Jieliang Chen

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.

Hepatitis B virus polymerase inhibits RIG-I- and Toll-like receptor 3-mediated beta interferon induction in human hepatocytes through interference with interferon regulatory factor 3 activation and dampening of the interaction between TBK1/IKKε and DDX3

Hepatitis B virus (HBV) infection remains one of the most serious health problems worldwide. Whilst studies have shown that HBV impairs interferon (IFN) production from dendritic cells in chronic hepatitis B patients, it remains unknown whether HBV inhibits IFN production in human hepatocytes. Using transient transfection assays in a primary human hepatocyte cell line (PH5CH8), this study demonstrated that HBV polymerase inhibits IFN-beta promoter activity induced by Newcastle disease virus, Sendai virus or poly(I : C) in a dose-dependent manner, whilst ectopic expression of the HBV core and X proteins had no effect on IFN-beta promoter activity. In addition, HBV polymerase blocked cellular IFN-beta expression and consequent antiviral immunity revealed by an infection protection assay. Furthermore, overexpression of key molecules on the IFN-beta induction axis, together with HBV polymerase, resulted in a block of IFN-beta promoter activity triggered by RIG-I, IPS-1, TRIF, TBK1 and IKKepsilon, but not by an IFN regulatory factor 3 dominant-positive mutant (IRF3-5D), suggesting that HBV polymerase prevents IFN-beta expression at the TBK1/IKKepsilon level. Further studies showed that HBV polymerase inhibited phosphorylation, dimerization and nuclear translocation of IRF3, in response to Sendai virus infection. Finally, it was shown that HBV polymerase-mediated dampening of the interaction between TBK1/IKKepsilon and DDX3 may be involved in the inhibitory effect on IFN-beta induction. Taken together, these findings reveal a novel role of HBV polymerase in HBV counteraction of IFN-beta production in human hepatocytes.

An Efficient Antiviral Strategy for Targeting Hepatitis B Virus Genome Using Transcription Activator-Like Effector Nucleases

The hepatitis B virus (HBV) is a DNA virus that can cause chronic hepatitis B (CHB) in humans. Current therapies for CHB infection are limited in efficacy and do not target the pre-existing viral genomic DNA, which are present in the nucleus as a covalently closed circular DNA (cccDNA) form. The transcription activator-like (TAL) effector nucleases (TALENs) are newly developed enzymes that can cleave sequence-specific DNA targets. Here, TALENs targeting the conserved regions of the viral genomic DNA among different HBV genotypes were constructed. The expression of TALENs in Huh7 cells transfected with monomeric linear full-length HBV DNA significantly reduced the viral production of HBeAg, HBsAg, HBcAg, and pgRNA, resulted in a decreased cccDNA level and misrepaired cccDNAs without apparent cytotoxic effects. The anti-HBV effect of TALENs was further demonstrated in a hydrodynamic injection-based mouse model. In addition, an enhanced antiviral effect with combinations of TALENs and interferon-α (IFN-α) treatment was observed and expression of TALENs restored HBV suppressed IFN-stimulated response element-directed transcription. Taken together, these data indicate that TALENs can specifically target and successfully inactivate the HBV genome and are potently synergistic with IFN-α, thus providing a potential therapeutic strategy for treating CHB infection.

Superior removal of arsenic from water with zirconium metal-organic framework UiO-66

In this study, water stable zirconium metal-organic framework (UiO-66) has been synthesized and for the first time applied as an adsorbent to remove aquatic arsenic contamination. The as-synthesized UiO-66 adsorbent functions excellently across a broad pH range of 1 to 10, and achieves a remarkable arsenate uptake capacity of 303 mg/g at the optimal pH, i.e., pH = 2. To the best of our knowledge, this is the highest arsenate As(V) adsorption capacity ever reported, much higher than that of currently available adsorbents (5–280 mg/g, generally less than 100 mg/g). The superior arsenic uptake performance of UiO-66 adsorbent could be attributed to the highly porous crystalline structure containing zirconium oxide clusters, which provides a large contact area and plenty of active sites in unit space. Two binding sites within the adsorbent framework are proposed for arsenic species, i.e., hydroxyl group and benzenedicarboxylate ligand. At equilibrium, seven equivalent arsenic species can be captured by one Zr6 cluster through the formation of Zr-O-As coordination bonds.

