Molecular Mechanisms Of Hepatitis B Viral infection, Pathogenesis And Persistence

HBV基因型A有两个主要亚型，A1（常见于非洲）和A2（常见于欧洲）。感染这两种亚型的个体表现出不同的临床表现和病毒学特征。这种差异是由病毒还是宿主造成的还没有确定。在HepG2细胞中，用含有抗生素标记物的质粒转染具有复制能力的1.3倍长度的HBV基因组，产生了具有A1和A2基因型的高滴度HBV （HBVcc）的稳定细胞系。用抗生素选择转染的HepG2细胞，并评估培养基中HBeAg和HBV DNA的水平。选择具有最高水平HBV DNA和/或HBeAg的克隆并扩增用于HBVcc的大规模纯化。在小鼠传代的原代人肝细胞（PXB细胞）和人源化嵌合小鼠模型中检测基因型A1和A2的HBVcc。在PXB细胞中，感染HBV亚型A2的细胞比感染亚型A1的细胞产生更高的HBV病毒和HBV抗原。两种亚型感染人源嵌合小鼠的HBVcc均表现良好，A2亚型在感染早期似乎也表现出较高的病毒血症和抗原血症的动力学。HBVcc在感染PXB细胞的人源化嵌合小鼠（HBVmp）中传代的效率远远高于原始HBVcc病毒库。在PXB细胞中，A2亚型HBVmp再次显示出比A1亚型更高的病毒复制水平。密度梯度分析和HBVmp的感染研究进一步支持A2的高复制表型。机制研究表明A2亚型在转录上比A1更活跃，这可能解释了基因型A2感染患者中较高的病毒血症。在感染的PXB细胞和小鼠中，这两种亚型对人ifn - α表现出相似的反应，同时降低HBV DNA、HBeAg和HBsAg。在这里，我们证明了HBV亚型A1和A2的HBVcc可以在体外和体内传代，并且在这些模型中对人类IFN-治疗的反应同样良好。A2亚型可能具有较高复制的固有表型，这可能解释了两种亚型患者在临床表现上的差异。

HBV genotype A have two major subtypes, A1 (commonly in Africa) and A2 (commonly in Europe). Individuals infected with these two subtypes appear to have different clinical manifestations and virologic features. Whether such a difference results from the virus or host has not been established.Stable cell lines producing high titer of HBV (HBVcc) with genotype A1 and A2 were generated by transfecting replication-competent 1.3X length HBV genome in a plasmid containing an antibiotic marker in HepG2 cells that can support HBV replication. Transfected HepG2 cells were selected with antibiotics and isolated clones were assessed for levels of HBeAg and HBV DNA in the media. Clones with highest levels of HBV DNA and/or HBeAg were selected and expanded for large-scale purification of HBVcc. HBVcc of genotype A1 and A2 were tested in mouse-passaged primary human hepatocytes (PXB cells) and humanized chimeric mouse model. In PXB cells, infection with HBVcc subtype A2 showed a higher production of HBV virus and HBV antigens than subtype A1 infection. HBVcc of both subtypes infected humanized chimeric mice equally well, and subtype A2 also seemed to show a kinetics of higher viremia and antigenemia in early stage of infection. HBVcc passaged in humanized chimeric mice (HBVmp) infected PXB cells much more efficiently than that of the original HBVcc viral stock. The HBVmp of subtype A2 again showed a higher viral replication level than that of subtype A1 in PXB cells. Density gradient analysis and infection study of HBVmp further supported a higher replication phenotype of A2. Mechanistic investigations suggested that subtype A2 is transcriptionally more active than A1, potentially explaining the higher viremia in genotype A2-infected patients. The two subtypes exhibited similar responses to human IFN-alfa with reduction of HBV DNA, HBeAg and HBsAg in both infected PXB cells and mice. Here we demonstrate that HBVcc of HBV subtype A1 and A2 can be passaged in vitro and in vivo and respond equally well to human IFN- treatment in these models. Subtype A2 may have an inherent phenotype of higher replication, possibly explaining the reported difference in clinical manifestations of the two subtypes in patients. HBV infects hepatocytes and establishes itself within the nucleus as a mini-chromosome referred to as covalently closed circular DNA (cccDNA), which serves as the transcriptional template for all viral products and is capable of replenishing itself. Its stability accounts for the long-term persistence of HBV in the liver of infected patients treated with nucleos(t)ide inhibitors, which are effective in blocking viral replication but have little or no impact on eliminating HBV cccDNA. Therefore, identifying cccDNA-associated host factors and their functions is not only important in understanding the basic biology of HBV but also relevant to designing novel strategies to target cccDNA for potential curative therapy. Recently we isolated and purified cccDNA-associated host proteins from HBV-infected cells and identified by mass spec Nucleolin as a key cccDNA-binding protein that plays an important role in HBV infection. Nucleolin is an integral component of the HBV cccDNA mini-chromosome and exerts epigenetic regulation of HBV transcription. The mechanism of epigenetic regulation whereby Nucleolin