SIRT3激动剂对乙肝病毒转录和复制的影响

该文探讨了SIRT3激动剂(Honokiol,HKL)对乙肝病毒(Hepatitis B virus,HBV)转录和复制的影响。培养HepG2-NTCP和人原代肝细胞(primary human hepatocytes,PHH),感染HBV颗粒后,用Honokiol(5μmol/L、10μmol/L、20μmol/L)处理细胞后继续培养10天,通过荧光定量PCR检测细胞内HBV DNA、cccDNA和HBV RNAs水平,Southern blot实验进一步检测胞内HBV DNA水平。构建SIRT3-KO细胞,检测敲除SIRT3后,Honokiol对细胞内HBV DNA、cccDNA和HBV RNAs的影响。通过小鼠尾静脉高压注射pCMV-KRAB-Cre质粒和precccDNA质粒构建持续感染小鼠模型,一周后腹腔注射Honokiol持续20天。荧光定量PCR检测小鼠血清中HBV DNA拷贝数,肝组织内HBV DNA、cccDNA和HBV RNAs水平。结果表明,Honokiol浓度依赖性地抑制HepG2-NTCP和PHH细胞内HBV DNA以及HBV RNAs水平,此外,Honokiol可以降低cccDNA的转录活性;敲除SIRT3后,Honokiol不能发挥抗病毒作用;小鼠模型中,Honokiol能够降低血清中HBV DNA和肝组织内HBV DNA拷贝数,以及能够显著抑制肝组织内HBV RNAs水平和cccDNA的转录活性。该研究结果表明,Honokiol能够抑制乙肝病毒转录和复制。     

Transforming growth factor-β1 suppresses hepatitis B virus replication by the reduction of hepatocyte nuclear factor-4α expression

Several studies have demonstrated that cytokine-mediated noncytopathic suppression of hepatitis B virus (HBV) replication may provide an alternative therapeutic strategy for the treatment of chronic hepatitis B infection. In our previous study, we showed that transforming growth factor-beta1 (TGF-β1) could effectively suppress HBV replication at physiological concentrations. Here, we provide more evidence that TGF-β1 specifically diminishes HBV core promoter activity, which subsequently results in a reduction in the level of viral pregenomic RNA (pgRNA), core protein (HBc), nucleocapsid, and consequently suppresses HBV replication. The hepatocyte nuclear factor 4alpha (HNF-4α) binding element(s) within the HBV core promoter region was characterized to be responsive for the inhibitory effect of TGF-β1 on HBV regulation. Furthermore, we found that TGF-β1 treatment significantly repressed HNF-4α expression at both mRNA and protein levels. We demonstrated that RNAi-mediated depletion of HNF-4α was sufficient to reduce HBc synthesis as TGF-β1 did. Prevention of HNF-4α degradation by treating with proteasome inhibitor MG132 also prevented the inhibitory effect of TGF-β1. Finally, we confirmed that HBV replication could be rescued by ectopic expression of HNF-4α in TGF-β1-treated cells. Our data clarify the mechanism by which TGF-β1 suppresses HBV replication, primarily through modulating the expression of HNF-4α gene.     

Induction of hepatitis B virus gene expression at low temperature

There is a limited understanding of the cellular regulation of HBV gene expression in differentiated hepatocytes. We previously demonstrated that HBV replication inversely correlates with cell proliferation and DNA synthesis. In this report, temperature-induced modulation of cell growth was used as a novel approach to study HBV gene expression in the absence of indirect effects from drugs or serum deprivation. We observed markedly elevated levels of hepatic HBV mRNA expression from integrated and episomal HBV DNA at 32 degrees C. Additionally, hepatoblastoma cells cultured at 32 degrees C expressed increased levels of albumin mRNA and decreased levels of c-myc mRNA, which demonstrates that liver-derived cells cultured at low temperature exhibit characteristics of functional and differentiated hepatocytes. In transiently transfected HepG2 cells cultured at 32 degrees C, the HBV enhancer 1 activated the X promoter and core/pregenomic promoter by 7.3- and 28-fold, respectively. In the absence of enhancer 1, core/pregenomic promoter activity was 2.4-fold higher than the X promoter in HepG2 cells at 32 degrees C. In contrast, enhancer 1 exclusively activated the X promoter in transfected non-liver cells at 32 degrees C. Therefore, the core/pregenomic promoter exhibits strict liver-specificity at low temperature. This work supports the hypothesis that HBV replication and gene expression are optimal in non-activated hepatocytes, and provides a novel system for delineating molecular aspects of the HBV replication process.     

