showed that HBV small surface protein (SHBs) was required for HBV to induce autophagy. The overexpression of SHBs was sufficient to induce autophagy. Furthermore, SHBs could trigger unfolded protein responses (UPR), and the blockage of UPR signaling pathways abrogated the SHB-induced lipidation of LC3-I. Meanwhile, the role of the autophagosome in HBV replication was examined. The inhibition of autophagosome formation by the autophagy inhibitor 3-methyladenine (3-MA) or small interfering RNA duplexes targeting the genes critical for autophagosome formation (Beclin1 and ATG5 genes) markedly inhibited HBV production, and the induction of autophagy by rapamycin or starvation greatly contributed to HBV production. Furthermore, evidence was provided to suggest that the autophagy machinery was required for HBV envelopment but not for the efficiency of HBV release. Finally, SHBs partially colocalized and interacted with autophagy protein LC3. Taken together, these results suggest that the hosts autophagy machinery is activated during HBV infection to enhance HBV replication.

PRMT5 restricts hepatitis B virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic RNA encapsidation.

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. The covalently closed circular DNA (cccDNA) minichromosome, which serves as the template for the transcription of viral RNAs, plays a key role in viral persistence. While accumulating evidence suggests that cccDNA transcription is regulated by epigenetic machinery, particularly the acetylation of cccDNA‐bound histone 3 (H3) and H4, the potential contributions of histone methylation and related host factors remain obscure. Here, by screening a series of methyltransferases and demethylases, we identified protein arginine methyltransferase 5 (PRMT5) as an effective restrictor of HBV transcription and replication. In cell culture–based models for HBV infection and in liver tissues of patients with chronic HBV infection, we found that symmetric dimethylation of arginine 3 on H4 on cccDNA was a repressive marker of cccDNA transcription and was regulated by PRMT5 depending on its methyltransferase domain. Moreover, PRMT5‐triggered symmetric dimethylation of arginine 3 on H4 on the cccDNA minichromosome involved an interaction with the HBV core protein and the Brg1‐based human SWI/SNF chromatin remodeler, which resulted in down‐regulation of the binding of RNA polymerase II to cccDNA. In addition to the inhibitory effect on cccDNA transcription, PRMT5 inhibited HBV core particle DNA production independently of its methyltransferase activity. Further study revealed that PRMT5 interfered with pregenomic RNA encapsidation by preventing its interaction with viral polymerase protein through binding to the reverse transcriptase–ribonuclease H region of polymerase, which is crucial for the polymerase–pregenomic RNA interaction. Conclusion: PRMT5 restricts HBV replication through a two‐part mechanism including epigenetic suppression of cccDNA transcription and interference with pregenomic RNA encapsidation; these findings improve the understanding of epigenetic regulation of HBV transcription and host–HBV interaction, thus providing new insights into targeted therapeutic intervention. (Hepatology 2017;66:398–415).

DNA-dependent activator of interferon-regulatory factors inhibits hepatitis B virus replication

AIM To investigate whether DNA-dependent activator of interferon-regulatory factors (DAI) inhibits hepatitis B virus (HBV) replication and what the mechanism is. METHODS After the human hepatoma cell line Huh7 was cotransfected with DAI and HBV expressing plasmid, viral protein (HBV surface antigen and HBV e antigen) secretion was detected by enzyme-linked immunosorbent assay, and HBV RNA was analyzed by real-time polymerase chain reaction and Northern blotting, and viral DNA replicative intermediates were examined by Southern blotting. Interferon regulatory factor 3 (IRF3) phosphorylation and nuclear translocation were analyzed via Western blotting and immunofluorescence staining respectively. Nuclear factor-κB (NF-κB) activity induced by DAI was detected by immunofluorescence staining of P65 and dual luciferase reporter assay. Transwell co-culture experiment was performed in order to investigate whether the antiviral effects of DAI were dependent on the secreted cytokines. RESULTS Viral protein secretion was significantly reduced by 57% (P < 0.05), and the level of total HBV RNA was reduced by 67% (P < 0.05). The viral core particle-associated DNA was also dramatically down-regulated in DAI-expressing Huh7 cells. Analysis of involved signaling pathways revealed that activation of NF-κB signaling was essential for DAI to elicit antiviral response in Huh7 cells. When the NF-κB signaling pathway was blocked by a NF-κB signaling suppressor (IκBα-SR), the anti-HBV activity of DAI was remarkably abrogated. The inhibitory effect of DAI was independent of IRF3 signaling and secreted cytokines. CONCLUSION This study demonstrates that DAI can inhibit HBV replication and the inhibitory effect is associated with activation of NF-κB but independent of IRF3 and secreted cytokines.

