Fang Shen

HBsAg inhibits TLR9-mediated activation and IFN-α production in plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs), the professional producers of type I interferons (IFN-alpha/beta), play a pivotal role in innate and adaptive immune responses against viral infections. Although functional impairment of circulating pDCs in chronic hepatitis B (CHB) patients has been reported previously, the mechanism responsible for these defects remains unclear. We hypothesize that HBsAg circulating in high amounts during HBV infection may interact with pDC and contribute to pDC dysfunction. In support of this hypothesis we show that pDCs treated with HBsAg secreted much less IFN-alpha than control pDCs. Furthermore, suppression is specific for TLR9, with no effects upon TLR7-mediated IFN-alpha secretion. HBsAg inhibited TLR9-mediated IRF-7 expression and nuclear translocation, which are important for induction of IFN-alpha gene transcription. HBsAg upregulated the SOCS-1 expression and bound to BDCA-2 receptors on the plasma membrane of pDCs, resulting in the inhibition of the IFN-alpha production. In conclusion, the above data suggested that HBsAg may directly interfere with the function of pDC through HBsAg-mediated upregulation of SOCS-1 expression and BDCA-2 ligation, which could partially explain how HBV evades the immune system to establish a persistent infection.

Expression profiles and function of Toll-like receptors 2 and 4 in peripheral blood mononuclear cells of chronic hepatitis B patients.

Toll-like receptors (TLRs) play a central role in sensing and initiating innate antiviral response. In this study, we first investigated the expression of TLR1-10 mRNA transcripts in peripheral blood mononuclear cells (PBMCs) from chronic HBV-infected (CHB) patients and healthy donors by quantitative real-time PCR. The expression of TLR1, TLR2, TLR4 and TLR6 transcripts was significantly lower in PBMCs from CHB patients, and the down-regulation of TLR2 was related to HBV genotype C. Flow cytometric analysis showed that the expression of TLR2 on PBMCs was significantly decreased in CHB patients. Furthermore, impaired cytokine production was observed in PBMCs from CHB patients after challenged with TLR2 and TLR4 ligands and was correlated with the levels of plasma hepatitis B virus surface antigen (HBsAg). In conclusion, our study reveals a possible interaction between HBsAg, TLR signaling and the innate immune response, which may partially explain the mechanism of HBV infection induced immuno-tolerance.

Heteroaryldihydropyrimidine (HAP) and Sulfamoylbenzamide (SBA) Inhibit Hepatitis B Virus Replication by Different Molecular Mechanisms

Heteroaryldihydropyrimidine (HAP) and sulfamoylbenzamide (SBA) are promising non-nucleos(t)ide HBV replication inhibitors. HAPs are known to promote core protein mis-assembly, but the molecular mechanism of abnormal assembly is still elusive. Likewise, the assembly status of core protein induced by SBA remains unknown. Here we show that SBA, unlike HAP, does not promote core protein mis-assembly. Interestingly, two reference compounds HAP_R01 and SBA_R01 bind to the same pocket at the dimer-dimer interface in the crystal structures of core protein Y132A hexamer. The striking difference lies in a unique hydrophobic subpocket that is occupied by the thiazole group of HAP_R01, but is unperturbed by SBA_R01. Photoaffinity labeling confirms the HAP_R01 binding pose at the dimer-dimer interface on capsid and suggests a new mechanism of HAP-induced mis-assembly. Based on the common features in crystal structures we predict that T33 mutations generate similar susceptibility changes to both compounds. In contrast, mutations at positions in close contact with HAP-specific groups (P25A, P25S, or V124F) only reduce susceptibility to HAP_R01, but not to SBA_R01. Thus, HAP and SBA are likely to have distinctive resistance profiles. Notably, P25S and V124F substitutions exist in low-abundance quasispecies in treatment-naïve patients, suggesting potential clinical relevance.

