Mechanisms of HBV cccDNA transcriptional regulation in persons with and without HIV

HIV感染者和未感染者的HBV cccDNA转录调控机制

• 批准号: 10882261
• 负责人: ASHWIN BALAGOPAL
• 金额: $ 81.1万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-14 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10882261
• 关键词: Anti-Retroviral Agents; Binding; Biological Assay; CD4 Positive T Lymphocytes; Candidate Disease Gene; Cause of Death; Cell Count; Cells; Cessation of life; Characteristics; Chronic; Chronic Hepatitis B; Circular DNA; Clinical; Complementary DNA; CpG Islands; CpG dinucleotide; Cytoplasm; DNA Methylation; Disease Outcome; Epigenetic Process; Funding; Gene Expression Profiling; Gene Silencing; Genes; Genetic; Genetic Transcription; Goals; HIV; HepG2; Hepatitis; Hepatitis B Therapy; Hepatitis B Virus; Hepatocyte; Human; Immune; Immune response; In Vitro; Individual; Integration Host Factors; Interruption; Knowledge; Liver; Liver diseases; Measurable; Measures; Messenger RNA; Methods; Modeling; Modification; Mutation; Nonsense Codon; Pathway interactions; Persons; Plasma; Primary carcinoma of the liver cells; Process; RNA; RNA Splicing; Resolution; Reverse Transcription; Reverse Transcription Inhibition; Sampling; T-Cell Depletion; Techniques; Testing; Therapeutic; Tissues; Transcript; Transcriptional Regulation; Use of New Techniques; Variant; Viral; Viral Genes; Viral hepatitis; Virion; Virus Diseases; Virus Replication; analog; analytical method; cDNA Library; circular RNA; co-infection; digital; end stage liver disease; immune activation; in vitro testing; indexing; insertion/deletion mutation; interest; intrahepatic; laser capture microdissection; multidisciplinary; nanopore; next generation sequencing; novel; response; therapy development; tool; transcriptome; transcriptome sequencing; viral DNA; viral RNA; viral rebound; virus genetics

Hepatitis B virus (HBV) infects >300 million people chronically and is the leading cause of end-stage liver disease and hepatocellular carcinoma (HCC), resulting in ~1 million deaths annually. HIV is a common co- infection in people with chronic hepatitis B (CHB) and worsens HBV and liver disease outcomes. Liver disease is a leading cause of death among PWH in the era of antiretrovirals, mostly due to viral hepatitis. Antiviral treatment with nucleos(t)ide analogues (NUCs) suppresses plasma HBV DNA but does not eliminate the covalently closed circular DNA (cccDNA) HBV template that resides in every infected cell. NUC interruption leads to HBV DNA rebound and clinical hepatitis. Therefore, finding an HBV cure is of major importance for HBV mono- and HIV/HBV co-infection. In the first funding cycle, we applied single-cell methods to HBV/HIV co-infected liver tissues and demonstrated that cccDNA is transcriptionally suppressed during NUCs. We now propose to understand the mechanism underlying this finding since permanently exploiting this mechanism therapeutically can lead to a functional cure. We propose an intensive study of NUC-associated cccDNA transcriptional suppression comparing people with HBV/HIV co- and HBV mono-infection. In aim one, we will quantify intrahepatic viral DNA and RNA quantities in hepatocytes from HBV mono- and HBV/HIV co-infected individuals using single-cell laser capture microdissection and our novel droplet digital PCR assays. We will quantify and compare the cccDNA transcriptional index (cccDNA TI), a measure of cccDNA transcription, in HBV mono- and HBV/HIV co- infection. In aim two we will use novel techniques to test whether cccDNA transcriptional suppression is due to genetic or epigenetic modifications of HBV using HBV mono- and HBV/HIV co-infected liver tissues from aim one. We will use Cas9-Nanopore sequencing to examine epigenetic modifications of CpG dinucleotides in cccDNA. We will use the NovaSeq platform to perform next-generation sequencing of cccDNA to look for mutations that may be associated with reduced transcription and to quantify global cccDNA transcriptional suppression of canonical viral genes and splice variants. In aim three we will test the hypothesis that the host response contributes to cccDNA transcriptional suppression and compare this response between HBV mono- and HBV/HIV co-infection. We will apply the novel 10X Visium spatial gene expression analysis (10x VSGEA) platform to liver tissue from Aim one to compare hepatocyte host transcriptomes in tissues with high vs. low cccDNA TI, directly interrogating HBV transcription using the same platform. We will confirm genes identified with 10X VSGEA using Tecan RNA sequencing, enriching for cells with high vs. low cccDNA TI. We will conduct in vitro studies to prove that genes of interest are associated with cccDNA transcriptional suppression, focusing on innate immune genes and HIV-associated immune activation. Our proposal fills knowledge gaps in understanding how to transform reversible cccDNA transcriptional suppression to a functional cure.

