Chromosome remodeling related to hepatitis B virus replication in HepG2 cells

Hepatitis B Virus (HBV) covalently closed circular DNA (cccDNA) is the main replicative intermediate of HBV and is organized into minichromosomes by the interaction with histone and nonhistone proteins. The remodeling of HBV minichromosomes such as post-translational modifications of histone proteins plays an important role in regulating HBV replication. To determine whether other remodeling occurs in addition to acetylation of cccDNA-bound H3 histones in the presence of HBV replication, a cell culture replication model of HBV was used to assess the dynamic status of acetylation, phosphorylation, and methylation of cccDNA-bound H3 histones at various times after transient transfection of linear HBV DNA into human hepatoma, HepG2 cells. H3 histones bound to cccDNA were found to be phosphorylated, mono-methylated, and acetylated in HepG2 cells containing replicating HBV. The acetylation and methylation status of H3 histones bound to cccDNA paralleled HBV replication. Our results demonstrate that phosphorylation and methylation occur in the remodeling of HBV minichromosomes during HBV replication. The modifications of cccDNA-bound H3 histones were associated with the level of HBV replication. These findings suggest that alterations in the extent of minichromosome remodeling might be a potential target to inhibit HBV replication in the development of effective novel antiviral agents.     

Inhibition of Hepatitis B Virus cccDNA by siRNA in Transgenic Mice

The elimination of viral covalently closed circular DNA (cccDNA) from the nucleus of infected hepatocytes is an obstacle to achieving sustained viral clearance during antiviral therapy of chronic hepatitis B virus (HBV) infection. The aim of our study was to determine whether treatment with siRNA is able to suppress viral cccDNA amplification using a HBV-transgenic mice model. The experimental results revealed that siRNAs can serve as efficient alternative anti-HBV agents, because they showed better inhibitory effect on viral replication and antigen expression in transgenic mice. More importantly, the siRNA markedly inhibited HBV cccDNA amplification.     

Small Molecular Inhibitors of DNA Double Strand Break Repair Pathways Increase the ANTI-HBV Activity of CRISPR/Cas9

Molecular Biology - The CRISPR/Cas9 nuclease system can effectively suppress the replication of the hepatitis B virus (HBV), while covalently closed circular DNA (cccDNA), a highly resistant form...     

AAV-Mediated Delivery of Zinc Finger Nucleases Targeting Hepatitis B Virus Inhibits Active Replication

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.     

化学发光Southern blot法检测HBV体外复制及其应用于药物对HBV的抑制分析

目的：建立化学发光Southern blot检测细胞内HBV DNA的方法,同时检测3种不同靶点抗乙肝药物的体外作用。方法：用地高辛标记HBV探针,优化杂交条件,检测来自HepG2及HepG2.2.15 HBV DNA复制中间体;利用建立的化学发光Southern blot检测HBV DNA的方法检测经拉米夫定、Bay41-4109、α-Galcer以不同药物浓度处理的HepG2.2.15HBV DNA复制中间体的水平。结果：（1）标记的HBV探针的检测灵敏度为0.1pg,杂交系统的检测灵敏度为1pg,可检测到HepG2.2.15细胞内的HBV DNA特异性信号;（2）以该法检测胞内HBV DNA可见3种药物都有明显的抑制作用,其半数有效量（IC50）分别为1.53μmol/L、0.41μmol/L、0.01μmol/L。结论：胞质HBV DNA的水平能准确地反映不同靶点抗HBV药物的抗病毒效果,建议在观察药物特别是中药抗病毒研究中采用。     