Hepatitis B Virus Surface Antigen Selectively Inhibits TLR2 Ligand–Induced IL-12 Production in Monocytes/Macrophages by Interfering with JNK Activation

It is widely accepted that chronic hepatitis B virus (HBV) infection is the result of an ineffective antiviral immune response against HBV infection. Our previous study found that the hepatitis B surface Ag (HBsAg) was related to decreased cytokine production induced by the TLR2 ligand (Pam3csk4) in PBMCs from chronic hepatitis B patients. In this study, we further explored the mechanism involved in the inhibitory effect of HBsAg on the TLR2 signaling pathway. The results showed that both Pam3csk4-triggered IL-12p40 mRNA expression and IL-12 production in PMA-differentiated THP-1 macrophage were inhibited by HBsAg in a dose-dependent manner, but the production of IL-1β, IL-6, IL-8, IL-10, and TNF-α was not influenced. The Pam3csk4-induced activation of NF-κB and MAPK signaling were further examined. The phosphorylation of JNK-1/2 and c-Jun was impaired in the presence of HBsAg, whereas the degradation of IκB-α, the nuclear translocation of p65, and the phosphorylation of p38 and ERK-1/2 were not affected. Moreover, the inhibition of JNK phosphorylation and IL-12 production in response to Pam3csk was observed in HBsAg-treated monocytes/macrophages (M/MΦs) from the healthy donors and the PBMCs and CD14-positive M/MΦs from chronic hepatitis B patients. Taken together, these results demonstrate that HBsAg selectively inhibits Pam3csk4- stimulated IL-12 production in M/MΦs by blocking the JNK–MAPK pathway and provide a mechanism by which HBV evades immunity and maintains its persistence.

Hepatitis B virus polymerase disrupts K63-linked ubiquitination of STING to block innate cytosolic DNA-sensing pathways

ABSTRACT The cellular innate immune system recognizing pathogen infection is essential for host defense against viruses. In parallel, viruses have developed a variety of strategies to evade the innate immunity. The hepatitis B virus (HBV), a DNA virus that causes chronic hepatitis, has been shown to inhibit RNA helicase RIG-I-mediated interferon (IFN) induction. However, it is still unknown whether HBV could affect the host DNA-sensing pathways. Here we report that in transiently HBV-transfected Huh7 cells, the stably HBV-producing cell line HepAD38, and HBV-infected HepaRG cells and primary human hepatocytes, HBV markedly interfered with IFN-β induction and antiviral immunity mediated by the stimulator of interferon genes (STING), which has been identified as a central factor in foreign DNA recognition and antiviral innate immunity. Screening analysis demonstrated that the viral polymerase (Pol), but not other HBV-encoded proteins, was able to inhibit STING-stimulated interferon regulatory factor 3 (IRF3) activation and IFN-β induction. Moreover, the reverse transcriptase (RT) and the RNase H (RH) domains of Pol were identified to be responsible for the inhibitory effects. Furthermore, Pol was shown to physically associate with STING and dramatically decrease the K63-linked polyubiquitination of STING via its RT domain without altering the expression level of STING. Taken together, these observations suggest that besides its inherent catalytic function, Pol has a role in suppression of IFN-β production by direct interaction with STING and subsequent disruption of its K63-linked ubiquitination, providing a new mechanism for HBV to counteract the innate DNA-sensing pathways. IMPORTANCE Although whether and how HBV infection induces the innate immune responses are still controversial, it has become increasingly clear that HBV has developed strategies to counteract the pattern recognition receptor-mediated signaling pathways. Previous studies have shown that type I IFN induction activated by the host RNA sensors could be inhibited by HBV. However, it remains unknown whether HBV as a DNA virus utilizes evasion mechanisms against foreign DNA-elicited antiviral signaling. In recent years, the cytosolic DNA sensor and key adaptor STING has been demonstrated to be essential in multiple foreign DNA-elicited innate immune signalings. Here, for the first time, we report STING as a new target of HBV to antagonize IFN induction and identify the viral polymerase responsible for the inhibitory effect, thus providing an additional molecular mechanism by which HBV evades the innate immunity; this implies that in addition to its inherent catalytic function, HBV polymerase is a multifunctional immunomodulatory protein.