Development and validation of a liquid chromatography–mass spectrometry metabonomic platform in human plasma of liver failure caused by hepatitis B virus

This paper presents an liquid chromatography (LC)/mass spectrometry (MS)-based metabonomic platform that combined the discovery of differential metabolites through principal component analysis (PCA) with the verification by selective multiple reaction monitoring (MRM). These methods were applied to analyze plasma samples from liver disease patients and healthy donors. LC-MS raw data (about 1000 compounds), from the plasma of liver failure patients (n = 26) and healthy controls (n = 16), were analyzed through the PCA method and a pattern recognition profile that had significant difference between liver failure patients and healthy controls (P < 0.05) was established. The profile was verified in 165 clinical subjects. The specificity and sensitivity of this model in predicting liver failure were 94.3 and 100.0%, respectively. The differential ions with m/z of 414.5, 432.0, 520.5, and 775.0 were verified to be consistent with the results from PCA by MRM mode in 40 clinical samples, and were proved not to be caused by the medicines taken by patients through rat model experiments. The compound with m/z of 520.5 was identified to be 1-Linoleoylglycerophosphocholine or 1-Linoleoylphosphatidylcholine through exact mass measurements performed using Ion Trap-Time-of-Flight MS and METLIN Metabolite Database search. In all, it was the first time to integrate metabonomic study and MRM relative quantification of differential peaks in a large number of clinical samples. Thereafter, a rat model was used to exclude drug effects on the abundance of differential ion peaks. 1-Linoleoylglycerophosphocholine or 1-Linoleoylphosphatidylcholine, a potential biomarker, was identified. The LC/MS-based metabonomic platform could be a powerful tool for the metabonomic screening of plasma biomarkers.

Characterization of Nucleosome Positioning in Hepadnaviral Covalently Closed Circular DNA Minichromosomes

ABSTRACT Hepadnaviral covalently closed circular DNA (cccDNA) exists as an episomal minichromosome in the nucleus of virus-infected hepatocytes, and serves as the transcriptional template for the synthesis of viral mRNAs. To obtain insight on the structure of hepadnaviral cccDNA minichromosomes, we utilized ducks infected with the duck hepatitis B virus (DHBV) as a model and determined the in vivo nucleosome distribution pattern on viral cccDNA by the micrococcal nuclease (MNase) mapping and genome-wide PCR amplification of isolated mononucleosomal DHBV DNA. Several nucleosome-protected sites in a region of the DHBV genome [nucleotides (nt) 2000 to 2700], known to harbor various cis transcription regulatory elements, were consistently identified in all DHBV-positive liver samples. In addition, we observed other nucleosome protection sites in DHBV minichromosomes that may vary among individual ducks, but the pattern of MNase mapping in those regions is transmittable from the adult ducks to the newly infected ducklings. These results imply that the nucleosomes along viral cccDNA in the minichromosomes are not random but sequence-specifically positioned. Furthermore, we showed in ducklings that a significant portion of cccDNA possesses a few negative superhelical turns, suggesting the presence of intermediates of viral minichromosomes assembled in the liver, where dynamic hepatocyte growth and cccDNA formation occur. This study supplies the initial framework for the understanding of the overall complete structure of hepadnaviral cccDNA minichromosomes.

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).

Discovery and Pre-Clinical Characterization of Third-Generation 4-H Heteroaryldihydropyrimidine (HAP) Analogues as Hepatitis B Virus (HBV) Capsid Inhibitors

Described herein are the discovery and structure-activity relationship (SAR) studies of the third-generation 4-H heteroaryldihydropyrimidines (4-H HAPs) featuring the introduction of a C6 carboxyl group as novel HBV capsid inhibitors. This new series of 4-H HAPs showed improved anti-HBV activity and better drug-like properties compared to the first- and second-generation 4-H HAPs. X-ray crystallographic study of analogue 12 (HAP_R01) with Cp149 Y132A mutant hexamer clearly elucidated the role of C6 carboxyl group played for the increased binding affinity, which formed strong hydrogen bonding interactions with capsid protein and coordinated waters. The representative analogue 10 (HAP_R10) was extensively characterized in vitro (ADMET) and in vivo (mouse PK and PD) and subsequently selected for further development as oral anti-HBV infection agent.

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).

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.