interacts with and functionally modulates the epigenetic machinery of cccDNA remains to be further elucidated. p97/VCP (valosin-containing protein), a cellular ATPase protein, had previously been shown to play a role in the life cycle of many viruses. To date, both of its biological functions and proviral role appear to be dependent on its ATPase function. We hypothesize that p97 plays an important role in HBV life cycle. Primary human hepatocytes were treated with nontargeting control (siNT) and p97 siRNAs and infected with HBV or transfected with HBV constructs. Cells infected with HBV were treated with various inhibitors targeting the ATPase function of p97. HBV markers (HBeAg, HBsAg, HBV DNA and intracellular HBV RNA) were monitored post-infection. sip97 treatment resulted in more than 80% (?) knockdown of p97 RNA and protein levels. HBV-infected cells treated with sip97 showed significant reduction of all HBV markers without cell toxicity. Various HBV constructs were used to further explore the target and mechanism of the p97 knockdown effect. Use of a cccDNA HBV reporter system (mcHBV-Gluc, ref) showed a similar effect of p97 knockdown, supporting a role of p97 in HBV gene expression. The activity of an HBV enhancers/core promoter reporter construct (Enh1Cp-Fluc), however, was not affected by sip97 treatment. We also showed that p97 knockdown had no effect on a CMV promoter-driven reporter construct, suggesting the effect is not on transcription. Using a HBV pgRNA expression plasmid driven by the CMV promoter (production of HBV pgRNA only, ref), we showed that HBV pgRNA and HBc protein levels were reduced by sip97 treatment, suggesting a role of p97 in post-transcriptional regulation of HBV RNA levels. p97 ATPase inhibitors had no effect on HBV transcription and replication in HBV-infected cells. Here, we demonstrate for the first time a novel role of p97 that is not associated with its ATPase function and critical for HBV replication. Our mechanistic studies suggest a novel role of p97 in regulating HBV gene expression at the post-transcriptional level. p97 may serve as a novel target for HBV therapeutic development. We conducted a high-throughput, whole-genome siRNA screening aimed at identifying host factors regulating HBV infection. Using HepG2-NTCP cells, we identified 3810 host genes involved in HBV infection. For validation, we selected a subgroup of hits (n=80) based on bioinformatic analyses and literature for standard infection assay. By assessing the intracellular and extracellular HBV antigen expression and nucleic acid levels using ELISA, PCR, Western blot and fluorescence staining, we confirmed that nuclear receptor coactivator 5 (NCOA5) or chromodomain-helicase-DNA-binding protein 4 (CHD4) was required for HBV replication as their knocking down (KD) led to reduced HBV replication in both HepG2-NTCP and primary human hepatocyte (PHH). On the other hand, neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS) was confirmed to be an antiviral host factor as its KD profoundly increased all the HBV markers in HBV-infected HepG2-NTCP cells. Similar results were obtained by using alternative models like minicircle HBV (mcHBV) transfection or Huh7-NTCP cells. To exclude the potential off-target effects of siRNA, NRAS knocking out (KO) HepG2-NTCP cells were constructed and HBV infection exhibited remarkable increase in the KO cells compared to control cells. The effects of different isoforms of RAS on HBV infection were also evaluated. The results showed that KRAS but not HRAS KD in HepG2-NTCP boosted HBV infection. Mechanistic study demonstrated reduced NRAS activation was associated with altered cell cycle progression and enhanced lipogenesis of the hepatocyte, which could be beneficial for HBV infection. More investigations uncovering the molecular details are ongoing. Hence, our screening identified multiple novel host factors involved in HBV life cycle. Our study emphasized the close dependence of HBV on host functions and provided potential means of intervention of host functions that are implicated in HBV infection for therapeutic development.