Hepatitis B virus polymerase impairs interferon-α-induced STA T activation through inhibition of importin-α5 and protein kinase C-δ.

Treatment with exogenous interferon (IFN)‐α is not effective in the majority of patients with chronic hepatitis B virus (HBV) infection. Recent evidence suggests that HBV has evolved strategies to block the nuclear translocation of signal transducer and activator of transcription (STAT) 1 to limit IFN‐α–induced cellular antiviral responses. However, it remains unclear whether STAT1 translocation is impaired in chronic hepatitis B patients and what mechanisms are involved. Here we report that the expression of HBV polymerase (Pol) in human hepatic cell lines inhibited induction of IFN‐stimulated genes and resulted in a weakened antiviral activity of IFN‐α. Ectopic expression of Pol suppressed IFN‐α–induced STAT1 serine 727 phosphorylation and STAT1/2 nuclear accumulation, whereas STAT1 tyrosine 701 phosphorylation, and STAT1‐STAT2 heterodimer formation were not affected. Further studies demonstrated that Pol interacted with the catalytic domain of protein kinase C‐δ (PKC‐δ) and perturbed PKC‐δ phosphorylation and its association with STAT1, which resulted in the suppression of STAT1 Ser727 phosphorylation. Moreover, Pol was found to interfere with nuclear transportation of STAT1/2 by competitively binding to the region of importin‐α5 required for STAT1/2 recruitment. Truncation analysis suggested that the terminal protein and RNase H domains of Pol were able to bind to PKC‐δ and importin‐α5, respectively, and were responsible for the inhibition of IFN‐α signaling. More importantly, the inhibition of STAT1 and PKC‐δ phosphorylation were confirmed in a hydrodynamic‐based HBV mouse model, and the blockage of IFN‐α–induced STAT1/2 nuclear translocation was observed in HBV‐infected cells from liver biopsies of chronic HBV patients. Conclusions: These results demonstrate a role for Pol in HBV‐mediated antagonization of IFN‐α signaling and provide a possible molecular mechanism by which HBV resists the IFN therapy and maintains its persistence. (HEPATOLOGY 2013;)

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).

Interplay between hepatitis B virus and the innate immune responses：implications for new therapeutic strategies

Hepatitis B virus (HBV) infection is still a worldwide health problem; however, the current antiviral therapies for chronic hepatitis B are limited in efficacy. The outcome of HBV infection is thought to be the result of complex interactions between the HBV and the host immune system. While the role of the adaptive immune responses in the resolution of HBV infection has been well characterized, the contribution of innate immune mechanisms remains elusive until recent evidence implicates that HBV appears to activate the innate immune response and this response is important for controlling HBV infection. Here, we review our current understanding of innate immune responses to HBV infection and the multifaceted evasion by the virus and discuss the potential strategies to combat chronic HBV infection via induction and restoration of host innate antiviral responses.

Hepatitis B virus spliced variants are associated with an impaired response to interferon therapy.

During hepatitis B virus (HBV) replication, spliced HBV genomes and splice-generated proteins have been widely described, however, their biological and clinical significance remains to be defined. Here, an elevation of the proportion of HBV spliced variants in the sera of patients with chronic hepatitis B (CHB) is shown to correlate with an impaired respond to interferon-α (IFN-α) therapy. Transfection of the constructs encoding the three most dominant species of spliced variants into cells or ectopic expression of the two major spliced protein including HBSP and N-terminal-truncated viral polymerase protein result in strong suppression of IFN-α signaling transduction, while mutation of the major splicing-related sites of HBV attenuates the viral anti-IFN activities in both cell and mouse models. These results have associated the productions of HBV spliced variants with the failure response to IFN therapy and illuminate a novel mechanism where spliced viral products are employed to resist IFN-mediated host defense.