Xuejun Wang

MicroRNA-141 represses HBV replication by targeting PPARA.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression primarily at the post-transcriptional level and play critical roles in a variety of physiological and pathological processes. In this report, miR-141 was identified to repress HBV expression by screening a small miRNA expressing library and synthetic miR-141 mimics could also significantly suppress HBV expression and replication in HepG2 cells. Bioinformatic analysis and experiment assays indicate that peroxisome proliferator-activated receptor alpha (PPARA) was the target of hsa-miR-141 during this process. Furthermore, knockdown of PPARA by small interfering RNA (siRNA) inhibited HBV replication similar to levels observed for miR-141. Promoter functional analysis indicated that repression of HBV replication by miR-141 mimics or siRNA was mediated by interfering with the HBV promoter functions, consistent with previous studies demonstrating that PPARA regulated HBV gene expression through interactions with HBV promoter regulatory elements. Our results suggest that miR-141 suppressed HBV replication by reducing HBV promoter activities by down-regulating PPARA. This study provides new insights into the molecular mechanisms associated with HBV-host interactions. Furthermore, this information may facilitate the development of novel anti-HBV therapeutic strategies.

Inhibition of Hepatitis B virus replication by phospholipid scramblase 1 in vitro and in vivo.

Human Phospholipid scramblase 1 (PLSCR1) is an α/β interferon-inducible protein that mediates antiviral activity against RNA viruses including vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV). In the present study, we investigated the antiviral activity of PLSCR1 protein against HBV (Hepatitis B virus). Firstly, PLSCR1 mRNA and protein expression was found to be downregulated in HepG2 cells after HBV infection. Then by performing co-transient-transfection experiments in cells and hydrodynamics-based transfection experiments in mice using a HBV expression plasmid and a PLSCR1 expression plasmid, we found that PLSCR1 inhibited HBV replication in vitro and in vivo through a significant reduction in the synthesis of viral proteins, DNA replicative intermediates and HBV RNAs. We also demonstrated that the antiviral action of PLSCR1 against HBV occurs, partly at least, by activating the Jak/Stat pathway. In conclusion, our results suggest that the expression of PLSCR1 is involved in HBV replication and that PLSCR1 has antiviral activity against HBV.

Irbesartan, an FDA approved drug for hypertension and diabetic nephropathy, is a potent inhibitor for hepatitis B virus entry by disturbing Na+-dependent taurocholate cotransporting polypeptide activity

The liver-specific Na(+)-dependent taurocholate cotransporting polypeptide (NTCP) was recently identified as an entry receptor for hepatitis B virus (HBV) hepatotropic infection. In this study, an NTCP-overexpressing HepG2 cell line named HepG2.N9 susceptible to HBV infection was established using transcription activator-like effector nucleases (TALEN) technology. Using this cell line, irbesartan, the new NTCP-interfering molecule reported recently, was demonstrated here to effectively inhibit HBV infection with an IC50 of 3.3μM for hepatitis B e antigen (HBeAg) expression and exhibited no obvious cytotoxicity up to 1000μM. Irbesartan suppressed HBV uptake weakly but inhibited HBV covalently closed circular DNA (cccDNA) formation efficiently at physiological temperature. These results suggested that irbesartan targeted HBV infection at a post-uptake prior to cccDNA formation step such as the cell membrane fusion. Based on these findings, irbesartan, an FDA approved drug for hypertension and diabetic nephropathy, could be a potential candidate for treatment of HBV infection although further in vivo experiments are required.

Inhibition of Dengue Virus 2 Replication by Artificial MicroRNAs Targeting the Conserved Regions

Dengue virus (DENV), a mosquito-borne flavivirus, causes serious diseases and threatens public health in tropical and subtropical areas worldwide. RNA interference (RNAi) is a prevailing strategy for antiviral therapy. In this paper, 6 single artificial microRNAs (amiRNAs) targeting the highly conserved regions of the DENV-2 genome were identified and inhibited virus replication efficiently. Then, effective tandem amiRNAs targeting 2 different DENV-2 genome regions were constructed and expressed simultaneously from a single microRNA-like polycistron to avoid virus variation or mutation escape. Finally, the most high-performance tandem amiRNA was embedded in a lenti-viral vector and inhibited DENV-2 virus replication stably and dose-dependently. Overall, these results indicated that RNAi based on multiple amiRNAs targeting viral conserved regions was an effective approach for improvements of nucleic acid inhibitors of DENV and provided a new therapeutic strategy for DENV infection in humans.