Spinoculation Enhances HBV Infection in NTCP-Reconstituted Hepatocytes

Hepatitis B virus (HBV) infection and its sequelae remain a major public health burden, but both HBV basic research and the development of antiviral therapeutics have been hindered by the lack of an efficient in vitro infection system. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the HBV receptor. We herein report that we established a NTCP-complemented HepG2 cell line (HepG2-NTCP12) that supports HBV infection, albeit at a low infectivity level following the reported infection procedures. In our attempts to optimize the infection conditions, we found that the centrifugation of HepG2-NTCP12 cells during HBV inoculation (termed “spinoculation”) significantly enhanced the virus infectivity. Moreover, the infection level gradually increased with accelerated speed of spinoculation up to 1,000g tested. However, the enhancement of HBV infection was not significantly dependent upon the duration of centrifugation. Furthermore, covalently closed circular (ccc) DNA was detected in infected cells under optimized infection condition by conventional Southern blot, suggesting a successful establishment of HBV infection after spinoculation. Finally, the parental HepG2 cells remained uninfected under HBV spinoculation, and HBV entry inhibitors targeting NTCP blocked HBV infection when cells were spinoculated, suggesting the authentic virus entry mechanism is unaltered under centrifugal inoculation. Our data suggest that spinoculation could serve as a standard protocol for enhancing the efficiency of HBV infection in vitro.     

Alpha/beta interferon differentially modulates the clearance of cytoplasmic encapsidated replication intermediates and nuclear covalently closed circular hepatitis B virus (HBV) DNA from the livers of hepatocyte nuclear factor 1alpha-null HBV transgenic mice

Treatment with alpha interferon is a standard therapy for patients with chronic hepatitis B virus (HBV) infections. This treatment can reduce virus load and ameliorate disease symptoms. However, in the majority of cases, alpha interferon therapy fails to resolve the chronic HBV infection. The reason alpha interferon therapy is inefficient at resolving chronic HBV infections is assumed to be because it fails to eliminate covalently closed circular (CCC) HBV DNA from the nuclei of infected hepatocytes. In an attempt to address this issue, the stability of HBV CCC DNA in response to alpha/beta interferon induction was examined in HNF1alpha-null HBV transgenic mice. Alpha/beta interferon induction by polyinosinic-polycytidylic acid [poly(I-C)] treatment efficiently eliminated encapsidated cytoplasmic HBV replication intermediates while only modestly reducing nuclear HBV CCC DNA. These observations indicate that nuclear HBV CCC DNA is more stable than cytoplasmic replication intermediates in response to alpha/beta interferon induction. Consequently it appears that for therapies to resolve chronic HBV infection efficiently, they will have to target the elimination of the most stable HBV replication intermediate, nuclear HBV CCC DNA.     

Baculovirus Mediated Production of Infectious Hepatitis C Virus in Human Hepatoma Cells Stably Expressing T7 RNA Polymerase

Although much has been learned about Hepatitis C virus (HCV), research progress has been hindered by the lack of a suitable cell culture system supporting its replication. Recently, a unique HCV strain JFH1 has been found to replicate efficiently in cell culture with production of infectious HCV (HCVcc). Baculovirus vectors were found to be efficient delivery vehicles and a HBV recombinant baculovirus/HepG2 system efficiently delivered the HBV genome into HepG2 resulting in HBV replication. In this study, we developed a recombinant baculovirus expression system to generate infectious HCV particles in hepatoma cell line Huh7-lunetT7 by using cDNA from the HCV JFH1 genotype. Results show that HCV positive, negative RNA strands and proteins were produced in this system. Furthermore, HCV particles were produced and secreted into the culture medium. Sucrose density gradient centrifugation of the culture medium revealed co-localization of HCV RNA and structural proteins in the fraction with a density of 1.08–1.13 g/ml. Electron microscopy (EM) showed viral particles approximately 55 nm in diameter, which could be recognized by anti-HCV E2 antibodies. Real-time RT-PCR detected that the level of HCV vRNA in the supernatant was 10⁷ copies/ml at 72 h post-transduction (hpt). In addition, the JFH1 virus produced by the recombinant baculovirus was confirmed to be infectious in vitro. In summary, this system provides a novel tool not only for the analysis of the replication and pathogenesis of HCV but also to screen for potential therapeutic targets.