Hyper-activated IRF-1 and STAT1 contribute to enhanced Interferon stimulated gene (ISG) expression by Interferon α and γ co-treatment in human hepatoma cells

Abstract Previous reports suggest that type I and type II Interferon can co-operatively inhibit some virus replication, e.g. HCV, SARS-CoV, HSV-1. To find out the molecular mechanism underlying this phenomenon, we analyzed the transcription profile stimulated by IFN-α and IFN-γ in Huh-7 cells and found that the transcription of a subset of IFN stimulated genes (ISGs) including BclG, XAF1, TRAIL and TAP1 was enhanced when IFN-α and γ were both present. Promoter analysis of BclG revealed that IRF-1 and STAT1 were both required in this process. Enhanced IRF-1/DNA complex formation was observed in interferon co-treatment group by gel shift analysis. Furthermore, IRF-1 activation was found to be generally required in this cluster of ISGs. STAT1 tyrosine phosphorylation was elevated by IFN combination treatment, however, only the hyper-transactivation of GAS but not ISRE was observed. In conclusion, hyper-activation of IRF-1 and elevated STAT1 dimer formation may be two general switches which contribute to a much more robust antiviral symphony against virus replication when type I and type II IFNs are co-administered.

Extracellular HBV RNAs are heterogeneous in length and circulate as virions and capsid-antibody-complexes in chronic hepatitis B patients

Extracellular HBV RNA has been detected in both HBV-replicating cell culture media and sera from chronic hepatitis B (CHB) patients, but its exact origin and composition remain controversial. Here, we demonstrated that extracellular HBV RNA species were of heterogeneous lengths, ranging from the length of pregenomic RNA to a few hundred nucleotides. In cell models, these RNAs were predominantly associated with naked capsids although virions also harbored a minority of them. Moreover, HBV RNAs in hepatitis B patients’ blood circulation were localized in unenveloped capsids in the form of capsid-antibody-complexes (CACs) and in virions. Furthermore, we showed that extracellular HBV RNAs could serve as template for viral DNA synthesis. In conclusion, extracellular HBV RNAs mainly consist of pgRNA or the pgRNA species degraded by the RNase H domain of the polymerase in the process of viral DNA synthesis and circulate as CACs and virions. Their presence in the blood circulation of CHB patients may be exploited to develop novel biomarkers for HBV persistence.

Hepatitis B Virus Sensitivity to Interferon-α in Hepatocytes is More Associated with Cellular Interferon Response than with Viral Genotype

Interferon‐α (IFN‐α) is used to treat chronic hepatitis B virus (HBV) infection, but only 20%‐40% of patients respond well. Clinical observations have suggested that HBV genotype is associated with the response to IFN therapy; however, its role in viral responsiveness to IFN in HBV‐infected hepatocytes remains unclear. Here, we produced infectious virions of HBV genotypes A to D to infect three well‐recognized cell–culture–based HBV infection systems, including primary human hepatocytes (PHH), differentiated HepaRG (dHepaRG), and HepG2‐NTCP cells to quantitatively compare the antiviral effect of IFN‐α on HBV across genotypes and cell models. The efficacy of IFN‐α against HBV in hepatocytes was generally similar across genotypes A2, B5, C2, and D3; however, it was significantly different among the infection models given that the half maximal inhibitory concentration value of IFN‐α for inhibition of viral DNA replication in PHH (<20 U/mL) and dHepaRG cells were much lower than that in HepG2‐NTCP cells (>500 U/mL). Notably, even in PHH, IFN‐α did not reduce HBV covalently closed circular DNA at the concentrations for which viral antigens and DNA replication intermediates were strongly reduced. The three cell‐culture models exhibited differential cellular response to IFN‐α. The genes reported to be associated with responsiveness to IFN‐α in patients were robustly induced in PHH while weakly induced in HepG2‐NTCP cells upon IFN‐α treatment. Reduction or promotion of IFN response in PHH or HepG2‐NTCP cells significantly attenuated or improved the inhibitory capacity of IFN‐α on HBV replication, respectively. Conclusion: In the cell–culture–based HBV infection models, the sensitivity of HBV to IFN‐α in hepatocytes is determined more by the cell‐intrinsic IFN response than by viral genotype, and improvement of the IFN response in HepG2‐NTCP cells promotes the efficacy of IFN‐α against HBV. (Hepatology 2018;67:1237‐1252).