Fibronectin and asialoglyprotein receptor mediate hepatitis B surface antigen binding to the cell surface.

Both fibronectin and the asialoglycoprotein receptor (ASGPR) have been identified by some investigators as partners for hepatitis B virus (HBV) envelope proteins. Because fibronectin is a natural ligand for ASGPR, we speculated that HBV might attach to ASGPR expressed on the hepatocyte surface via fibronectin. To test this hypothesis, we first confirmed by co-immunoprecipitation that ASGPR, fibronectin and HBsAg bind to each other in HepG2.2.15 cells, and possible binding domains were identified by GST pull-down. In addition, by measuring binding of HBsAg to cells, we found that ASGPR and fibronectin enhanced the binding capability of HBsAg to HepG2 cells, and even to 293T and CHO cells, which normally do not bind HBV. In conclusion, our findings suggest that both fibronectin and ASGPR mediate HBsAg binding to the cell surface, which provides further evidence for the potential roles of these two proteins in mediating HBV binding to liver cells.

A Simple and Robust Vector-Based shRNA Expression System Used for RNA Interference

Background RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive. Results In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a>95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV) knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo. Conclusion This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

A simple and efficient strategy for the de novo construction of greater-than-genome-length hepatitis B virus replicons.

Hepatitis B virus (HBV) uses small covalently closed circular DNA for transcription and replication; linearization at any site would destruct at least one HBV open reading frame and interrupt the virus life cycle. Therefore, greater-than-genome-length (GGL) HBV replicons have been widely used for HBV replication studies. However, the existing strategies for the de novo construction of GGL HBV replicons are too complex to implement, especially when multiple replicons need to be cloned. In this study, the pHBV-basic plasmid was constructed for efficient cloning of GGL HBV replicons; changing the orientation of the site for type IIs restriction enzyme SapI in this plasmid reduced the de novo construction of various GGL HBV replicons to only one to three steps. Furthermore, Q5 high-fidelity DNA polymerase was found to be ideal for HBV genome amplification. In vitro experiments showed that the HBV replicon containing 1.31 genome copies replicated with better efficiency as evidenced by the titers of HBV DNA and HBsAg and HBeAg markers. The vector described in this study could serve as a powerful vehicle for in vitro and in vivo investigation of HBV replication.

CpG oligodeoxynucleotide inhibits HBV replication in a hydrodynamic injection murine model.

BACKGROUND Chronic HBV infection is a significant public health problem and one major cause of liver cirrhosis and hepatocellular carcinoma (HCC). HBV impairs the host immune system and results in immunotolerance, which is a major obstacle to HBV therapy. CpG oligodeoxynucleotide (ODN) is a strong immunostimulant which activates the innate immune response rapidly and has been shown to be an efficient therapy agent in viral infection treatment. Here, we report the anti-HBV activity of CpG-1826 in a hydrodynamic injection murine model. METHODS CpG-1826 was administrated intraperitoneally every other day in HBV carrier mice established by tail vein hydrodynamic injection of HBV plasmids. The serum concentrations of HBV surface antigen (HBsAg), HBV e antigen (HBeAg), HBV surface antibody (HBsAb), HBV core antibody (HBcAb), interferon-α (IFN-α) and interferon-γ (IFN-γ) were measured by enzyme-linked immunosorbent assay (ELISA). The activities of alanine aminotransferase (ALT) were determined by ALT kit using a Spectramax Plus spectrophotometer. Hepatic HBV DNA was quantified by quantitative real-time PCR. The expression of HBV core antigen (HBcAg) in liver was detected by immunohistochemistry. Drug toxicity of CpG-1826 was evaluated by body weighting and liver histopathology confirmation. RESULTS CpG-1826 administration inhibited HBV replication efficiently with significant reduction of serum HBsAg and HBeAg, hepatic HBcAg and HBV DNA levels. The serum levels of IFN-α, IFN-γ and HBsAb increased but the HBcAb level declined in the CpG-1826 group compared to CpG-1982 and PBS control groups. Results of ALT activity indicated no significant difference among CpG-1826 group, CpG-1982 and PBS control groups. Body weighting and histopathology examination showed no obvious toxicity. CONCLUSIONS Given the stimulation activity of a host immune system, CpG ODN is a promising strategy for HBV therapy with more relevant research needed.