Molecular Mechanisms Of Hepatitis B Viral infection, Pathogenesis And Persistence

乙型肝炎病毒感染、发病机制和持续性的分子机制

• 批准号: 10697773
• 负责人: T. Jake Liang
• 金额: $ 170.73万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10697773
• 关键词: ATP phosphohydrolase; Affect; Africa; Antibiotics; Antiviral Response; Bioinformatics; Biological Assay; Biological Models; Biological Process; Biology; CHD4 gene; CMV promoter; Cell Cycle Progression; Cell Nucleus; Cells; Circular DNA; Clinical; Complex; DNA-Binding Proteins; Dependence; Enhancers; Enzyme-Linked Immunosorbent Assay; Epigenetic Process; Europe; Exhibits; Fluorescence; Gene Expression; Genes; Genetic Transcription; Genotype; HBV Genotype; HepG2; Hepatitis; Hepatitis B; Hepatitis B Infection; Hepatitis B Surface Antigens; Hepatitis B Virus; Hepatitis B e Antigens; Hepatocyte; Homologous Gene; Human; Immune; In Vitro; Individual; Infection; Integration Host Factors; Interferons; Intervention; Investigation; KRAS2 gene; Kinetics; Knock-in; Knock-out; Length; Life Cycle Stages; Literature; Liver; Liver diseases; Modeling; Molecular; Monitor; Mus; Natural Immunity; Neuroblastoma; Nuclear Receptors; Nucleic Acids; Pathogenesis; Patients; Persons; Phenotype; Plasmids; Play; Post-Transcriptional Regulation; Process; Production; Protein Isoforms; Proteins; RNA; Reporter; Reporting; Role; Small Interfering RNA; Stains; Subgroup; System; T-Lymphocyte; Testing; Time; Toxic effect; Transfection; United States; Validation; Viral; Viral Antigens; Viral Genome; Viremia; Virus; Virus Diseases; Virus Replication; Virus-Cell Membrane Interaction; Western Blotting; base; curative treatments; density; design; epigenetic regulation; extracellular; in vivo; inhibitor; knock-down; lipid biosynthesis; mouse model; novel; novel strategies; nucleolin; p97 ATPase; pgRNA; promoter; ras Oncogene; response; screening; stable cell line; therapeutic development; valosin-containing protein; viral DNA; viral RNA; whole genome

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.