Protective Role of Sirtuin3 (SIRT3) in Oxidative Stress Mediated by Hepatitis B Virus X Protein Expression

Background/Aim

The hepatitis B virus (HBV) infection is accompanied by the induction of oxidative stress, especially mediated by HBV X protein (HBx). Oxidative stress has been implicated in a series of pathological states, such as DNA damage, cell survival and apoptosis. However, the host factor by which cells protect themselves under this oxidative stress is poorly understood.

Methodology/Principal Findings

In this study, we first confirmed that HBV infection significantly induced oxidative stress. Moreover, viral protein HBx plays a major role in the oxidative stress induced by HBV. Importantly, we found that mitochondrial protein SIRT3 overexpression could decrease reactive oxygen species (ROS) induced by HBx while SIRT3 knockdown increased HBx-induced ROS. Importantly, SIRT3 overexpression abolished oxidative damage of HBx-expressing cells as evidenced by γH₂AX and AP sites measurements. In contrast, SIRT3 knockdown promoted HBx-induced oxidative damage. In addition, we also observed that oxidant H₂O₂ markedly promoted HBV replication while the antioxidant N-acetyl-L-cysteine (NAC) inhibited HBV replication. Significantly, SIRT3 overexpression inhibited HBV replication by reducing cellular ROS level.

Conclusions/Significance

Collectively, these data suggest HBx expression induces oxidative stress, which promotes cellular oxidative damage and viral replication during HBV pathogenesis. Mitochondrial protein SIRT3 protected HBx expressing-cells from oxidative damage and inhibited HBV replication possibly by decreased cellular ROS level. These studies shed new light on the physiological significance of SIRT3 on HBx-induced oxidative stress, which can contribute to the liver pathogenesis.     

Pregenomic RNA encapsidation analysis of eleven missense and nonsense polymerase mutants of human hepatitis B virus.

We characterized 11 DNA polymerase mutants of human hepatitis B virus (HBV) which contain single missense or nonsense mutations in the various domains within this gene. Except for mutant 738, a tight association between DNA replication and RNA packaging of these missense pol mutants was observed. Further analysis of HBV core particle-associated RNA indicated that only the 3.5-kb core-specific RNA, but not the precore-specific RNA, is selectively packaged in this tissue culture system. Previously, we have demonstrated that only the 3.5-kb core-specific RNA can serve as an efficient template for pol translation. Taken together, our results suggest that selectivity of HBV RNA packaging occurs as a result of selective translation of pol-containing mRNAs. Furthermore, our data suggest that the RNA encapsidation domain of pol overlaps with all of the domains of pol involved in the synthesis of terminal protein, as well as DNA replication. Finally, on the basis of gradient centrifugation analysis, a pol defect appeared to have no negative effect on the assembly or stability of core particles. A new method to assay RNA encapsidation, as well as potential RNase H activity, is reported.     

ATP1B3 Restricts Hepatitis B Virus Replication Via Reducing the Expression of the Envelope Proteins

Our recent study reported that ATP1B3 inhibits hepatitis B virus(HBV) replication via inducing NF-κB activation.However, ATP1B3 mutants which were defective in NF-κB activation still maintained the moderate degree of suppression on HBV replication, suggesting that another uncharacterized mechanism is also responsible for ATP1B3-mediated HBV suppression. Here, we demonstrated that ATP1B3 reduced the expression of HBV envelope proteins LHBs, MHBs and SHBs, but had no effect on intracellular HBV DNA, RNA levels as well as HBV promoter activities. Further investigation showed that proteasome inhibitor MG132 rescued ATP1B3-mediated envelope proteins degradation, demonstrating that proteasome-dependent pathway is involved in ATP1B3-induced degradation of envelope proteins. Co-IP showed that ATP1B3 interacts with LHBs and MHBs and induces LHBs and MHBs polyubiquitination. Immunofluorescence colocalization analysis confirmed LHBs and MHBs colocalized with ATP1B3 together. Our work provides important information for targeting ATP1B3 as a potential therapeutic molecule for HBV infection.