乙肝病毒(乙肝病毒)慢性感染3亿人，是终末期肝病的主要原因 疾病和肝细胞癌，每年导致约100万人死亡。艾滋病毒是一种常见的共同疾病 慢性乙型肝炎(CHB)患者的感染会加重乙肝病毒和肝脏疾病的结局。肝病 在抗逆转录病毒药物时代，是PWH死亡的主要原因，主要是由病毒性肝炎引起的。抗病毒药物 用核素类似物(NUCS)治疗可抑制血浆HBVDNA，但不能消除 共价闭合环状DNA(CccDNA)驻留在每个受感染细胞中的HBV模板。NUC中断 导致HBVDNA反弹和临床肝炎。因此，找到一种治愈乙肝病毒的方法对 乙肝病毒单一感染和艾滋病毒/乙肝病毒混合感染。 在第一个资助周期中，我们将单细胞方法应用于乙肝病毒/艾滋病病毒混合感染的肝组织，并展示了 该cccDNA在核转录过程中被抑制。我们现在建议了解这一机制 这一发现的基础，因为永久地利用这一机制在治疗上可以导致功能性 解药。我们建议深入研究与NUC相关的cccDNA转录抑制的比较 乙肝病毒/艾滋病病毒混合感染者和乙肝病毒单一感染者。在第一个目标中，我们将量化肝内病毒DNA和 单细胞激光检测乙肝病毒单一感染者和乙肝病毒/艾滋病病毒混合感染者肝细胞的RNA含量 捕获显微切割和我们的新型液滴数字聚合酶链式反应分析。我们将对cccDNA进行量化和比较 转录指数(CcCDNATI)，一种衡量cccDNA转录的指标，在乙肝病毒单基因和乙肝病毒/艾滋病病毒联合感染中 感染。在第二个目标中，我们将使用新技术来测试cccDNA转录抑制是否是由于 利用来自AIM的HBV单感染和HBVHIV混合感染的肝组织进行乙肝病毒的遗传或表观遗传修饰 一。我们将使用Cas9-Nanopore测序来检测CpG二核苷酸的表观遗传修饰 CccDNA。我们将使用NovaSeq平台对cccDNA进行下一代测序，以寻找 可能与转录减少相关的突变，并量化全球cccDNA转录 抑制典型病毒基因和剪接变异体。在第三个目标中，我们将测试宿主的假设 反应有助于cccDNA转录抑制，并比较这种反应在乙肝病毒单抗和 和乙肝病毒/艾滋病病毒混合感染。我们将应用新的10倍空间基因表达分析(10倍VSGEA) 从Aim One到肝组织的平台，以比较高和低组织中的肝细胞宿主转录 CccDNATI，在同一个平台上直接查询乙肝病毒转录。我们将确认已确认的基因 用10倍VSGEA进行Tecan RNA测序，丰富了高ccCDNATI与低cccCDNATI的细胞。我们会 进行体外研究，以证明感兴趣的基因与cccDNA转录抑制有关， 关注先天免疫基因和艾滋病毒相关免疫激活。我们的建议填补了以下方面的知识空白 了解如何将可逆的cccDNA转录抑制转化为功能治愈。