A 基因型 HBV 有两种主要亚型：A1（常见于非洲）和 A2（常见于欧洲）。感染这两种亚型的个体似乎具有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尚未确定。产生高滴度 HBV (HBVcc) 基因型 A1 和 A2 的稳定细胞系是通过将具有复制能力的 1.3X 长度 HBV 基因组转染到 HepG2 细胞中含有抗生素标记的质粒中而产生的，该质粒可支持 HBV 复制。用抗生素选择转染的 HepG2 细胞，并评估分离的克隆的培养基中的 HBeAg 和 HBV DNA 水平。选择并扩增具有最高水平 HBV DNA 和/或 HBeAg 的克隆，用于大规模纯化 HBVcc。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测试了基因型 A1 和 A2 的 HBVcc。在 PXB 细胞中，HBVcc A2 亚型感染显示出比 A1 亚型感染更高的 HBV 病毒和 HBV 抗原产量。两种亚型的HBVcc对人源化嵌合小鼠的感染效果相同，A2亚型似乎在感染早期也表现出较高的病毒血症和抗原血症动力学。 HBVcc 在感染 PXB 细胞的人源化嵌合小鼠 (HBVmp) 中的传代效率比原始 HBVcc 病毒储液的传代效率高得多。在 PXB 细胞中，A2 亚型的 HBVmp 再次表现出比 A1 亚型更高的病毒复制水平。 HBVmp 的密度梯度分析和感染研究进一步支持 A2 具有更高的复制表型。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 感染患者病毒血症较高的原因。这两种亚型对人 IFN-α 表现出相似的反应，在受感染的 PXB 细胞和小鼠中，HBV DNA、HBeAg 和 HBsAg 均减少。在这里，我们证明了 HBV A1 和 A2 亚型的 HBVcc 可以在体外和体内传代，并且在这些模型中对人干扰素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，这可能解释了所报告的两种亚型患者临床表现的差异。 HBV 感染肝细胞，并在细胞核内以微型染色体的形式建立，称为共价闭合环状 DNA (cccDNA)，它作为所有病毒产物的转录模板，并且能够自我补充。其稳定性是乙肝病毒在接受核苷（酸）抑制剂治疗的感染患者肝脏中长期持续存在的原因，核苷（酸）抑制剂可有效阻止病毒复制，但对消除乙肝病毒cccDNA影响很小或没有影响。因此，识别 cccDNA 相关宿主因子及其功能不仅对于了解 HBV 的基础生物学很重要，而且还有助于设计针对 cccDNA 进行潜在治疗的新策略。最近，我们从 HBV 感染的细胞中分离和纯化了 cccDNA 相关宿主蛋白，并通过质谱鉴定 Nucleolin 是关键的 cccDNA 结合蛋白，在 HBV 感染中发挥着重要作用。核仁素是 HBV cccDNA 微型染色体的组成部分，对 HBV 转录发挥表观遗传调控作用。 Nucleolin 与 cccDNA 的表观遗传机制相互作用并对其进行功能性调节的表观遗传调控机制仍有待进一步阐明。 p97/VCP（含缬氨肽的蛋白）是一种细胞 ATP 酶蛋白，此前已被证明在许多病毒的生命周期中发挥作用。迄今为止，其生物学功能和原病毒作用似乎都依赖于其 ATP 酶功能。我们假设 p97 在 HBV 生命周期中发挥重要作用。用非靶向对照 (siNT) 和 p97 siRNA 处理原代人肝细胞，并用 HBV 感染或用 HBV 构建体转染。用多种针对 p97 ATP 酶功能的抑制剂处理感染 HBV 的细胞。感染后监测 HBV 标志物（HBeAg、HBsAg、HBV DNA 和细胞内 HBV RNA）。 sip97 处理导致 p97 RNA 和蛋白质水平降低超过 80% (?)。用 sip97 处理的 HBV 感染细胞显示所有 HBV 标志物显着降低，且没有细胞毒性。使用各种HBV构建体进一步探讨p97敲低效应的靶点和机制。使用 cccDNA HBV 报告系统（mcHBV-Gluc，参考文献）显示了 p97 敲低的类似效果，支持 p97 在 HBV 基因表达中的作用。然而，HBV 增强子/核心启动子报告构建体 (Enh1Cp-Fluc) 的活性不受 sip97 处理的影响。我们还表明 p97 敲低对 CMV 启动子驱动的报告构建体没有影响，表明该影响不影响转录。使用由 CMV 启动子驱动的 HBV pgRNA 表达质粒（仅产生 HBV pgRNA，参考文献），我们发现 sip97 处理可降低 HBV pgRNA 和 HBc 蛋白水平，表明 p97 在 HBV RNA 水平转录后调节中的作用。 p97 ATPase 抑制剂对 HBV 感染细胞中的 HBV 转录和复制没有影响。在这里，我们首次证明了 p97 的新作用，与其 ATP 酶功能无关，但对 HBV 复制至关重要。我们的机制研究表明 p97 在转录后水平调节 HBV 基因表达中具有新作用。 p97 可能作为 HBV 治疗开发的新靶点。 我们进行了高通量全基因组 siRNA 筛选，旨在确定调节 HBV 感染的宿主因素。使用 HepG2-NTCP 细胞，我们鉴定了 3810 个与 HBV 感染有关的宿主基因。为了进行验证，我们根据生物信息学分析和标准感染测定文献选择了一个命中子组 (n=80)。通过使用 ELISA、PCR、蛋白质印迹和荧光染色评估细胞内和细胞外 HBV 抗原表达和核酸水平，我们证实核受体辅激活因子 5 (NCOA5) 或染色结构域解旋酶 DNA 结合蛋白 4 (CHD4) 是 HBV 复制所必需的，因为它们的敲除 (KD) 会导致 HBV 复制减少。 HepG2-NTCP 和原代人肝细胞 (PHH)。另一方面，神经母细胞瘤RAS病毒（v-ras）癌基因同源物（NRAS）被证实是一种抗病毒宿主因子，因为其KD显着增加了HBV感染的HepG2-NTCP细胞中的所有HBV标志物。通过使用小环 HBV (mcHBV) 转染或 Huh7-NTCP 细胞等替代模型也获得了类似的结果。为了排除 siRNA 潜在的脱靶效应，构建了 NRAS 敲除 (KO) HepG2-NTCP 细胞，与对照细胞相比，KO 细胞中的 HBV 感染表现出显着增加。还评估了 RAS 不同亚型对 HBV 感染的影响。结果表明，HepG2-NTCP 中的 KRAS 而不是 HRAS KD 会增强 HBV 感染。机制研究表明，NRAS 激活减少与细胞周期进程改变和肝细胞脂肪生成增强有关，这可能有利于 HBV 感染。更多揭示分子细节的研究正在进行中。因此，我们的筛查发现了多种参与乙型肝炎病毒生命周期的新宿主因素。我们的研究强调了 HBV 对宿主功能的密切依赖性，并为治疗开发提供了干预与 HBV 感染相关的宿主功能的潜在方法。